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Sugawara R, Hamada K, Ito H, Scala M, Ueda H, Tabata H, Ogata K, Nagata KI. A p.N92K variant of the GTPase RAC3 disrupts cortical neuron migration and axon elongation. J Biol Chem 2025; 301:108346. [PMID: 40015633 PMCID: PMC11968283 DOI: 10.1016/j.jbc.2025.108346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Revised: 02/05/2025] [Accepted: 02/19/2025] [Indexed: 03/01/2025] Open
Abstract
RAC3 encodes a small GTPase of the Rho family, crucial for actin cytoskeleton organization and signaling pathways. De novo deleterious variants in RAC3 cause neurodevelopmental disorder with structural brain anomalies and dysmorphic facies (NEDBAF). Disease-causing variants thus far reported are thought to impact key conserved regions within RAC3, such as the P-loop, switch I/II, and G boxes, which are essential for the interaction with regulatory proteins and effectors. Recently, however, a novel variant, c.276T > A, p.N92K, was identified in a prenatal case with complex brain malformations. This variant, located outside the core functional regions, represents a unique class of RAC3 pathogenic mutations. We investigated the variant's effects using in vitro, in silico, and in vivo approaches. Overexpression of RAC3-N92K in primary hippocampal neurons impaired differentiation, leading to round cell shape with lamellipodia, suggesting that RAC3-N92K is active. Biochemical studies showed that RAC3-N92K is (1) resistant to GAP-mediated inactivation, (2) responsive to GEF activation, and (3) capable of interacting with RAC effectors PAK1 and MLK2, as well as Rho-kinase 1, activating gene expression through SRF, NFκB, and AP1 pathways. Structural analyses suggest that N92K disrupts GAP interactions but preserves interactions with GEF, PAK1, and MLK2. In vivo, RAC3-N92K expression in embryonic mouse cortical neurons led to migration defects and periventricular clustering during corticogenesis, along with impaired axon elongation. These findings indicate that RAC3-N92K's activated state significantly disrupts cortical development, expanding the genetic and pathophysiological spectrum of NEDBAF.
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Affiliation(s)
- Ryota Sugawara
- Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan
| | - Keisuke Hamada
- Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan
| | - Hidenori Ito
- Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan
| | - Marcello Scala
- Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy; Unit of Medical Genetics, IRCCS Giannina Gaslini Institute, Genova, Italy
| | - Hiroshi Ueda
- United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan; Center for One Medicine Innovative Translational Research (COMIT), Gifu University, Gifu, Japan
| | - Hidenori Tabata
- Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan
| | - Kazuhiro Ogata
- Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan.
| | - Koh-Ichi Nagata
- Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan; Department of Neurochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan.
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Burguera S, Vidal L, Bauzá A. Aluminum Fluorides as Noncovalent Lewis Acids in Proteins: The Case of Phosphoryl Transfer Enzymes. Chempluschem 2025; 90:e202400578. [PMID: 39363715 DOI: 10.1002/cplu.202400578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Revised: 10/02/2024] [Accepted: 10/02/2024] [Indexed: 10/05/2024]
Abstract
The Protein Data Bank (PDB) was scrutinized for the presence of noncovalent O ⋅ ⋅ ⋅ Al Triel Bonding (TrB) interactions, involving protein residues (e. g. GLU and GLN), adenosine/guanine diphosphate moieties (ADP and GDP), water molecules and two aluminum fluorides (AlF3 and AlF4 -). The results were statistically analyzed, revealing a vast number of O ⋅ ⋅ ⋅ Al contacts in the active sites of phosphoryl transfer enzymes, with a marked directionality towards the Al σ-/π-hole. The physical nature of the TrBs studied herein was analyzed using Molecular Electrostatic Potential (MEP) maps, the Quantum Theory of Atoms in Molecules (QTAIM), the Non Covalent Interaction plot (NCIplot) visual index and Natural Bonding Orbital (NBO) studies. As far as our knowledge extends, it is the first time that O ⋅ ⋅ ⋅ Al TrBs are analyzed within a biological context, participating in protein trapping mechanisms related to phosphoryl transfer enzymes. Moreover, since they are involved in the stabilization of aluminum fluorides inside the protein's active site, we believe the results reported herein will be valuable for those scientists working in supramolecular chemistry, catalysis and rational drug design.
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Affiliation(s)
- Sergi Burguera
- Department of Chemistry, Universitat de les Illes Balears, Ctra. de Valldemossa, km. 7.5, 07122, Palma de Mallorca, Islas Baleares, Spain
| | - Lenin Vidal
- Department of Chemistry, Universitat de les Illes Balears, Ctra. de Valldemossa, km. 7.5, 07122, Palma de Mallorca, Islas Baleares, Spain
| | - Antonio Bauzá
- Department of Chemistry, Universitat de les Illes Balears, Ctra. de Valldemossa, km. 7.5, 07122, Palma de Mallorca, Islas Baleares, Spain
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3
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Murayama K, Kato-Murayama M, Hosaka T, Kitamura T, Yokoyama S, Shirouzu M. Structural basis for the effects of Ser387 phosphorylation of MgcRacGAP on its GTPase-activating activities for CDC42 and RHOA. J Struct Biol 2024; 216:108151. [PMID: 39522789 DOI: 10.1016/j.jsb.2024.108151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Revised: 11/01/2024] [Accepted: 11/07/2024] [Indexed: 11/16/2024]
Abstract
MgcRacGAP is a GTPase-activating protein (GAP) for the Rho family GTPases. During cytokinesis, MgcRacGAP localizes to the midbody, where it activates the GTPase activity of Rho family GTPases to facilitate cytokinesis. In the midbody, Aurora B phosphorylates Ser387 within the GAP domain of human MgcRacGAP, a modification that is suggested to influence GTPase preference. However, there are conflicting reports, with some studies indicating that Ser387 phosphorylation does not alter the GTPase preference of MgcRacGAP. This controversy highlights the need for a deeper understanding of the molecular interactions involved, which can be clarified through structural analyses. In the present study, we determined the crystal structures of the wild-type MgcRacGAP GAP domain complexed with CDC42•GDP•AlF4- and the S378D phosphomimetic mutant GAP domain fused with RHOA•GDP•AlF4-. Additionally, crystal structures of the GAP domains were determined for the S387D and S387A mutants. Our analysis revealed that neither GTPase binding nor S387D mutation affected the overall structure of the GAP domain. However, comparison of the CDC42•MgcRacGAP (wild-type) complex with the RHOA-MgcRacGAP(S378D) fusion protein structure indicated that the S387D mutation caused positional shifts in both CDC42 and RHOA relative to MgcRacGAP. These shifts reduced interactions with CDC42 more severely than those with RHOA. In fact, the S387D mutation decreased the GTPase-activating activity of MgcRacGAP toward CDC42, while its impact on RHOA was only moderate. This difference in the rate of activity reduction may play an important role in GTPase preference.
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Affiliation(s)
- Kazutaka Murayama
- Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, 2-1 Seiryo, Aoba, Sendai 980-8575, Japan; RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
| | - Miyuki Kato-Murayama
- RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
| | - Toshiaki Hosaka
- RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
| | - Toshio Kitamura
- Division of Cellular Therapy, Institute of Medical Sciences, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
| | - Shigeyuki Yokoyama
- RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
| | - Mikako Shirouzu
- RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
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González B, Aldea M, Cullen PJ. Chaperone-Dependent Degradation of Cdc42 Promotes Cell Polarity and Shields the Protein from Aggregation. Mol Cell Biol 2023; 43:200-222. [PMID: 37114947 PMCID: PMC10184603 DOI: 10.1080/10985549.2023.2198171] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 03/02/2023] [Accepted: 03/06/2023] [Indexed: 04/29/2023] Open
Abstract
Rho GTPases are global regulators of cell polarity and signaling. By exploring the turnover regulation of the yeast Rho GTPase Cdc42p, we identified new regulatory features surrounding the stability of the protein. We specifically show that Cdc42p is degraded at 37 °C by chaperones through lysine residues located in the C-terminus of the protein. Cdc42p turnover at 37 °C occurred by the 26S proteasome in an ESCRT-dependent manner in the lysosome/vacuole. By analyzing versions of Cdc42p that were defective for turnover, we show that turnover at 37 °C promoted cell polarity but was defective for sensitivity to mating pheromone, presumably mediated through a Cdc42p-dependent MAP kinase pathway. We also identified one residue (K16) in the P-loop of the protein that was critical for Cdc42p stability. Accumulation of Cdc42pK16R in some contexts led to the formation of protein aggregates, which were enriched in aging mother cells and cells undergoing proteostatic stress. Our study uncovers new aspects of protein turnover regulation of a Rho-type GTPase that may extend to other systems. Moreover, residues identified here that mediate Cdc42p turnover correlate with several human diseases, which may suggest that turnover regulation of Cdc42p is important to aspects of human health.
