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. 2011 Jan 6;469(7328):107-11.
doi: 10.1038/nature09593. Epub 2010 Dec 19.

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

Andrey S Selyunin  1 , Sarah E Sutton, Bethany A Weigele, L Evan Reddick, Robert C Orchard, Stefan M Bresson, Diana R Tomchick, Neal M Alto
Affiliations

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

Andrey S Selyunin et al. Nature. .

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.

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Figures

Fiure 1
Fiure 1. EspG inhibits endomembrane trafficking and disrupts Golgi architecture
a, hGH trafficking assay showing how the hGH–FKBP* (Phe 36 Met mutant) aggregates in the endoplasmic reticulum until drug application (AP21998), whereby hGH enters the general secretory pathway and is secreted into the culture medium. b, hGH release assay showing the effects of type III and type IV effector proteins on trafficking through the general secretory pathway (Methods). hGH was quantified by enzyme-linked immunosorbent assay and normalized to GFP control (Drug) experiments. The subcellular localization of eGFP-tagged effectors is indicated. ER, endoplasmic reticulum. c, Co-localization of eGFP–EspG (green) with cis-Golgi matrix protein GM130 (red). The Golgi in untransfected cells appears as tightly associated cisternae. d, Golgi and microtubule phenotypes induced by EspG protein microinjection (asterisk). The percentage of microinjected cells exhibiting each phenotype is indicated (n = 3, from >40 cells per experiment). e, f, ARF GTPase (e) and PAK isoforms (f) that interact with EspG by yeast two-hybrid. g, h, Glutathione pull-down of GST–ARF isoforms (g) and GST–PAK1 fragments (h) with recombinant MalE-tagged EspG.
Figure 2
Figure 2. The structure of EspG in complex with GTP-bound ARF6
a, The overall structure of EspG–ARF6GTP complex. EspG is shown in cyan and ARF6 in green. Switch I and switch II on ARF6 are coloured orange and red, respectively. b, EspG selectively binds the GTP-loaded ARF1 and ARF6 (GST tagged) in glutathione pull-down assays. The native lane represents ARF GTPases purified from bacteria without removing or loading specific nucleotides. c, Structural overlay of EspG–ARF6GTP and ASAP3(GAP)–ARF6GDP·AlFX (Protein Data Bank ID, 3LVQ) showing how EspG sterically hinders ARF binding to ASAP3–GAP. The catalytic Arg finger of ASAP3 is labelled. d, GTP hydrolysis assay showing that EspG inhibits GAP-assisted GTP hydrolysis on ARF1. The rate of γ32P[GTP] hydrolysis was measured as the percentage of γ32P[GTP] remaining on ARF1 over time. Intrinsic ARF1 GTPase activity (control, green), GAP-stimulated activity (GAP, blue triangle), and EspG inhibition of GAP activity (EspG + GAP, open diamond) or mutant EspG Glu 392 Arg (open circle) are shown. e, Time course of the Golgi disruption phenotype presented as the percentage of microinjected cells with altered Golgi morphology as shown in Fig. 1d. At least 45 microinjected cells were scored in each trial for a Golgi disruption phenotype, and the data are representative of three experimental trials. BFA, brefeldin A.
Figure 3
Figure 3. The structure of EspG in complex with PAK2 Iα3 peptide
a, The overall EspG–PAK2Iα3 complex with EspG oriented and coloured as in Fig. 2a. The PAK2 Iα3 peptide (residues 123–134) are shown in magenta. b, Detailed interactions between EspG and PAK2Iα3. Key binding residues from EspG (blue labels) and PAK2 (black labels) are shown. c, Close-up view of autoinhibited PAK1 homodimer (Protein Data Bank ID, 1F3M) focused on chain B (kinase domain, blue) and chain D (autoinhibitory domain, yellow). The Iα3-helix inhibitory functions are labelled (i)–(iii) corresponding with those outlined in the results section. The Iα3-helix extracted from the PAK1 structure (numbering corresponds to PAK2 for ease of comparison) is shown at the upper right. The corresponding PAK2 Iα3-helix extracted from the EspG structure (lower right) is oriented by the amino-terminal helical residues 123–127. KI loop, kinase inhibitory loop. CRIB, Cdc42/Rac1 interacting binding. d, e, PAK2 kinase assays comparing 2 µM EspG with equimolar GTPγS-loaded Cdc42 (d) and the indicated EHEC type III effectors (e). Phosphorylation of myelin basic protein (MBP) substrate, input levels of PAK2 and quantification of each experiment are shown. f, PAK2 kinase assays comparing autoinhibited wild-type (WT) PAK2 with PAK2 mutants Val 123 Asp and Phe 129 Asp. Data are presented as in d.
Figure 4
Figure 4. EspG functions as a catalytic scaffold at membrane organelles
a, Structural overlay of EspG–ARF6GTP and EspG–PAK2Iα3 highlighting the close association between ARF and PAK on the surface of EspG. Colours are as in Figs 2a and 3a except that EspG from the PAK2 structure is coloured purple. b, Golgi-mimetic-liposome-binding assays showing that EspG nucleates a trimeric complex between ARF1 and PAK2 on membrane surfaces. After centrifugation, proteins remaining in the supernatant (S) or those associated with liposomes in the pellet (P) are indicated. c, HEK239A cells co-transfected with the indicated constructs showing that eGFP–EspG co-localizes with ARF1–mCherry and recruits a PAK activity probe (mCherry–PAK2121–136) to Golgi membranes. The percentage of cells exhibiting co-localized EspG with mCherry-tagged proteins (n = 3) is shown in the upper right of the merged micrographs. d, Model of the dual function of EspG as an inhibitor of membrane trafficking and as a catalytic scaffold that assembles a GTPase–kinase signalling complex at cellular membranes. GEF, guanine nucleotide exchange factor.
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