Solution Structure, Backbone Dynamics, and Interaction with Cdc42 of Salmonella Guanine Nucleotide Exchange Factor SopE2,

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Abstract
SopE and SopE2 are delivered by the Salmonella type III secretion system into eukaryotic cells to promote cell invasion. SopE and SopE2 are potent guanine nucleotide exchange factors (GEFs) for Rho GTPases Cdc42 and Rac1 and constitute a novel class of Rho GEFs. Although the sequence of SopE-like GEFs is not at all homologous to those of the Dbl homology domain-containing eukaryotic GEFs, the mechanism of nucleotide release seems to have significant similarities. We have determined the solution structure of the catalytic domain (residues 69−240) of SopE2, showing that SopE269-240 comprises two three-helix bundles (α1α4α5 and α2α3α6) arranged in a Λ shape. Compared to the crystal structure of SopE78-240 in complex with Cdc42, SopE269-240 exhibits a less open Λ shape due to movement of SopE78-240 helices α2 and α5 to accommodate binding to the Cdc42 switch regions. In an NMR titration to investigate the SopE269-240−Cdc42 interaction, the SopE269-240 residues affected by binding Cdc42 were very similar to the SopE78-240 residues that contact Cdc42 in the SopE78-240−Cdc42 complex. Analysis of the backbone 15N dynamics of SopE269-240 revealed flexibility in residues that link the two three-helix bundles, including the α3−α4 linker that incorporates a β-hairpin and the catalytic loop, and the α5−α6 loop, and flexibility in residues involved in interaction with Cdc42. Together, these observations provide experimental evidence of a previously proposed mechanism of GEF-mediated nucleotide exchange based on the Rac1−Tiam1 complex structure, with SopE/E2 flexibility, particularly in the interbundle loops, enabling conformational rearrangements of the nucleotide binding region of Cdc42 through an induced fit type of binding. Such flexibility in SopE/E2 may also facilitate interaction through adaptive binding with alternative target proteins such as Rab5, allograft inflammatory factor 1, and apolipoprotein A-1.