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Affiliation(s)
- Beatriz González
- Department of Biological Sciences, State University of New York at Buffalo, New York, USA
| | - Martí Aldea
- Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona, Spain
| | - Paul J. Cullen
- Department of Biological Sciences, State University of New York at Buffalo, New York, USA
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González B, Cullen PJ. Regulation of Cdc42 protein turnover modulates the filamentous growth MAPK pathway. J Cell Biol 2022; 221:213675. [PMID: 36350310 PMCID: PMC9811999 DOI: 10.1083/jcb.202112100] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 05/25/2022] [Accepted: 09/19/2022] [Indexed: 11/11/2022] Open
Abstract
Rho GTPases are central regulators of cell polarity and signaling. How Rho GTPases are directed to function in certain settings remains unclear. Here, we show the protein levels of the yeast Rho GTPase Cdc42p are regulated, which impacts a subset of its biological functions. Specifically, the active conformation of Cdc42p was ubiquitinated by the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones and turned over in the proteasome. A GTP-locked (Q61L) turnover-defective (TD) version, Cdc42pQ61L+TD, hyperactivated the MAPK pathway that regulates filamentous growth (fMAPK). Cdc42pQ61L+TD did not influence the activity of the mating pathway, which shares components with the fMAPK pathway. The fMAPK pathway adaptor, Bem4p, stabilized Cdc42p levels, which resulted in elevated fMAPK pathway signaling. Our results identify Cdc42p turnover regulation as being critical for the regulation of a MAPK pathway. The control of Rho GTPase levels by stabilization and turnover may be a general feature of signaling pathway regulation, which can result in the execution of a specific developmental program.
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Affiliation(s)
- Beatriz González
- Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY
| | - Paul J. Cullen
- Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY,Correspondence to Paul J. Cullen:
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Huang Q, Xie J, Seetharaman J. Crystal Structure of Schizosaccharomyces pombe Rho1 Reveals Its Evolutionary Relationship with Other Rho GTPases. BIOLOGY 2022; 11:biology11111627. [PMID: 36358328 PMCID: PMC9687936 DOI: 10.3390/biology11111627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 10/23/2022] [Accepted: 11/01/2022] [Indexed: 11/09/2022]
Abstract
Simple Summary Rho family of proteins are involved in cytoskeletal organization, cell mobility and polarity, and are implicated in cancer morphogenesis. The structure and function of the Rho homologs from higher-level organisms are well studied, but not from the lower-level organisms. Such as over 95% of the known structures of Rho GTPases are from higher-order mammalian organisms, with only three structures of Rho homologs reported to date from lower-level, single-celled organisms. In this paper we report the crystal structure of Rho1 from Schizosaccharomyces pombe, also called fission yeast (SpRho1), in complex with GDP in the presence of Mg2+ at 2.63-Å resolution, to broaden our understanding of Rho homologs in lower-level organisms. Although the overall structure is similar to that of known Rho homologs, we observed subtle differences at the Switch I and II regions, in β2 and β3, and in the Rho insert domain and loop from Phe107 to Pro112. Combined with literature and sequence analyses, we suggest that the Switch regions and Rho insert domain may contribute to downstream kinase activation in different species through their interactions with different effectors and regulators; and the conservation and divergence of Rho GTPases among difference species and provide evolutionary insight for SpRho1. While many studies have reported the evolutionary development of Rho GTPases based on their amino acid sequences, the present study, for the first time, explores these evolutionary aspects based on structure. Our analysis indicates that SpRho is evolutionarily closer to HsRhoC than HsRhoA, as previously believed. Abstract The Rho protein, a homolog of Ras, is a member of the Ras superfamily of small GTPases. Rho family proteins are involved in cytoskeletal organization, cell mobility, and polarity, and are implicated in cancer morphogenesis. Although Rho homologs from higher-order mammalian organisms are well studied, there are few studies examining Rho proteins in lower-level single-celled organisms. Here, we report on the crystal structure of Rho1 from Schizosaccharomyces pombe (SpRho1) in complex with GDP in the presence of Mg2+ at a 2.78 Å resolution. The overall structure is similar to that of known Rho homologs, including human RhoA, human RhoC, and Aspergillus fumigatus Rho1 (AfRho1), with some exceptions. We observed subtle differences at the Switch I and II regions, in β2 and β3, and in the Rho insert domain and loop from Phe107 to Pro112. Our analysis suggests that SpRho is evolutionarily closer to HsRhoC than HsRhoA, as previously believed.
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Affiliation(s)
- Qingqing Huang
- Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
| | - Jiarong Xie
- Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
| | - Jayaraman Seetharaman
- Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
- Correspondence:
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Kozlova MI, Shalaeva DN, Dibrova DV, Mulkidjanian AY. Common Mechanism of Activated Catalysis in P-loop Fold Nucleoside Triphosphatases-United in Diversity. Biomolecules 2022; 12:1346. [PMID: 36291556 PMCID: PMC9599734 DOI: 10.3390/biom12101346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2022] [Revised: 08/20/2022] [Accepted: 09/14/2022] [Indexed: 11/16/2022] Open
Abstract
To clarify the obscure hydrolysis mechanism of ubiquitous P-loop-fold nucleoside triphosphatases (Walker NTPases), we analysed the structures of 3136 catalytic sites with bound Mg-NTP complexes or their analogues. Our results are presented in two articles; here, in the second of them, we elucidated whether the Walker A and Walker B sequence motifs-common to all P-loop NTPases-could be directly involved in catalysis. We found that the hydrogen bonds (H-bonds) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and aspartate of the Walker B motif (AspWB) are particularly short (even as short as 2.4 ångströms) in the structures with bound transition state (TS) analogues. Given that a short H-bond implies parity in the pKa values of the H-bond partners, we suggest that, in response to the interactions of a P-loop NTPase with its cognate activating partner, a proton relocates from [Ser/Thr]WA to AspWB. The resulting anionic [Ser/Thr]WA alkoxide withdraws a proton from the catalytic water molecule, and the nascent hydroxyl attacks the gamma phosphate of NTP. When the gamma-phosphate breaks away, the trapped proton at AspWB passes by the Grotthuss relay via [Ser/Thr]WA to beta-phosphate and compensates for its developing negative charge that is thought to be responsible for the activation barrier of hydrolysis.
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Affiliation(s)
- Maria I. Kozlova
- School of Physics, Osnabrueck University, D-49069 Osnabrueck, Germany
| | - Daria N. Shalaeva
- School of Physics, Osnabrueck University, D-49069 Osnabrueck, Germany
| | - Daria V. Dibrova
- School of Physics, Osnabrueck University, D-49069 Osnabrueck, Germany
| | - Armen Y. Mulkidjanian
- School of Physics, Osnabrueck University, D-49069 Osnabrueck, Germany
- Center of Cellular Nanoanalytics, Osnabrueck University, D-49069 Osnabrueck, Germany
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CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain. Int J Mol Sci 2022; 23:ijms23168842. [PMID: 36012107 PMCID: PMC9408373 DOI: 10.3390/ijms23168842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 08/05/2022] [Accepted: 08/06/2022] [Indexed: 11/17/2022] Open
Abstract
The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.
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9
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Chen S, Shu L, Zhao R, Zhao Y. Molecular dynamics simulations reveal the activation mechanism of mutations G12V and Q61L of Cdc42. Proteins 2022; 90:1376-1389. [PMID: 35152498 DOI: 10.1002/prot.26320] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 01/26/2022] [Accepted: 02/09/2022] [Indexed: 12/12/2022]
Abstract
Cell division control protein 42 homolog (Cdc42), which contributes to multiple cellular processes including cell proliferation and migration, is a potential target for cancer therapy, especially in the intervention of tumor migration. Cdc42's mutants G12V and Q61L are discovered constitutively active, and the overexpression of them exhibits oncogenic activities. Here, using molecular dynamics (MD) simulations and dynamic analysis, we illustrated the activation mechanism of Cdc42G12V and Cdc42Q61L . Without GAP, the two mutations differently elicited state transition from the wild-type's open "inactive" state 1 to the closed "active" state 2, induced by the introduction of a newly formed water-mediated T35-γ-phosphate hydrogen bond in G12V system and the additional hydrophobic interactions between L61 and T35 together with the direct T35-γ-phosphate hydrogen bond in Q61L system. When binding with GAP, both mutations weakened the hydrogen bond interactions between Cdc42-GTP and GAP's finger loop, and disturbed the catalytically competent organizations of GAP's catalytic R305/R306 and Cdc42's Q61, thereby impairing the GAP-mediated GTP hydrolysis. Our findings first reveal the activation mechanism of Cdc42's G12V and Q61L mutants on a molecular basis, which provide new insights into the structural and dynamical characteristics of Cdc42 and its mutants and can be exploited in the further development of novel therapies targeting Cdc42-related cancers.
