Book of Abstracts: Albany 2007
June 19-23 2007
A Systems Structural Approach to Molecular Recognition in the Rab GTPase Family
Rab GTPases comprise the largest and most diverse GTPase family with approximately 60 proteins encoded in mammalian genomes. As critical regulators/mediators of membrane trafficking, Rab GTPases cycle between active (GTP-bound) and inactive (GDP-bound) conformations. GTP-bound Rab GTPases mediate selective interactions with effectors, including cargo sorting complexes, motor proteins, tethering factors, and lipid kinases as well as proteins implicated in signal transduction, cytoskeletal dynamics, and cytokinesis. The interconversion between states is tightly regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). The majority of Rab GTPases have spatially and temporally overlapping distributions within the dynamic network of tubular-vesicular organelles that represent endosomal and biosynthetic systems. Accumulating experimental evidence supports the hypothesis that the spatiotemporal distributions, activation status, and functional outputs of Rab GTPases are coordinated through molecular mechanisms involving large multidomain proteins and hetero-oligomeric complexes that contain multiple distinct binding sites and/or catalytic activities for Rab GTPases. It is further likely that an additional layer of coordination occurs at the level of individual binding/catalytic domains, which have the capacity for selective recognition of individual Rab GTPases or small subsets of Rab GTPases.
We are attempting to unravel the non-phylogenetic encoding of specificity that allows homologous as well as non-homologous effectors and regulatory factors to achieve highly selective recognition of small, and in some cases overlapping, subsets of Rab GTPases. The overall approach combines: (i) quantitative profiling of interactions with the Rab GTPase family; (ii) elucidation of the underlying structural bases; and (iii) identification of positive and negative determinants of the observed interaction profiles. We have successfully utilized this approach to determine how Vps9 domain GEFs achieve highly selective catalysis of GDP/GTP exchange for two members (Rab5 and Rab21) of subfamily V (Rabs 5, 21, and 22), how structurally similar binding sites in the multivalent endosomal effector Rabenosyn-5 distinguish between structurally related subsets of Rab GTPases (Rabs 5 and 22 vs. Rabs 4 and 14), and how TBC domain GAPs accelerate GTP hydrolysis by an unconventional dual catalytic finger mechanism. The results of these studies provide strong evidence for the encoding of both positive and negative recognition determinants at the family and sub-family levels. Recognition modalities cross phylogenetic boundaries and reflect an elaborate but decipherable interplay of tertiary structural similarities/variations, positive/negative sequence determinants and, in some cases, structural plasticity.
1Program in Molecular Medicine