Identification and Characterization of a Bacterial Catalytic Scaffold with Specificity for Host Endomembrane Traffic

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signaling 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 signaling networks through enzyme scaffolding, and the identity of the effectors or their mechanisms of action are poorly understood. Here, we used a functional screen to identify the EHEC O157:H7 type III effector EspG as a regulator of endomembrane trafficking and we report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAK) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GAP-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signaling inhibition at membrane organelles. 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 PAK are organized on adjacent surfaces of EspG, suggesting its dual role as a “catalytic scaffold” that effectively reprograms cellular events through the functional assembly of GTPase-kinase signaling complex. Bidirectional vesicular transport between ER and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood, in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E.coli Secreted Protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi Intermediate Compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. Mechanistic insights presented in this study establish the effectiveness of a small bacterial catalytic scaffold in studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.