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Affiliation(s)
- Shiyao Chen
- School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
| | - Liang Shu
- Department of Neurology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Rong Zhao
- Department of Neurology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Yaxue Zhao
- School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
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10
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Jung G, Pan M, Alexander C, Jin T, Hammer JA. Dual regulation of the actin cytoskeleton by CARMIL-GAP. J Cell Sci 2022; 135:275754. [PMID: 35583107 PMCID: PMC9270954 DOI: 10.1242/jcs.258704] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2021] [Accepted: 05/09/2022] [Indexed: 11/29/2022] Open
Abstract
Capping protein Arp2/3 myosin I linker (CARMIL) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of capping protein (CP). Here, we characterize CARMIL-GAP (GAP for GTPase-activating protein), a Dictyostelium CARMIL isoform that contains a ∼130 residue insert that, by homology, confers GTPase-activating properties for Rho-related GTPases. Consistent with this idea, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP-null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP-null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that, although both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the nucleotide state of Rho GTPases is often required to drive Rho-dependent biological processes. Summary:Dictyostelium CARMIL-GAP supports phagocytosis and chemotaxis by regulating both capping protein and Rac1.
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Affiliation(s)
- Goeh Jung
- Cell and Developmental Biology Center, National Heart lung and Blood Institute, National Institutes of Health, USA
| | - Miao Pan
- Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, National Institutes of Health, USA
| | - Chris Alexander
- Cell and Developmental Biology Center, National Heart lung and Blood Institute, National Institutes of Health, USA
| | - Tian Jin
- Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, National Institutes of Health, USA
| | - John A Hammer
- Cell and Developmental Biology Center, National Heart lung and Blood Institute, National Institutes of Health, USA
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11
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Smith KP, Lee W, Tonelli M, Lee Y, Light SH, Cornilescu G, Chakravarthy S. Solution structure and dynamics of the mitochondrial-targeted GTPase-activating protein (GAP) VopE by an integrated NMR/SAXS approach. Protein Sci 2022; 31:e4282. [PMID: 35137487 PMCID: PMC9047041 DOI: 10.1002/pro.4282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Revised: 01/17/2022] [Accepted: 01/27/2022] [Indexed: 11/11/2022]
Abstract
The bacterial pathogen Vibrio cholerae use a type III secretion system to inject effector proteins into a host cell. Recently, a putative Toxic GTPase Activating Protein (ToxGAP) called Vibrio outer protein E (VopE) was identified as a T3SS substrate and virulence factor that affected host mitochondrial dynamics and immune response. However, biophysical and structural characterization has been absent. Here, we describe solution NMR structure of the putative GTPase-activating protein (GAP) domain (73-204) of VopE. Using size exclusion chromatography coupled with small-angle x-ray scattering and residual dipolar coupling data, we restrained the MD process to efficiently determine the overall fold and improve the quality of the output calculated structures. Comparing the structure of VopE with other ToxGAP's revealed a similar overall fold with several features unique to VopE. Specifically, the "Bulge 1," α1 helix, and noteworthy "backside linker" elements on the N-terminus are dissimilar to the other ToxGAP's. By using NMR relaxation dispersion experiments, we demonstrate that these regions undergo motions on a > 6 s-1 timescale. Based on the disposition of these mobile regions relative to the putative catalytic arginine residue, we hypothesize that the protein may undergo structural changes to bind cognate GTPases.
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Affiliation(s)
- Kyle P. Smith
- Department of Cell & Developmental BiologyNorthwestern University ChicagoIllinoisUSA
- Xilio TherapeuticsWalthamMassachusettsUSA
| | - Woonghee Lee
- Department of ChemistryUniversity of Colorado‐DenverDenverColoradoUSA
| | - Marco Tonelli
- National Magnetic Resonance Facility at Madison, Department of BiochemistryUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Yeongjoon Lee
- Department of ChemistryUniversity of Colorado‐DenverDenverColoradoUSA
| | - Samuel H. Light
- Department of MicrobiologyUniversity of ChicagoChicagoIllinoisUSA
| | - Gabriel Cornilescu
- Advanced Technology Research Facility, Frederick National Laboratory for Cancer ResearchLeidos Biomedical Research, Inc., National Cancer Institute, National Institutes of HealthFrederickMarylandUSA
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12
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Wontakal SN, Britto M, Zhang H, Han Y, Gao C, Tannenbaum S, Durham BH, Lee MT, An X, Mishima M. RACGAP1 variants in a sporadic case of CDA III implicate the dysfunction of centralspindlin as the basis of the disease. Blood 2022; 139:1413-1418. [PMID: 34818416 PMCID: PMC8900277 DOI: 10.1182/blood.2021012334] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 11/15/2021] [Indexed: 11/20/2022] Open
Affiliation(s)
- Sandeep N Wontakal
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY
| | - Mishan Britto
- Centre for Mechanochemical Cell Biology & Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom
| | - Huan Zhang
- Laboratory of Membrane Biology, New York Blood Center, New York, NY
| | - Yongshuai Han
- Laboratory of Membrane Biology, New York Blood Center, New York, NY
| | - Chengjie Gao
- Laboratory of Membrane Biology, New York Blood Center, New York, NY
| | - Sarah Tannenbaum
- Department of Pediatrics, Columbia University Irving Medical Center, New York, NY; and
| | - Benjamin H Durham
- Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY
| | - Margaret T Lee
- Department of Pediatrics, Columbia University Irving Medical Center, New York, NY; and
| | - Xiuli An
- Laboratory of Membrane Biology, New York Blood Center, New York, NY
| | - Masanori Mishima
- Centre for Mechanochemical Cell Biology & Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, United Kingdom
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13
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Fic W, Bastock R, Raimondi F, Los E, Inoue Y, Gallop JL, Russell RB, St Johnston D. RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion. J Cell Biol 2021; 220:211832. [PMID: 33646271 PMCID: PMC7927664 DOI: 10.1083/jcb.202009116] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 12/04/2020] [Accepted: 01/14/2021] [Indexed: 01/01/2023] Open
Abstract
Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. We used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion.
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Affiliation(s)
- Weronika Fic
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Genetics, University of Cambridge, Cambridge, UK
| | - Rebecca Bastock
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Genetics, University of Cambridge, Cambridge, UK
| | - Francesco Raimondi
- BioQuant and Biochemie Zentrum Heidelberg, Heidelberg University, Heidelberg, Germany
| | - Erinn Los
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Genetics, University of Cambridge, Cambridge, UK
| | - Yoshiko Inoue
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Jennifer L. Gallop
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Robert B. Russell
- BioQuant and Biochemie Zentrum Heidelberg, Heidelberg University, Heidelberg, Germany
| | - Daniel St Johnston
- Gurdon Institute, University of Cambridge, Cambridge, UK,Department of Genetics, University of Cambridge, Cambridge, UK,Correspondence to Daniel St Johnston:
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14
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Functional and Therapeutic Relevance of Rho GTPases in Innate Immune Cell Migration and Function during Inflammation: An In Silico Perspective. Mediators Inflamm 2021; 2021:6655412. [PMID: 33628114 PMCID: PMC7896857 DOI: 10.1155/2021/6655412] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Revised: 01/23/2021] [Accepted: 02/01/2021] [Indexed: 12/17/2022] Open
Abstract
Systematic regulation of leukocyte migration to the site of infection is a vital step during immunological responses. Improper migration and localization of immune cells could be associated with disease pathology as seen in systemic inflammation. Rho GTPases act as molecular switches during inflammatory cell migration by cycling between Rho-GDP (inactive) to Rho-GTP (active) forms and play an essential role in the precise regulation of actin cytoskeletal dynamics as well as other immunological functions of leukocytes. Available reports suggest that the dysregulation of Rho GTPase signaling is associated with various inflammatory diseases ranging from mild to life-threatening conditions. Therefore, it is crucial to understand the step-by-step activation and inactivation of GTPases and the functioning of different Guanine Nucleotide Exchange Factors (GEFs) and GTPase-Activating Proteins (GAPs) that regulate the conversion of GDP to GTP and GTP to GDP exchange reactions, respectively. Here, we describe the molecular organization and activation of various domains of crucial elements associated with the activation of Rho GTPases using solved PDB structures. We will also present the latest evidence available on the relevance of Rho GTPases in the migration and function of innate immune cells during inflammation. This knowledge will help scientists design promising drug candidates against the Rho-GTPase-centric regulatory molecules regulating inflammatory cell migration.
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15
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Arrington ME, Temple B, Schaefer A, Campbell SL. The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2. J Biol Chem 2020; 295:12130-12142. [PMID: 32636302 PMCID: PMC7443499 DOI: 10.1074/jbc.ra120.012915] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Revised: 07/02/2020] [Indexed: 12/20/2022] Open
Abstract
The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.
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Affiliation(s)
- Megan E Arrington
- Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Brenda Temple
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA; R. L. Juliano Structural Bioinformatics Core Facility, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Antje Schaefer
- Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Sharon L Campbell
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.
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16
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Basu S, González B, Li B, Kimble G, Kozminski KG, Cullen PJ. Functions for Cdc42p BEM adaptors in regulating a differentiation-type MAP kinase pathway. Mol Biol Cell 2020; 31:491-510. [PMID: 31940256 PMCID: PMC7185891 DOI: 10.1091/mbc.e19-08-0441] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Ras homology (Rho) GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous (Σ1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared with the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. Versions of Bem1p defective for recruitment of Ste20p to the plasma membrane, intramolecular interactions, and interaction with the GEF, Cdc24p, were defective for fMAPK pathway signaling. Bem1p also regulated effector pathways in different ways. In some pathways, multiple domains of the protein were required for its function, whereas in other pathways, a single domain or function was needed. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.
