Mechanism of SspH1: A Bacterial Effector Ubiquitin Ligase Hijacks the Eukaryotic Ubiquitylation Pathway

Abstract

Bacterial effector proteins promote the pathogenicity of bacteria by interacting with host cell proteins and modulating signaling pathways in the host cell. Some bacterial effectors hijack the eukaryotic ubiquitylation pathway to promote pathogenicity, despite the absence of the pathway in prokaryotes. One family of bacterial effectors, the IpaH-SspH family of E3 ligases, are unrelated in sequence or structure to eukaryotic E3s. This work elucidates key aspects in the structure and mechanism of one member of the IpaH-SspH family from Salmonella typhimurium, SspH1. Biophysical and biochemical methods were used to examine the structure of SspH1 in solution and the interactions between SspH1 and components of the eukaryotic ubiquitylation pathway, with an emphasis on the mechanisms of ubiquitin transfer from the E2 active site to the SspH1 active site, and from the SspH1 active site to substrate. Important results of this work include 1) a reanalysis of existing crystal structures of IpaH-SspH1 E3 domains leading to a new way of characterizing the structural organization of the SspH1 E3 domain, revealing two independent subdomains. 2) Identification of the E2~Ub conjugate binding region and how E2~Ub engages SspH1 in a productive complex. 3) Generation of a chemically stable mimic of SspH1~Ub allowing for structural characterization of the second stage of Ub transfer, from SspH1~Ub to substrate. 4) Development of a model in which E2~Ub not only is involved in the transfer of Ub to the SspH1 active site, but also drives the transfer of Ub from the SspH1 active site to substrate.