Mechanism of catalysis, E2 recognition, and autoinhibition for the IpaH family of bacterial E3 ubiquitin ligases

Abstract

IpaH enzymes are secreted bacterial effectors that function within host cells as E3 ubiquitin (Ub) ligases. Catalytic activity is imparted by a conserved novel E3 ligase (NEL) domain that is unique to Gram-negative pathogens and whose activity is repressed by a flanking substrate-binding leucine-rich repeat (LRR) domain when substrate is absent. How the NEL domain catalyzes the conjugation of Ub onto substrates, recognizes host E2s, and maintains its autoinhibited state remain poorly understood. Here we used mutagenesis and enzyme kinetic analyses to address these gaps in knowledge. Mutagenesis of conserved residues on two remote surfaces of the NEL domain identified functional clusters proximal to and distal to the active site cysteine. By analyzing the kinetics of Ub charging and discharging, we identified proximal active site residues that function as either the catalytic acid or catalytic base for aminolysis. Further analysis revealed that distal site residues mediate the direct binding of E2. In studying the full-length protein, we also have uncovered that IpaH family autoinhibition is achieved by a short-circuiting mechanism wherein the LRR domain selectively blocks productive aminolysis, but not the nonproductive discharge of Ub from the E3 to solvent. This mode of autoinhibition, which is not shared by the HECT domain ligase Smurf2, leads to the unanticipated depletion of E2∼Ub and thus a concomitant dominant-negative effect on other E3s in vitro, raising the possibility that short circuiting also may serve to restrict the function of host E3s in cells.IpaH family | bacterial E3 ubiquitin ligase | ubiquitin | host-pathogen P osttranslational modification by the covalent attachment of the small protein ubiquitin (Ub) onto lysine side chains of target proteins is an essential eukaryotic modification that imparts a wide range of consequences to the substrate, from targeting to the proteasome for degradation to the assembly of signaling complexes (1, 2). Ubiquitination begins with an E1 enzyme, which activates Ub through the consumption of ATP to form a thioester E1∼Ub adduct on its catalytic cysteine. The E1∼Ub conjugate then transthiolates Ub to the active-site cysteine of an E2 enzyme to form an E2∼Ub adduct. Finally, the charged E2∼Ub then collaborates with a diverse group of E3 ligases to catalyze the stable attachment of Ub to the e-amino group of side-chain lysine residues or, in some cases, the N-terminal amino group of protein substrates via an aminolysis reaction.E3 ligases can be divided into two groups based on whether or not they use a catalytic cysteine to form an E3∼Ub intermediate. The HECT (homologous to E6AP C-terminus), RBR (RING-inbetween-RING), and IpaH (invasion-plasmid antigen H) families of structurally distinct E3 ligases use a catalytic cysteine residue to accept activated Ub in the form of an E3∼Ub thioester adduct. The E3 itself then catalyzes the aminolysis reaction to form an isopeptide bond between Ub and the acceptor lysine substrate. In contrast, the RING (really interesting ...

Exploitation of the host cell ubiquitin machinery by microbial effector proteins

“…2). The LRR domain is connected to the NEL domain through a flexible linker and inhibits its ubiquitin ligase activity in the absence of substrate proteins (Chou et al, 2012;Quezada et al, 2009;Keszei and Sicheri, 2017). Removal of the LRR domain from SspH1, SspH2, SlrP or IpaH9.8 increases their E3 ligase activity (Quezada et al, 2009;Chou et al, 2012;Zouhir et al, 2014).…”

“…A structural study of SspH1 showed that the binding of the host protein kinase N1 (PKN1) to the LRR domain releases its inhibitory effect on the NEL domain, which promotes the ubiquitylation and degradation of PKN1, and leads to the attenuation of the androgen response (Haraga and Miller, 2006;Keszei et al, 2014). Site-directed mutagenesis suggests that the LRR domain inhibits the activity of NELs not by preventing the catalytic cysteine from forming an intermediate with ubiquitin, but by selectively blocking the transfer of ubiquitin onto substrate proteins (Keszei and Sicheri, 2017).…”

A generalin vitroassay to study enzymatic activities of the ubiquitin system

“…Most previous measurements of ubiquitination rates relied on quantification of protein bands from Western blots (e.g. [21][22][23][24]). While reliable, SDS-PAGE and antibody detection steps are time-consuming and expensive.…”

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

“…Despite structural differences, SspH/IpaH E3s share important functional similarities with their eukaryotic HECT counterparts. Like HECT E3s, SspH/IpaH E3s form an E3ϳUb intermediate, use specific host E2ϳUbs, and target specific host proteins (12)(13)(14)(15)(16).…”

“…Although the structure of the SspH1 E3 domain has not yet been solved, those of several E3s, including the closely related SspH2 from Salmonella (77.6% identical in the E3 domain), are available. In addition, several studies have shown that SspH/IpaH E3s use E2ϳUbs from the Ube2D (UbcH5) family as a source of activated Ub (12)(13)(14)(15)(16)20). Thus, SspH1 is a structurally tractable system for investigating protein interactions and domain motions that occur during substrate ubiquitylation.…”