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Affiliation(s)
- Sukanya Basu
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260
| | - Beatriz González
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260
| | - Boyang Li
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260
| | - Garrett Kimble
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260
| | - Keith G Kozminski
- Departments of Biology and Cell Biology, University of Virginia, Charlottesville, VA 22904
| | - Paul J Cullen
- Department of Biological Sciences, University at Buffalo, Buffalo, NY 14260
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17
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Wang E, Weng G, Sun H, Du H, Zhu F, Chen F, Wang Z, Hou T. Assessing the performance of the MM/PBSA and MM/GBSA methods. 10. Impacts of enhanced sampling and variable dielectric model on protein-protein Interactions. Phys Chem Chem Phys 2019; 21:18958-18969. [PMID: 31453590 DOI: 10.1039/c9cp04096j] [Citation(s) in RCA: 83] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Enhanced sampling has been extensively used to capture the conformational transitions in protein folding, but it attracts much less attention in the studies of protein-protein recognition. In this study, we evaluated the impact of enhanced sampling methods and solute dielectric constants on the overall accuracy of the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics/generalized Born surface area (MM/GBSA) approaches for the protein-protein binding free energy calculations. Here, two widely used enhanced sampling methods, including aMD and GaMD, and conventional molecular dynamics (cMD) simulations with two AMBER force fields (ff03 and ff14SB) were used to sample the conformations for 21 protein-protein complexes. The MM/PBSA and MM/GBSA calculation results illustrate that the standard MM/GBSA based on the cMD simulations yields the best Pearson correlation (rp = -0.523) between the predicted binding affinities and the experimental data, which is much higher than that given by MM/PBSA (rp = -0.212). Two enhanced sampling methods (aMD and GaMD) are indeed more efficient for conformational sampling, but they did not improve the binding affinity predictions for protein-protein systems, suggesting that the aMD or GaMD sampling (at least in short timescale simulations) may not be a good choice for the MM/PBSA and MM/GBSA predictions of protein-protein complexes. The solute dielectric constant of 1.0 is recommended to MM/GBSA, but a higher solute dielectric constant is recommended to MM/PBSA, especially for the systems with higher polarity on the protein-protein binding interfaces. Then, a preliminary assessment of the MM/GBSA calculations based on a variable dielectric generalized Born (VDGB) model was conducted. The results highlight the potential power of VDGB in the free energy predictions for protein-protein systems, but more thorough studies should be done in the future.
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Affiliation(s)
- Ercheng Wang
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Gaoqi Weng
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Huiyong Sun
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Hongyan Du
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Feng Zhu
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Fu Chen
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Zhe Wang
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Tingjun Hou
- Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. and State Key Lab of CAD&CG, Zhejiang University, Hangzhou, Zhejiang 310058, China
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18
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Scheffzek K, Shivalingaiah G. Ras-Specific GTPase-Activating Proteins-Structures, Mechanisms, and Interactions. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a031500. [PMID: 30104198 DOI: 10.1101/cshperspect.a031500] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.
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Affiliation(s)
- Klaus Scheffzek
- Division of Biological Chemistry (Biocenter), Medical University of Innsbruck, A-6020 Innsbruck, Austria
| | - Giridhar Shivalingaiah
- Division of Biological Chemistry (Biocenter), Medical University of Innsbruck, A-6020 Innsbruck, Austria
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19
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Mellor P, Marshall JDS, Ruan X, Whitecross DE, Ross RL, Knowles MA, Moore SA, Anderson DH. Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation. Sci Rep 2018; 8:7108. [PMID: 29740032 PMCID: PMC5940657 DOI: 10.1038/s41598-018-25487-5] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Accepted: 04/20/2018] [Indexed: 12/11/2022] Open
Abstract
The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.
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Affiliation(s)
- Paul Mellor
- Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada
| | - Jeremy D S Marshall
- Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.,Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada
| | - Xuan Ruan
- Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada
| | - Dielle E Whitecross
- Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada
| | - Rebecca L Ross
- Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James's University Hospital, Leeds, United Kingdom
| | - Margaret A Knowles
- Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James's University Hospital, Leeds, United Kingdom
| | - Stanley A Moore
- Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada
| | - Deborah H Anderson
- Cancer Research Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada. .,Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada. .,Cancer Research, Saskatchewan Cancer Agency, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
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20
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Chen F, Liu H, Sun H, Pan P, Li Y, Li D, Hou T. Assessing the performance of the MM/PBSA and MM/GBSA methods. 6. Capability to predict protein-protein binding free energies and re-rank binding poses generated by protein-protein docking. Phys Chem Chem Phys 2018; 18:22129-39. [PMID: 27444142 DOI: 10.1039/c6cp03670h] [Citation(s) in RCA: 347] [Impact Index Per Article: 49.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Understanding protein-protein interactions (PPIs) is quite important to elucidate crucial biological processes and even design compounds that interfere with PPIs with pharmaceutical significance. Protein-protein docking can afford the atomic structural details of protein-protein complexes, but the accurate prediction of the three-dimensional structures for protein-protein systems is still notoriously difficult due in part to the lack of an ideal scoring function for protein-protein docking. Compared with most scoring functions used in protein-protein docking, the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) methodologies are more theoretically rigorous, but their overall performance for the predictions of binding affinities and binding poses for protein-protein systems has not been systematically evaluated. In this study, we first evaluated the performance of MM/PBSA and MM/GBSA to predict the binding affinities for 46 protein-protein complexes. On the whole, different force fields, solvation models, and interior dielectric constants have obvious impacts on the prediction accuracy of MM/GBSA and MM/PBSA. The MM/GBSA calculations based on the ff02 force field, the GB model developed by Onufriev et al. and a low interior dielectric constant (εin = 1) yield the best correlation between the predicted binding affinities and the experimental data (rp = -0.647), which is better than MM/PBSA (rp = -0.523) and a number of empirical scoring functions used in protein-protein docking (rp = -0.141 to -0.529). Then, we examined the capability of MM/GBSA to identify the possible near-native binding structures from the decoys generated by ZDOCK for 43 protein-protein systems. The results illustrate that the MM/GBSA rescoring has better capability to distinguish the correct binding structures from the decoys than the ZDOCK scoring. Besides, the optimal interior dielectric constant of MM/GBSA for re-ranking docking poses may be determined by analyzing the characteristics of protein-protein binding interfaces. Considering the relatively high prediction accuracy and low computational cost, MM/GBSA may be a good choice for predicting the binding affinities and identifying correct binding structures for protein-protein systems.
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Affiliation(s)
- Fu Chen
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Hui Liu
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Huiyong Sun
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Peichen Pan
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Youyong Li
- Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123, China
| | - Dan Li
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
| | - Tingjun Hou
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. and State Key Lab of CAD&CG, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China
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21
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Jin Y, Molt RW, Pellegrini E, Cliff MJ, Bowler MW, Richards NGJ, Blackburn GM, Waltho JP. Assessing the Influence of Mutation on GTPase Transition States by Using X-ray Crystallography, 19 F NMR, and DFT Approaches. Angew Chem Int Ed Engl 2017; 56:9732-9735. [PMID: 28498638 PMCID: PMC5575484 DOI: 10.1002/anie.201703074] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2017] [Indexed: 11/08/2022]
Abstract
We report X-ray crystallographic and 19 F NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 F NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.
