The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42

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Abstract
BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78–261) by NMR spectroscopy and it shows that BopE78–261 comprises two three-helix bundles (α1α4α5 and α2α3α6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE78–240 and SopE269–240 form the arms of a ‘Λ’ shape, BopE78–261 adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE78–240–Cdc42 complex crystal structure indicates that, in a BopE–Cdc42 interaction, the closed conformation of BopE78–261 would engender steric clashes with the Cdc42 switch regions. This implies that BopE78–261 must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE78–261–Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE78–261 indicates that BopE78–261 does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE78–261 catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10–40-fold lower kcat of BopE compared with SopE and SopE2.