GTP-binding proteins (G proteins) mediate critical steps in a variety of signaling and regulatory pathways. The function of G proteins in diverse biological processes depends on their ability to cycle between inactive (GDP-bound) and active (GTP-bound) conformations in a precisely regulated manner. Aberrant forms of G proteins have been implicated in the pathogenesis of variety of disease states including cancer. Consequently, G proteins and the factors that regulate their activity are potential targets for pharmacological interdiction. The experiments described in this proposal address the mechanism by which Mss4, a 13 kDa exchange factor selective for a subset of Rab family GTPases, regulates the activation of the monomeric G protein Rab3a which functions in neuronal signaling. The experimental approach will combine high resolution structural studies by X-ray crystallography with biochemical and mutational experiments in order to identify with the principal stereochemical determinants of Rab3a activation and regulation by Mss4 as well as the inactive (GDP-bound) and active (GMPPNP-bound) forms of Rab3a, the respective crystal structures will be solved and refined at high resolution. These studies will reveal the conformational changes associated with activation and provide a structural basis for the function of Mss4 and Rab3a in neuronal signaling. In parallel, mutations in Rab3a and Mss4 will be introduced to probe the binding interaction and determine the basis for the selectivity in the interaction between Mss4 and Rab family GTPases. The results, when interpreted in the context of the Mss4 and Raba3a crystal structures, will provide a clear picture of the structural and functional determinants that govern specificity and lead to the release of nucleotide. Finally, screens will be conducted for co-crystals of Mss4 bound to Rab3a or other Rav family proteins. Comparison with structural and biochemical data for other systems including EF-Tu/EF-Ts will distinguish features unique to the Mss4/Rab3a system from those that may be global determinants for nucleotide exchange. In addition, the structure of the GMPPNP-bound form of Rab3a will provide a basis for exploring the regulatory interaction between the active form of Rav proteins and specific effector proteins in the fusion/docking complex which mediates vesicle fusion with target membranes in exocytosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM056324-01A1
Application #
2630956
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1998-05-01
Project End
2003-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Malaby, Andrew W; Das, Sanchaita; Chakravarthy, Srinivas et al. (2018) Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors. Structure 26:106-117.e6
Mishra, Ashwini K; Lambright, David G (2016) Invited review: Small GTPases and their GAPs. Biopolymers 105:431-48
Malaby, Andrew W; Chakravarthy, Srinivas; Irving, Thomas C et al. (2015) Methods for analysis of size-exclusion chromatography-small-angle X-ray scattering and reconstruction of protein scattering. J Appl Crystallogr 48:1102-1113
Kahn, Richard A; Lambright, David G (2015) A PH Domain with Dual Phospholipid Binding Sites Regulates the ARF GAP, ASAP1. Structure 23:1971-3
Priya, Amulya; Kalaidzidis, Inna V; Kalaidzidis, Yannis et al. (2015) Molecular insights into Rab7-mediated endosomal recruitment of core retromer: deciphering the role of Vps26 and Vps35. Traffic 16:68-84
Mishra, Ashwini K; Lambright, David G (2015) High-throughput assay for profiling the substrate specificity of Rab GTPase-activating proteins. Methods Mol Biol 1298:47-60
Del Campo, Claudia M; Mishra, Ashwini K; Wang, Yu-Hsiu et al. (2014) Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/SidM. Structure 22:397-408
Mishra, Ashwini K; Del Campo, Claudia M; Collins, Robert E et al. (2013) The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism. J Biol Chem 288:24000-11
Malaby, Andrew W; van den Berg, Bert; Lambright, David G (2013) Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors. Proc Natl Acad Sci U S A 110:14213-8
Davey, Jonathan R; Humphrey, Sean J; Junutula, Jagath R et al. (2012) TBC1D13 is a RAB35 specific GAP that plays an important role in GLUT4 trafficking in adipocytes. Traffic 13:1429-41

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