title: The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold.
abstract: The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, 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.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816, USA.
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Selyunin
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Sutton
authors.forename: Bethany A
authors.initials: BA
authors.lastname: Weigele
authors.forename: L Evan
authors.initials: LE
authors.lastname: Reddick
authors.forename: Robert C
authors.initials: RC
authors.lastname: Orchard
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Bresson
authors.forename: Diana R
authors.initials: DR
authors.lastname: Tomchick
authors.forename: Neal M
authors.initials: NM
authors.lastname: Alto
mesh_terms: D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D055162:Biocatalysis; D001692:Biological Transport; D020134:Catalytic Domain; D002460:Cell Line; D018360:Crystallography, X-Ray; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D019453:Escherichia coli O157 / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D058767:Protein Unfolding; D051381:Rats; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D054462:p21-Activated Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C429282:EspG protein, E coli; D006160:Guanosine Triphosphate; D054462:p21-Activated Kinases; D020727:ADP-Ribosylation Factors