Structure and mechanism of a type III secretion protease, NleC

Abstract

NleC is one of the virulence factors that is injected into infected host cells by enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) via a needle-like protein complex called the type III secretion system (T3SS). The cytosolic NleC specifically cleaves the p65 subunit of NF-κB in the p65-p50 heterodimeric complex just after the Cys38 site in its N-terminal domain. The degradation of the remainder of the p65 C-terminal domain by the proteasome disrupts the NF-κB signalling pathway, thus dampening the host inflammatory response. Here, the crystal structure of NleC is reported at 1.55 Å resolution. In conjunction with biochemical analyses, the structure reveals that NleC is a member of the zincin zinc protease family and that the configuration of the NleC active site resembles that of the metzincin clan of metallopeptidases but without the canonical Met turn of astacin. The extended zinc-binding motif of NleC (HEXXHXXTXXXD) includes three metal ligands. The fifth zinc ligand, a conserved tyrosine (a bound water molecule is the fourth ligand), lies 45 residues downstream of the zincin motif. Furthermore, the electrostatic potential complementarity between NleC and p65 also contributes to the cleavage activity of the protease. These results not only provide important insights into the mechanism of how NleC recognizes its substrates, but also shed light on the design of new antibiotics for the food-borne diseases arising from EPEC and EHEC.

Metalloprotease NleC Suppresses Host NF-κB/Inflammatory Responses by Cleaving p65 and Interfering with the p65/RPS3 Interaction

“…suggests that NleC could be specific for p65, as recombinant NleC could not digest p50 in cell lysates [16]. Moreover, two cleavage site(s) on p65 by NleC have been identified as between proline 10 and alanine 11 (P10/A11) [16] or cysteine 38 and glutamic acid 39 (C38/E39) [41,44,45], although none of these studies ruled out the other cleavage site experimentally. The detailed mechanisms on how NleC specifically recognizes and cleaves p65 remain poorly understood.…”

The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK

“…Subsequently, both NleC and NleD were identified to be metalloprotease effectors. Whereas NleC and its cleavage of NF-B are very well characterized (9)(10)(11)(12)(13)(14)(15)22), to date only one study has examined the substrate specificity and enzymatic function of NleD (15). Baruch et al showed that NleD cleaved the MAP kinases p38 and JNK and that mutation of glutamate within the metalloprotease motif (H 142 ELLH 146 ) abolished the cleavage of JNK.…”

A three-dimensional ZnIIcoordination framework: poly[[μ2-(E)-1,2-bis(pyridin-4-yl)ethene][μ4-(E)-2,2′-(diazene-1,2-diyl)dibenzoato][μ2-(E)-2,2′-(diazene-1,2-diyl)dibenzoato]dizinc(II)]

“…Thus, in order to construct novel coordination polymers, many chemists now choose carboxylate-containing ligands with varied coordination modes as the main connectors, and mixed ligands with N-donor groups as ancillary bridges (Batten & Robson, 1998). The 1,2-bis(pyridin-4-yl)-ethene (bpe) ligand is effective in forming meaningful coordination frameworks because it can satisfy the coordination requirements of the metal centres (Liu & Li, 2012).…”