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.
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 BopE(78-261) comprises two three-helix bundles (alpha1alpha4alpha5 and alpha2alpha3alpha6). 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 SopE(78-240) and SopE2(69-240) form the arms of a 'Lambda' shape, BopE(78-261) 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 SopE(78-240)-Cdc42 complex crystal structure indicates that, in a BopE-Cdc42 interaction, the closed conformation of BopE(78-261) would engender steric clashes with the Cdc42 switch regions. This implies that BopE(78-261) must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE(78-261)-Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE(78-261) indicates that BopE(78-261) does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE(78-261) catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10-40-fold lower k(cat) of BopE compared with SopE and SopE2.
Upadhyay
Abhishek
A
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Wu
Huan-Lin
HL
Williams
Christopher
C
Field
Terry
T
Galyov
Edouard E
EE
van den Elsen
Jean M H
JM
Bagby
Stefan
S
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012475:Salmonella / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D017186:Titrimetry; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D020662:Guanine Nucleotide Exchange Factors; C103987:SopE protein, Salmonella; D020764:cdc42 GTP-Binding Protein