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Affiliation(s)
- Yi Jin
- Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield, S10 2TN, UK.,School of Chemistry, Cardiff University, Cardiff, CF10 3AT, UK
| | - Robert W Molt
- School of Chemistry, Cardiff University, Cardiff, CF10 3AT, UK.,Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.,ENSCO, Inc., Melbourne, FL, 32940, USA
| | - Erika Pellegrini
- Structural Biology Group, ESRF-The European Synchrotron, CS40220, 38043, Grenoble, Cedex 9, France
| | - Matthew J Cliff
- Manchester Institute of Biotechnology, Manchester, M1 7DN, UK
| | - Matthew W Bowler
- Structural Biology Group, ESRF-The European Synchrotron, CS40220, 38043, Grenoble, Cedex 9, France.,European Molecular Biology Laboratory, Grenoble Outstation CS90181, 38042, Grenoble, Cedex 9, France
| | | | - G Michael Blackburn
- Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield, S10 2TN, UK
| | - Jonathan P Waltho
- Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield, S10 2TN, UK.,Manchester Institute of Biotechnology, Manchester, M1 7DN, UK
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22
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Jin Y, Molt RW, Pellegrini E, Cliff MJ, Bowler MW, Richards NGJ, Blackburn GM, Waltho JP. Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography,
19
F NMR, and DFT Approaches. Angew Chem Int Ed Engl 2017. [DOI: 10.1002/ange.201703074] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Yi Jin
- Department of Molecular Biology and BiotechnologyKrebs InstituteUniversity of Sheffield Sheffield S10 2TN UK
- School of ChemistryCardiff University Cardiff CF10 3AT UK
| | - Robert W. Molt
- School of ChemistryCardiff University Cardiff CF10 3AT UK
- Department of Biochemistry and Molecular BiologyIndiana University School of Medicine Indianapolis IN 46202 USA
- ENSCO, Inc. Melbourne FL 32940 USA
| | - Erika Pellegrini
- Structural Biology GroupESRF-The European Synchrotron, CS40220 38043 Grenoble, Cedex 9 France
| | | | - Matthew W. Bowler
- Structural Biology GroupESRF-The European Synchrotron, CS40220 38043 Grenoble, Cedex 9 France
- European Molecular Biology Laboratory, Grenoble Outstation CS90181 38042 Grenoble, Cedex 9 France
| | | | - G. Michael Blackburn
- Department of Molecular Biology and BiotechnologyKrebs InstituteUniversity of Sheffield Sheffield S10 2TN UK
| | - Jonathan P. Waltho
- Department of Molecular Biology and BiotechnologyKrebs InstituteUniversity of Sheffield Sheffield S10 2TN UK
- Manchester Institute of Biotechnology Manchester M1 7DN UK
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23
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Betts MJ, Wichmann O, Utz M, Andre T, Petsalaki E, Minguez P, Parca L, Roth FP, Gavin AC, Bork P, Russell RB. Systematic identification of phosphorylation-mediated protein interaction switches. PLoS Comput Biol 2017; 13:e1005462. [PMID: 28346509 PMCID: PMC5386296 DOI: 10.1371/journal.pcbi.1005462] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Revised: 04/10/2017] [Accepted: 03/16/2017] [Indexed: 11/18/2022] Open
Abstract
Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation.
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Affiliation(s)
- Matthew J. Betts
- CellNetworks, Bioquant, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, Germany
- Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
| | - Oliver Wichmann
- CellNetworks, Bioquant, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, Germany
- Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
| | - Mathias Utz
- CellNetworks, Bioquant, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, Germany
- Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
| | - Timon Andre
- CellNetworks, Bioquant, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, Germany
- Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
| | - Evangelia Petsalaki
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada
| | - Pablo Minguez
- European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany
| | - Luca Parca
- European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany
| | - Frederick P. Roth
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada
- Donnelly Centre and Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Ontario, Canada
- Center for Cancer Systems Biology, Dana-Farber Cancer Institute, One Jimmy Fund Way, Boston, Massachusetts, United States
- Canadian Institute for Advanced Research, Toronto, Ontario, Canada
| | - Anne-Claude Gavin
- European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany
| | - Peer Bork
- European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany
| | - Robert B. Russell
- CellNetworks, Bioquant, University of Heidelberg, Im Neuenheimer Feld 267, Heidelberg, Germany
- Biochemie Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, Heidelberg, Germany
- * E-mail:
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24
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Cerione RA. The experiences of a biochemist in the evolving world of G protein-dependent signaling. Cell Signal 2017; 41:2-8. [PMID: 28214588 DOI: 10.1016/j.cellsig.2017.02.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Accepted: 02/14/2017] [Indexed: 12/24/2022]
Abstract
This review describes how a biochemist and basic researcher (i.e. myself) came to make a career in the area of receptor-coupled signal transduction and the roles cellular signaling activities play both in normal physiology and in disease. Much of what has been the best part of this research life is due to the time I spent with Bob Lefkowitz (1982-1985), during an extraordinary period in the emerging field of G-protein-coupled receptors. Among my laboratory colleagues were some truly outstanding scientists including Marc Caron, the late Jeffrey Stadel, Berta Strulovici, Jeff Benovic, Brian Kobilka, and Henrik Dohlman, as well as many more. I came to Bob's laboratory after being trained as a physical biochemist and enzymologist. Bob and his laboratory exposed me to a research style that made it possible to connect the kinds of fundamental biochemical and mechanistic questions that I loved to think about with a direct relevance to disease. Indeed, I owe Bob a great deal for having imparted a research style and philosophy that has remained with me throughout my career. Below, I describe how this has taken me on an interesting journey through various areas of cellular signaling, which have a direct relevance to the actions of one or another type of G-protein.
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Affiliation(s)
- Richard A Cerione
- Department of Molecular Medicine, Cornell University, Ithaca, NY 14853-6401, US.
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25
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Nouri K, Fansa EK, Amin E, Dvorsky R, Gremer L, Willbold D, Schmitt L, Timson DJ, Ahmadian MR. IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES. J Biol Chem 2016; 291:26364-26376. [PMID: 27815503 PMCID: PMC5159498 DOI: 10.1074/jbc.m116.752121] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Revised: 10/27/2016] [Indexed: 11/16/2022] Open
Abstract
IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.
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Affiliation(s)
- Kazem Nouri
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany
| | - Eyad K Fansa
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany
| | - Ehsan Amin
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany
| | - Radovan Dvorsky
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany
| | - Lothar Gremer
- the Institute of Physical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany.,Forschungszentrum Jülich, ICS-6, 52428 Jülich, Germany
| | - Dieter Willbold
- the Institute of Physical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany.,Forschungszentrum Jülich, ICS-6, 52428 Jülich, Germany
| | - Lutz Schmitt
- the Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany, and
| | - David J Timson
- the School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton BN2 4GJ, United Kingdom
| | - Mohammad R Ahmadian
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany,
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26
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Amin E, Jaiswal M, Derewenda U, Reis K, Nouri K, Koessmeier KT, Aspenström P, Somlyo AV, Dvorsky R, Ahmadian MR. Deciphering the Molecular and Functional Basis of RHOGAP Family Proteins: A SYSTEMATIC APPROACH TOWARD SELECTIVE INACTIVATION OF RHO FAMILY PROTEINS. J Biol Chem 2016; 291:20353-71. [PMID: 27481945 PMCID: PMC5034035 DOI: 10.1074/jbc.m116.736967] [Citation(s) in RCA: 77] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Revised: 07/15/2016] [Indexed: 12/30/2022] Open
Abstract
RHO GTPase-activating proteins (RHOGAPs) are one of the major classes of regulators of the RHO-related protein family that are crucial in many cellular processes, motility, contractility, growth, differentiation, and development. Using database searches, we extracted 66 distinct human RHOGAPs, from which 57 have a common catalytic domain capable of terminating RHO protein signaling by stimulating the slow intrinsic GTP hydrolysis (GTPase) reaction. The specificity of the majority of the members of RHOGAP family is largely uncharacterized. Here, we comprehensively investigated the sequence-structure-function relationship between RHOGAPs and RHO proteins by combining our in vitro data with in silico data. The activity of 14 representatives of the RHOGAP family toward 12 RHO family proteins was determined in real time. We identified and structurally verified hot spots in the interface between RHOGAPs and RHO proteins as critical determinants for binding and catalysis. We have found that the RHOGAP domain itself is nonselective and in some cases rather inefficient under cell-free conditions. Thus, we propose that other domains of RHOGAPs confer substrate specificity and fine-tune their catalytic efficiency in cells.
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Affiliation(s)
- Ehsan Amin
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany
| | - Mamta Jaiswal
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany
| | - Urszula Derewenda
- the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, and
| | - Katarina Reis
- the Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden
| | - Kazem Nouri
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany
| | - Katja T Koessmeier
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany
| | - Pontus Aspenström
- the Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden
| | - Avril V Somlyo
- the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, and
| | - Radovan Dvorsky
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany,
| | - Mohammad R Ahmadian
- From the Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, 40225 Düsseldorf, Germany,
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27
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The Structural Basis for Cdc42-Induced Dimerization of IQGAPs. Structure 2016; 24:1499-508. [PMID: 27524202 DOI: 10.1016/j.str.2016.06.016] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2016] [Revised: 06/18/2016] [Accepted: 06/20/2016] [Indexed: 01/18/2023]
Abstract
In signaling, Rho-family GTPases bind effector proteins and alter their behavior. Here we present the crystal structure of Cdc42·GTP bound to the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP2. Four molecules of Cdc42 are bound to two GRD molecules, which bind each other in a parallel dimer. Two Cdc42s bind very similarly to the Ras/RasGAP interaction, while the other two bind primarily to "extra domain" sequences from both GRDs, tying the GRDs together. Calorimetry confirms two-site binding of Cdc42·GTP for the GRDs of both IQGAP2 and IQGAP1. Mutation of important extra domain residues reduces binding to single-site and abrogates Cdc42 binding to a much larger IQGAP1 fragment. Importantly, Rac1·GTP displays only single-site binding to the GRDs, indicating that only Cdc42 promotes IQGAP dimerization. The structure identifies an unexpected role for Cdc42 in protein dimerization, thus expanding the repertoire of interactions of Ras family proteins with their targets.
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28
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Mishra AK, Lambright DG. Invited review: Small GTPases and their GAPs. Biopolymers 2016; 105:431-48. [PMID: 26972107 PMCID: PMC5439442 DOI: 10.1002/bip.22833] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Revised: 02/16/2016] [Accepted: 03/10/2016] [Indexed: 12/11/2022]
Abstract
Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 431-448, 2016.
