Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling

Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nuc leotide-dependent process. Using N-terminal peptide sequencing, matrix-assi sted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg -81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membra ne targeting. Topologically, Arg-81 lies within the conformationally variab le Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg- 81 side chain is buried in the Rab5 GTP conformation, but is solvent-access ible in the GDP conformation, explaining the dependence of proteolysis on n ucleotides. Peptide mapping of Rab4 was performed to take advantage of addi tional scissile bonds within Switch II to determine more precisely the limi ts of the nucleotide-dependent protease-accessible region. The Rab4 cleavag e sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an ei ght-residue surface-exposed conformationally variable region lying in the c entre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for i ts relative variability. C-terminal to Switch II(v) is a highly conserved R ab-specific YYRGA motif that we term Switch II(c) for its constant sequence . N-terminal to Switch II(v) is a sequence-invariant G-domain involved in n ucleotide binding and hydrolysis, We propose that the Rab Switch II(v) regi on imparts specificity to nucleotide-dependent protein-protein interactions .