Structral And Functional Study Of RIP1 And NleC,Two Protein Involved In NF-?B Signaling Pathway

Today, the study of NF-?B signaling is essentially an industry. The significance of NF-?B signaling research is huge for pharmaceutical business. NleC and RIP1 are two different types of enzyme, which both affect NF-?B signaling.NleC, a member of virulence factors injected into infected host cells by enteropathogenic and enterohaemorrhagic Escherichia coli, specifically cleaves the p65 subunit of NF-?B in the p65-p50 heterodimeric complex, resulting the disruption of host NF-?B signaling pathway, thus dampening the host inflammatory response.RIP1 not only plays an important role during the activation of IKK complex in NF-?B signaling pathway, also is an essential upstream signaling molecule in tumor-necrosis-factor-?-induced necroptosis. Necrostatins, a series of small-molecule inhibitors, suppress necroptosis by specifically inhibiting RIP1 kinase activity.We determined the crystal structure of NleC. Representing a brand new type of fold, which was never reported before, 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. In conjunction with biochemical and biophysical analyses, we verified the Cys38 cutting site position of p65 substrate. Furthermore, the electrostatic potential complementarity between NleC and p65 also contributes to the cleavage activity of the protease.We also get the crystal structures of the RIP1 kinase domain individually bound to necrostatin-1 analog, necrostatin-3 analog, and necrostatin-4. Necrostatin, caged in a hydrophobic pocket between the N- and C-lobes of the kinase domain, stabilizes RIP1 in an inactive conformation through interactions with highly conserved amino acids in the activation loop and the surrounding structural elements.Our study provides a structural basis for NleC substrate recognition as well as RIP1 inhibition by necrostatins, and in the mean time offers insights into potential structure-based drug design.