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Affiliation(s)
- Ashwini K Mishra
- Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605
| | - David G Lambright
- Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605
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29
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Sirota FL, Maurer-Stroh S, Eisenhaber B, Eisenhaber F. Single-residue posttranslational modification sites at the N-terminus, C-terminus or in-between: To be or not to be exposed for enzyme access. Proteomics 2016; 15:2525-46. [PMID: 26038108 PMCID: PMC4745020 DOI: 10.1002/pmic.201400633] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 04/17/2015] [Accepted: 05/29/2015] [Indexed: 11/30/2022]
Abstract
Many protein posttranslational modifications (PTMs) are the result of an enzymatic reaction. The modifying enzyme has to recognize the substrate protein's sequence motif containing the residue(s) to be modified; thus, the enzyme's catalytic cleft engulfs these residue(s) and the respective sequence environment. This residue accessibility condition principally limits the range where enzymatic PTMs can occur in the protein sequence. Non‐globular, flexible, intrinsically disordered segments or large loops/accessible long side chains should be preferred whereas residues buried in the core of structures should be void of what we call canonical, enzyme‐generated PTMs. We investigate whether PTM sites annotated in UniProtKB (with MOD_RES/LIPID keys) are situated within sequence ranges that can be mapped to known 3D structures. We find that N‐ or C‐termini harbor essentially exclusively canonical PTMs. We also find that the overwhelming majority of all other PTMs are also canonical though, later in the protein's life cycle, the PTM sites can become buried due to complex formation. Among the remaining cases, some can be explained (i) with autocatalysis, (ii) with modification before folding or after temporary unfolding, or (iii) as products of interaction with small, diffusible reactants. Others require further research how these PTMs are mechanistically generated in vivo.
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Affiliation(s)
- Fernanda L Sirota
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore
| | - Sebastian Maurer-Stroh
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore.,School of Biological Sciences (SBS), Nanyang Technological University (NTU), Singapore
| | - Birgit Eisenhaber
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore
| | - Frank Eisenhaber
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore.,Department of Biological Sciences (DBS), National University of Singapore (NUS), Singapore.,School of Computer Engineering (SCE), Nanyang Technological University (NTU), Singapore
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30
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Juvenile myelomonocytic leukemia displays mutations in components of the RAS pathway and the PRC2 network. Nat Genet 2015; 47:1334-40. [DOI: 10.1038/ng.3420] [Citation(s) in RCA: 123] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2015] [Accepted: 09/16/2015] [Indexed: 12/18/2022]
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31
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Sudha G, Singh P, Swapna LS, Srinivasan N. Weak conservation of structural features in the interfaces of homologous transient protein-protein complexes. Protein Sci 2015; 24:1856-73. [PMID: 26311309 DOI: 10.1002/pro.2792] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2015] [Revised: 08/13/2015] [Accepted: 08/17/2015] [Indexed: 12/21/2022]
Abstract
Residue types at the interface of protein-protein complexes (PPCs) are known to be reasonably well conserved. However, we show, using a dataset of known 3-D structures of homologous transient PPCs, that the 3-D location of interfacial residues and their interaction patterns are only moderately and poorly conserved, respectively. Another surprising observation is that a residue at the interface that is conserved is not necessarily in the interface in the homolog. Such differences in homologous complexes are manifested by substitution of the residues that are spatially proximal to the conserved residue and structural differences at the interfaces as well as differences in spatial orientations of the interacting proteins. Conservation of interface location and the interaction pattern at the core of the interfaces is higher than at the periphery of the interface patch. Extents of variability of various structural features reported here for homologous transient PPCs are higher than the variation in homologous permanent homomers. Our findings suggest that straightforward extrapolation of interfacial nature and inter-residue interaction patterns from template to target could lead to serious errors in the modeled complex structure. Understanding the evolution of interfaces provides insights to improve comparative modeling of PPC structures.
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Affiliation(s)
- Govindarajan Sudha
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Prashant Singh
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, Karnataka, India
| | - Lakshmipuram S Swapna
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, Karnataka, India
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32
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Wu D, Zhu X, Jimenez-Cowell K, Mold AJ, Sollecito CC, Lombana N, Jiao M, Wei Q. Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2. Exp Cell Res 2015; 333:80-92. [PMID: 25704760 PMCID: PMC4387072 DOI: 10.1016/j.yexcr.2015.02.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2014] [Revised: 01/17/2015] [Accepted: 02/10/2015] [Indexed: 10/25/2022]
Abstract
Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling.
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Affiliation(s)
- Di Wu
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States
| | - Xiaoxi Zhu
- Experimental and Clinical Research Center (ECRC), a Cooperation between Max Delbrück Center and Charité Universitätsmedizin Berlin, Campus Buch, Berlin, Germany
| | - Kevin Jimenez-Cowell
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States
| | - Alexander J Mold
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States
| | | | - Nicholas Lombana
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States
| | - Meng Jiao
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States
| | - Qize Wei
- Department of Biological Sciences, Fordham University, Bronx, NY 10458, United States.
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33
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Moorman VR, Valentine KG, Bédard S, Kasinath V, Dogan J, Love FM, Wand AJ. Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3. J Mol Biol 2014; 426:3520-38. [PMID: 25109462 DOI: 10.1016/j.jmb.2014.07.031] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2014] [Revised: 07/29/2014] [Accepted: 07/31/2014] [Indexed: 01/09/2023]
Abstract
Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type guanosine triphosphatase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21-activated kinase 3, is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation was measured to investigate the dynamical changes in activated GMPPCP·Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs-PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side-chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl-bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately -10kcalmol(-1) at 298K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs becomes more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring becomes more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding.
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Affiliation(s)
- Veronica R Moorman
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - Kathleen G Valentine
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - Sabrina Bédard
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - Vignesh Kasinath
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - Jakob Dogan
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - Fiona M Love
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
| | - A Joshua Wand
- Graduate Group in Biochemistry and Molecular Biophysics, Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059, USA.
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34
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Lőrincz ÁM, Szarvas G, Smith SME, Ligeti E. Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils. Free Radic Biol Med 2014; 68:65-71. [PMID: 24321316 DOI: 10.1016/j.freeradbiomed.2013.12.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2013] [Revised: 11/15/2013] [Accepted: 12/01/2013] [Indexed: 10/25/2022]
Abstract
Precise spatiotemporal regulation of O2(-)-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1-3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67(phox) and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O2(-)-generating system consisting of isolated membranes and recombinant p47(phox) and p67(phox) proteins. Addition of soluble RacGAPs decreased O2(-) production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O2(-) production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O2(-) production by Nox2, allowing selective regulation via different signaling pathways.
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Affiliation(s)
- Ákos M Lőrincz
- Department of Physiology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary
| | - Gábor Szarvas
- Department of Physiology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary
| | - Susan M E Smith
- Department of Biology and Physics, Kennesaw State University, 1000 Chastain Road, Building 12, Room 308, Kennesaw, GA 30144, USA
| | - Erzsébet Ligeti
- Department of Physiology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary.
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35
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Rajasekar K, Campbell L, Nietlispach D, Owen D, Mott H. The structure of the RLIP76 RhoGAP-Ral binding domain dyad: fixed position of the domains leads to dual engagement of small G proteins at the membrane. Structure 2013; 21:2131-42. [PMID: 24207123 PMCID: PMC3852207 DOI: 10.1016/j.str.2013.09.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2013] [Revised: 09/12/2013] [Accepted: 09/13/2013] [Indexed: 01/15/2023]
Abstract
RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.
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Affiliation(s)
- Karthik V. Rajasekar
- Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
| | - Louise J. Campbell
- Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
| | - Daniel Nietlispach
- Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
| | - Darerca Owen
- Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
| | - Helen R. Mott
- Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
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36
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Zhou Y, Johnson JL, Cerione RA, Erickson JW. Prenylation and membrane localization of Cdc42 are essential for activation by DOCK7. Biochemistry 2013; 52:4354-63. [PMID: 23718289 DOI: 10.1021/bi301688g] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The unconventional guanine nucleotide exchange factor (GEF) family comprising 11 DOCK180 related proteins is classified into four subfamilies, A through D, based on their relative GEF activity toward the closely related Rac and Cdc42 GTPases. DOCK proteins participate in the remodeling of the actin cytoskeleton and are key regulators of cell motility, phagocytosis, and adhesion. Here we show that the guanine nucleotide exchange domain of DOCK7, DHR2 (for DOCK homology region 2), is a potent GEF for prenylated Cdc42 and Rac1 in a model liposome system, demonstrating that the prenylation and membrane localization of Cdc42 or Rac1 are necessary for their activation by DOCK7. Additionally, we identify DOCK7 residues that confer GTPase GEF specificity. Finally, using our liposome reconstitution assay, we show that a more narrowly defined GEF domain of DHR2 (designated DHR2s) harbors an N-terminal site distinct from the GEF active site that binds preferentially to the active, GTP-bound forms of Cdc42 and Rac1 and thereby recruits free DHR2s from solution to the membrane surface. This recruitment results in a progressive increase in the effective concentration of DHR2s at the membrane surface that in turn provides for an accelerated rate of guanine nucleotide exchange on Cdc42. The positive cooperativity observed in our reconstituted system suggests that the action of DOCK7 in vivo may involve the coordinated integration of Cdc42/Rac signaling in the context of the membrane recruitment of a DOCK7 GEF complex.
