Structure and mechanism of a type III secretion protease, NleC

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.