Structure-function study of the IpaH family of bacterial E3 ubiquitin ligases

As a crucial mediator of the cellular response to infection, the ubiquitin system represents a major point of subversion by invasive pathogens. One mechanism of ubiquitin subversion shared among numerous Gram-negative bacteria is via the secretion of effector proteins belonging to the IpaH family. Once delivered into the host cell, IpaH effectors hijack the ubiquitin system by the action of a conserved C-terminal novel E3 ligase (NEL) domain, which catalyzes the covalent attachment of ubiquitin onto substrates bound at its N-terminal leucine-rich repeat (LRR) domain. When substrate is absent, IpaH enzymes are autoinhibited by inter-domain contacts between the LRR and NEL domains that prevent spurious catalytic activity and autoubiquitination. Although structures have revealed the unique fold of the NEL domain and conformations of the autoinhibited full-length enzyme, many questions remain as to how IpaH enzymes recognize substrates, how they catalyze ubiquitination, and how autoinhibition prevents catalytic activity. In this thesis, I use structural and biochemical methods to shed light on some of these questions. In chapter 1, I provide background on the ubiquitin system, the host-pathogen interface, the subversion of ubiquitin by bacterial effectors, and the IpaH family. In chapter 2, I present the first structure of an IpaH-substrate complex, resolved by X-ray crystallography. Using the structure as a guide, I probed the consequence of this interaction in cells. In chapter 3, I discuss my studies of the function of conserved residues in the NEL domain in which I identified two distinct surfaces that mediate either catalysis of ubiquitination or E2 binding. Also in this chapter, I explore the underlying mechanism of autoinhibition, revealing that the autoinhibited state permits charging and non-productive hydrolysis, but not aminolysis. In chapter 4, I present my efforts to stabilize IpaH catalytic intermediates for biochemical and structural studies. In addition, I show that the Shigella IpaH9.8 effector does not ubiquitinate its proposed substrate, NEMO, in vitro, indicating that substrate recognition is more complex than previously appreciated. Finally, in chapter 5, I explore future directions based on the work presented in this thesis.Ph.D