STRUCTURAL BASIS OF RAB FUNCTION

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

DESCRIPTION The Rab family of monomeric G proteins is comprised of more than forty functionally distinct members regulating discrete steps in vesicle trafficking pathways. Rab3a regulates the fusion of synaptic vesicles to presynaptic membranes during neurotransmitter release in neurons, whereas Rab5 is essential for endocytosis. The combined approaches of X-ray crystallographic and mutational analysis will be used to characterize in detail the stereochemical basis of Rab activation and regulation. We have recently determined the crystal structure of the active conformation of Rab3a. Conditions will be optimized to obtain crystals of the inactive conformation of a Rab protein that are suitable for structure/determination. These studies will identify residues that are important determinants of binding specificity, and will be essential for characterization of the molecular mechanism of regulated nucleotide exchange. Recently, we have demonstrated formation of a stable of a stable 1:1 complex of the nucleotide free form of Rab3A bound to Mss4, which we have isolated by gel filtration chromatography. Complexes of Mss4 bound to wild type Rab3A bound to Mss4, which we have isolated by gel filtration chromatography. Complexes of Mss4 bound to wild type Rab3A and dominant inhibitory Rab3A mutants that increase the stability of the complex are being screened to obtain Rab3a-Mss4 co-crystals. In addition, we will obtain co-crystals of a complex comprised of the endocytic Rab protein Rab5 and the Rab-binding domain of the effector Early Endosome-associated Antigen-1 (EEA-1), as well as screen for co- crystals of an EEA-1 phospholipid head group complex. Site-directed mutagenesis will be used to study intrinsic and GAP-mediated GTPase activity of Rab3a. Finally, we will isolate stable complexes formed by Rab3 and Rab3aGAP, and we will obtain co-crystals of the Rab3a- Rab3aGAP complexes.