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Affiliation(s)
- Yeyun Zhou
- Field of Biophysics/MacCHESS, Cornell High Energy Synchrotron Source, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, United States
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37
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Yu Q, Hu L, Yao Q, Zhu Y, Dong N, Wang DC, Shao F. Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP. Cell Res 2013; 23:775-87. [PMID: 23588383 PMCID: PMC3674391 DOI: 10.1038/cr.2013.54] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.
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Affiliation(s)
- Qin Yu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China,National Institute of Biological Sciences, #7 Science Park Rd, Zhongguancun Life Science Park, Beijing 102206, China,Graduate University of Chinese Academy of Sciences, Beijing 100049, China
| | - Liyan Hu
- National Institute of Biological Sciences, #7 Science Park Rd, Zhongguancun Life Science Park, Beijing 102206, China
| | - Qing Yao
- National Institute of Biological Sciences, #7 Science Park Rd, Zhongguancun Life Science Park, Beijing 102206, China
| | - Yongqun Zhu
- Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Na Dong
- National Institute of Biological Sciences, #7 Science Park Rd, Zhongguancun Life Science Park, Beijing 102206, China,E-mail:
| | - Da-Cheng Wang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China,E-mail:
| | - Feng Shao
- National Institute of Biological Sciences, #7 Science Park Rd, Zhongguancun Life Science Park, Beijing 102206, China,Tel: +86-10-80728593; Fax: +86-10-80728046
E-mail:
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38
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Xu J, Zhou X, Wang J, Li Z, Kong X, Qian J, Hu Y, Fang JY. RhoGAPs attenuate cell proliferation by direct interaction with p53 tetramerization domain. Cell Rep 2013; 3:1526-38. [PMID: 23684608 DOI: 10.1016/j.celrep.2013.04.017] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Revised: 03/13/2013] [Accepted: 04/21/2013] [Indexed: 12/15/2022] Open
Abstract
Many Rho GTPase activation proteins (RhoGAPs) are deleted or downregulated in cancers, but the functional consequences are still unclear. Here, we show that the RhoGAP ArhGAP11A induces cell-cycle arrest and apoptosis by binding to the tumor suppressor p53. The RhoGAP domain of ArhGAP11A binds to the tetramerization domain of p53, but not to its family members p63 or p73. The interaction stabilizes the tetrameric conformation of p53 and enhances its DNA-binding activity, thereby inducing cell-cycle arrest and apoptosis. Upon DNA damage stress, ArhGAP11A accumulates in the nucleus and interacts with p53, whereas knockdown of ArhGAP11A partially blocks p53 transcriptional activity. These findings explain why RhoGAPs are frequently deleted in cancers and suggest that the RhoGAP family sits at the crossroads between the cell-migration and proliferation pathways.
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Affiliation(s)
- Jie Xu
- State Key Laboratory for Oncogenes and Related Genes, Division of Gastroenterology and Hepatology, Renji Hospital, Shanghai Institute for Digestive Diseases, Shanghai Jiao-Tong University School of Medicine, Key Laboratory of Gastroenterology & Hepatology, Ministry of Health, 145 Middle Shandong Road, Shanghai 200001, China.
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39
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Matsuura A, Lee HH. Crystal structure of GTPase-activating domain from human MgcRacGAP. Biochem Biophys Res Commun 2013; 435:367-72. [PMID: 23665020 DOI: 10.1016/j.bbrc.2013.04.094] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2013] [Accepted: 04/22/2013] [Indexed: 11/29/2022]
Abstract
Cytokinesis in animal cells relies on a centralspindlin complex consisting of male germ cell RacGap (MgcRacGAP) and mitotic kinesin-like protein 1 (MKLP1). Rho GTPases act as molecular switches to regulate the actin cytoskeleton for cytokinesis, of which Rac1 is regulated by MgcRacGAP. In this study, we determined the crystal structure of the GTPase-activating protein (GAP) domain of MgcRacGAP at a resolution of 1.9Å. The conformation of Arg385, which is a key residue for GAP activity, was found to be different from that of previously reported GAP proteins, and MgcRacGAP (residues 348-546) was found to exist as a monomer in solution, according to Stokes radii. We also measured the GAP activity of MgcRacGAP mutants for Rac1.
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Affiliation(s)
- Atsushi Matsuura
- Department of Bio & Nano Chemistry, Kookmin University, Seoul 136-702, Republic of Korea
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40
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Csépányi-Kömi R, Sáfár D, Grósz V, Tarján ZL, Ligeti E. In silico tissue-distribution of human Rho family GTPase activating proteins. Small GTPases 2013; 4:90-101. [PMID: 23518456 PMCID: PMC3747261 DOI: 10.4161/sgtp.23708] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Rho family small GTPases are involved in the spatio-temporal regulation of several physiological processes. They operate as molecular switches based on their GTP- or GDP-bound state. Their GTPase activator proteins (Rho/Rac GAPs) are able to increase the GTP hydrolysis of small GTPases, which turns them to an inactive state. This regulatory step is a key element of signal termination. According to the human genome project the potential number of Rho family GAPs is approximately 70. Despite their significant role in cellular signaling our knowledge on their expression pattern is quite incomplete. In this study we tried to reveal the tissue-distribution of Rho/Rac GAPs based on expressed sequence tag (EST) database from healthy and tumor tissues and microarray experiments. Our accumulated data sets can provide important starting information for future research. However, the nomenclature of Rho family GAPs is quite heterogeneous. Therefore we collected the available names, abbreviations and aliases of human Rho/Rac GAPs in a useful nomenclature table. A phylogenetic tree and domain structure of 65 human RhoGAPs are also presented.
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41
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Csépányi-Kömi R, Lévay M, Ligeti E. Small G proteins and their regulators in cellular signalling. Mol Cell Endocrinol 2012; 353:10-20. [PMID: 22108439 DOI: 10.1016/j.mce.2011.11.005] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/30/2011] [Revised: 09/27/2011] [Accepted: 11/07/2011] [Indexed: 01/04/2023]
Abstract
Small molecular weight GTPases (small G proteins) are essential in the transduction of signals from different plasma membrane receptors. Due to their endogenous GTP-hydrolyzing activity, these proteins function as time-dependent biological switches controlling diverse cellular functions including cell shape and migration, cell proliferation, gene transcription, vesicular transport and membrane-trafficking. This review focuses on endocrine diseases linked to small G proteins. We provide examples for the regulation of the activity of small G proteins by various mechanisms such as posttranslational modifications, guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs). Finally we summarize endocrine diseases where small G proteins or their regulatory proteins have been revealed as the cause.
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Affiliation(s)
- Roland Csépányi-Kömi
- Department of Physiology, Semmelweis University, Tűzoltó u. 37-47, 1094 Budapest, Hungary
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42
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Elliott SF, Allen G, Timson DJ. Biochemical analysis of the interactions of IQGAP1 C-terminal domain with CDC42. World J Biol Chem 2012; 3:53-60. [PMID: 22451851 PMCID: PMC3312201 DOI: 10.4331/wjbc.v3.i3.53] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2011] [Revised: 01/31/2012] [Accepted: 02/07/2012] [Indexed: 02/05/2023] Open
Abstract
AIM: To understand the interaction of human IQGAP1 and CDC42, especially the effects of phosphorylation and a cancer-associated mutation.
METHODS: Recombinant CDC42 and a novel C-terminal fragment of IQGAP1 were expressed in, and purified from, Escherichia coli. Site directed mutagenesis was used to create coding sequences for three phosphomimicking variants (S1441E, S1443D and S1441E/S1443D) and to recapitulate a cancer-associated mutation (M1231I). These variant proteins were also expressed and purified. Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42. These interactions were quantified using surface plasmon resonance measurements. Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant.
RESULTS: The novel, C-terminal region of human IQGAP1 (residues 877-1558) is soluble following expression and purification. It is also capable of binding to CDC42, as judged by crosslinking experiments. Interaction appears to be strongest in the presence of added GTP. The three phosphomimicking mutants had different affinities for CDC42. S1441E had an approximately 200-fold reduction in affinity compared to wild type. This was caused largely by a dramatic reduction in the association rate constant. In contrast, both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type. The cancer-associated variant, M1231I, also had a similar affinity to wild type. However, in the case of this variant, both the association and dissociation rate constants were reduced approximately 10-fold. Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region, revealed no gross structural changes compared to wild type (root mean square deviation of 0.564 Å over 5556 equivalent atoms). However, predictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type. One such region is a loop linking the proposed CDC42 binding site with the helix containing the altered residue. It is suggested that this increase in rigidity is responsible for the observed changes in association and dissociation rate constants.
CONCLUSION: The consequences of introducing negative charge at Ser-1441 or Ser-1443 in IQGAP1 are different. The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.
