The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold

Abstract

Pathogenic strains of Escherichia coli translocate many proteins into the host cell to promote virulence. The structure of one of these proteins, EspG from E. coli O157:H7, has been determined in a complex with two host enzymes and its mechanism dissected. These structures reveal how EspG disrupts endomembrane trafficking pathways by specifically recognizing the GTP-bound active state of the host's ARF6 enzyme during the vesicle budding reaction at membrane organelles. EspG directly activates PAK kinase by trapping an unfolded transition state in the kinase activation cascade. Pathogenic Escherichia coli translocate many proteins into the host cell to promote virulence. It is now shown that one of these proteins, EspG, which is present in enterohaemorrhagic E. coli, interferes with the host signalling network. The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3,4,5,6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.