A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases

Abstract

Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.microbial pathogenesis ͉ type III secretion ͉ crystallography ͉ SspH2

The structure of the Slrp–Trx1 complex sheds light on the autoinhibition mechanism of the type III secretion system effectors of the NEL family

“…In both copies of the NEL domain there was no visible electron density for loop αM-αN (residues 709-728) of the C-terminal helix bundle. In contrast, the loop linking the LRR and NEL domains that was disordered in the published apo structures of IpaH3 [21] and SspH2 [22] is clearly defined in the Trx1 bound SlrP structure. The catalytic loop carrying the conserved C 546 XD 548 motif [24] is exposed to the solvent and accessible to the E2-Ub conjugate.…”

“…Previous studies demonstrated that removal of the LRR domain of SlrP (residues 1-457) resulted in dramatic increase of its ubiquitin E3 ligase activity [22]. These results suggest that the LRR domain, known as a versatile structural framework for protein-protein interaction [47], is not implicated in substrate binding but is rather involved in the regulation of the adjacent NEL catalytic domain [14].…”

“…Comparison of the SlrP/Trx1 complex with the apo structures of the NEL effectors IpaH3 from Shigella (PDB ID 3CVR) [21] and SspH2 from Salmonella (PDB ID 3G06) [22] shed light on the molecular mechanism of these ubiquitin E3 ligases.…”

“…In the SspH2 structure (PDB ID 3G06) [22], the SspH2 residue Leu638 corresponding to SlrP Phe604 ( Figure S2) is buried in a hydrophobic pocket formed by the other face of the -helix C-capping the LLR domain (SspH2 helix α4 = SlrP helix α3) and the N-terminal extremity of the linker loop. In this closed conformation, the NEL domain is turned toward the concave face of the LRR domain and the catalytic cysteine is buried in this interface ( Figure 6C).…”

“…The diffraction data were processed using the XDS package [29]. Molecular replacement performed using the program PHASER [30] failed to solve the crystallographic phase problem by using either of the LRR and NEL domains from the available structures of IpaH3 (PDB ID 3CVR) [21] or SspH2 (PDB ID 3G06) [22].…”