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Affiliation(s)
- Sarah F Elliott
- Sarah F Elliott, George Allen, David J Timson, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, BT9 7BL, United Kingdom
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43
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Ligeti E, Welti S, Scheffzek K. Inhibition and Termination of Physiological Responses by GTPase Activating Proteins. Physiol Rev 2012; 92:237-72. [DOI: 10.1152/physrev.00045.2010] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Physiological processes are strictly organized in space and time. However, in cell physiology research, more attention is given to the question of space rather than to time. To function as a signal, environmental changes must be restricted in time; they need not only be initiated but also terminated. In this review, we concentrate on the role of one specific protein family involved in biological signal termination. GTPase activating proteins (GAPs) accelerate the endogenously low GTP hydrolysis rate of monomeric guanine nucleotide-binding proteins (GNBPs), limiting thereby their prevalence in the active, GTP-bound form. We discuss cases where defective or excessive GAP activity of specific proteins causes significant alteration in the function of the nervous, endocrine, and hemopoietic systems, or contributes to development of infections and tumors. Biochemical and genetic data as well as observations from human pathology support the notion that GAPs represent vital elements in the spatiotemporal fine tuning of physiological processes.
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Affiliation(s)
- Erzsébet Ligeti
- Department of Physiology, Semmelweis University, Budapest, Hungary; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany; and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria
| | - Stefan Welti
- Department of Physiology, Semmelweis University, Budapest, Hungary; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany; and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria
| | - Klaus Scheffzek
- Department of Physiology, Semmelweis University, Budapest, Hungary; Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany; and Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria
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44
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Bosch DE, Wittchen ES, Qiu C, Burridge K, Siderovski DP. Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica. J Biol Chem 2011; 286:39236-46. [PMID: 21930699 PMCID: PMC3234748 DOI: 10.1074/jbc.m111.253898] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2011] [Revised: 09/13/2011] [Indexed: 01/28/2023] Open
Abstract
The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.
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Affiliation(s)
| | | | | | - Keith Burridge
- Lineberger Comprehensive Cancer Center and
- the Department of Cell and Developmental Biology
- University of North Carolina McAllister Heart Institute, The University of North Carolina, Chapel Hill, North Carolina 27599-7365
| | - David P. Siderovski
- From the Department of Pharmacology
- University of North Carolina Neuroscience Center
- Lineberger Comprehensive Cancer Center and
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45
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Fleishman SJ, Corn JE, Strauch EM, Whitehead TA, Karanicolas J, Baker D. Hotspot-centric de novo design of protein binders. J Mol Biol 2011; 413:1047-62. [PMID: 21945116 DOI: 10.1016/j.jmb.2011.09.001] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2011] [Revised: 08/31/2011] [Accepted: 09/02/2011] [Indexed: 11/24/2022]
Abstract
Protein-protein interactions play critical roles in biology, and computational design of interactions could be useful in a range of applications. We describe in detail a general approach to de novo design of protein interactions based on computed, energetically optimized interaction hotspots, which was recently used to produce high-affinity binders of influenza hemagglutinin. We present several alternative approaches to identify and build the key hotspot interactions within both core secondary structural elements and variable loop regions and evaluate the method's performance in natural-interface recapitulation. We show that the method generates binding surfaces that are more conformationally restricted than previous design methods, reducing opportunities for off-target interactions.
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Affiliation(s)
- Sarel J Fleishman
- Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
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46
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Chandrashekar R, Salem O, Krizova H, McFeeters R, Adams PD. A switch I mutant of Cdc42 exhibits less conformational freedom. Biochemistry 2011; 50:6196-207. [PMID: 21667996 DOI: 10.1021/bi2004284] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Cdc42 is a Ras-related small G-protein and functions as a molecular switch in signal transduction pathways linked with cell growth and differentiation. It is controlled by cycling between GTP-bound (active) and GDP-bound (inactive) forms. Nucleotide binding and hydrolysis are modulated by interactions with effectors and/or regulatory proteins. These interactions are centralized in two relatively flexible "Switch" regions as characterized by internal dynamics on multiple time scales [Loh, A. P., et al. (2001) Biochemistry 40, 4590-4600], and this flexibility may be essential for protein interactions. In the Switch I region, Thr(35) seems to be critical for function, as it is completely invariant in Ras-related proteins. To investigate the importance of conformational flexibility in Switch I of Cdc42, we mutated threonine to alanine, determined the solution structure, and characterized the backbone dynamics of the single-point mutant protein, Cdc42(T35A). Backbone dynamics data suggest that the mutation changes the time scale of the internal motions of several residues, with several resonances not being discernible in wild-type Cdc42 [Adams, P. D., and Oswald, R. E. (2007) Biomol. NMR Assignments 1, 225-227]. The mutation does not appear to affect the thermal stability of Cdc42, and chymotrypsin digestion data further suggest that changes in the conformational flexibility of Switch I slow proteolytic cleavage relative to that of the wild type. In vitro binding assays show less binding of Cdc42(T35A), relative to that of wild type, to a GTPase binding protein that inhibits GTP hydrolysis in Cdc42. These results suggest that the mutation of T(35) leads to the loss of conformational freedom in Switch I that could affect effector-regulatory protein interactions.
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Affiliation(s)
- Reena Chandrashekar
- Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States
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47
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Recognition of the F&H motif by the Lowe syndrome protein OCRL. Nat Struct Mol Biol 2011; 18:789-95. [PMID: 21666675 PMCID: PMC3130824 DOI: 10.1038/nsmb.2071] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2010] [Accepted: 04/15/2011] [Indexed: 12/25/2022]
Abstract
Lowe syndrome and Type 2 Dent disease are caused by defects in the inositol 5-phosphatase OCRL. Most missense mutations in the OCRL ASH-RhoGAP domain found in affected patients abolish interactions with the endocytic adaptors APPL1 and Ses (both Ses1 and Ses2), which bind OCRL through a short F&H motif. Using X-ray crystallography, we have identified the F&H motif binding site on the RhoGAP domain of OCRL. We further show that clinical mutations affect F&H binding indirectly by destabilizing the RhoGAP fold. In contrast, a clinical mutation that does not perturb F&H binding and ASH-RhoGAP stability disrupts OCRL's interaction with Rab5. Additionally, OCRL's F&H binding site is conserved even in species that do not express APPL or Ses. Our study predicts the existence of other OCRL binding partners and demonstrates the critical role of the perturbation of OCRL interactions in disease.
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48
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Kulkarni K, Yang J, Zhang Z, Barford D. Multiple factors confer specific Cdc42 and Rac protein activation by dedicator of cytokinesis (DOCK) nucleotide exchange factors. J Biol Chem 2011; 286:25341-51. [PMID: 21613211 DOI: 10.1074/jbc.m111.236455] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
DOCK (dedicator of cytokinesis) guanine nucleotide exchange factors (GEFs) activate the Rho-family GTPases Rac and Cdc42 to control cell migration, morphogenesis, and phagocytosis. The DOCK A and B subfamilies activate Rac, whereas the DOCK D subfamily activates Cdc42. Nucleotide exchange is catalyzed by a conserved DHR2 domain (DOCK(DHR2)). Although the molecular basis for DOCK(DHR2)-mediated GTPase activation has been elucidated through structures of a DOCK9(DHR2)-Cdc42 complex, the factors determining recognition of specific GTPases are unknown. To understand the molecular basis for DOCK-GTPase specificity, we have determined the crystal structure of DOCK2(DHR2) in complex with Rac1. DOCK2(DHR2) and DOCK9(DHR2) exhibit similar tertiary structures and homodimer interfaces and share a conserved GTPase-activating mechanism. Multiple structural differences between DOCK2(DHR2) and DOCK9(DHR2) account for their selectivity toward Rac1 and Cdc42. Key determinants of selectivity of Cdc42 and Rac for their cognate DOCK(DHR2) are a Phe or Trp residue within β3 (residue 56) and the ability of DOCK proteins to exploit differences in the GEF-induced conformational changes of switch 1 dependent on a divergent residue at position 27. DOCK proteins, therefore, differ from DH-PH GEFs that select their cognate GTPases through recognition of structural differences within the β2/β3 strands.
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Affiliation(s)
- Kiran Kulkarni
- Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom
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49
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Wu X, Ramachandran S, Lin MCJ, Cerione RA, Erickson JW. A minimal Rac activation domain in the unconventional guanine nucleotide exchange factor Dock180. Biochemistry 2011; 50:1070-80. [PMID: 21033699 DOI: 10.1021/bi100971y] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.
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Affiliation(s)
- Xin Wu
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
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Wu H, Brennwald P. The function of two Rho family GTPases is determined by distinct patterns of cell surface localization. Mol Cell Biol 2010; 30:5207-17. [PMID: 20823269 PMCID: PMC2953063 DOI: 10.1128/mcb.00366-10] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2010] [Revised: 05/24/2010] [Accepted: 08/25/2010] [Indexed: 11/20/2022] Open
Abstract
Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.
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Affiliation(s)
- Hao Wu
- Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7090
| | - Patrick Brennwald
- Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7090
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