Self-association of EPEC intimin mediated by the beta-barrel-containing anchor domain: a role in clustering of the Tir receptor.

Abstract

Outer membrane intimin directs attachment of enteropathogenic Escherichia coli (EPEC) via its Tir receptor in mammalian target cell membranes. Phosphorylation of Tir triggers local actin polymerization and the formation of 'pedestal-like' pseudopods. We demonstrate that the intimin protein contains three domains, a flexible N-terminus (residues 40-188), a central membrane-integrated beta-barrel (189-549), and a tightly folded Tir-binding domain (550-939). Intimin was shown by electron microscopy to form ring-like structures with an approximately 7 nm external diameter and an electron dense core, and to form channels of 50picoSiemens conductance in planar lipid bilayers. Gel filtration, multiangle light scattering and cross-linking showed that this central beta-barrel membrane-anchoring domain directs intimin dimerization. Isothermal titration calorimetry revealed a high affinity, single-binding site interaction of 2 : 1 stoichiometry between dimeric intimin and Tir, and modelling suggests that this interaction determines a reticular array-like superstructure underlying receptor clustering. In support of this model, actin rearrangement induced in Tir-primed cultured cells by intimin-containing proteoliposomes was dependent on the concentration of both intimin and Tir, and co-localized with clustered phosphorylated Tir.