RESUMEN
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.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Biocatálisis , Escherichia coli O157/química , Proteínas de Escherichia coli/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Factores de Ribosilacion-ADP/química , Regulación Alostérica , Animales , Transporte Biológico , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Activación Enzimática , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/química , Aparato de Golgi/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Membranas Intracelulares/metabolismo , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Desplegamiento Proteico , Ratas , Técnicas del Sistema de Dos Híbridos , Quinasas p21 Activadas/químicaRESUMEN
The Escherichia coli type III effector Map belongs to a large family of bacterial virulence factors that activate host Rho GTPase signaling pathways through an unknown molecular mechanism. Here we report direct evidence that Map functions as a potent and selective guanine-nucleotide exchange factor (GEF) for Cdc42. The 2.3-A structure of the Map-Cdc42 complex revealed that Map mimics the GEF strategy of the mammalian Dbl family but has a three-dimensional architecture that is nearly identical to the bacterial GEF Salmonella spp. SopE. A comparative analysis between human and bacterial GEFs revealed a previously uncharacterized pairing mechanism between Map and the variable beta2-3 interswitch region of Cdc42. We propose a GTPase selection model that is experimentally validated by the preferential activation Rac1 and RhoA by the Shigella spp. effectors IpgB1 and IpgB2, respectively. These results significantly expand the repertoire of bacterial GEF mimics and unify a GEF selection mechanism for host GTPase substrates.