Crystal Structure Of Outer Membrane Protein NMB0315from Neisseria Meningitidis

Neisseria meningitidis, a commensal bacteria of human nasopharynx, has drawn global health concern for its potential to cause meningitis and sepsis. The pandemic outbreaks of meningococcal disease (MD) can be devastating with a mortality rate approximately10%in many countries. Due to the rapid progress of the disease after its onset, an early prevention seems to be indispensable, leading to a global research effort on vaccine creation. The outer membrane proteins (OMPs) of N. meningitidis, which have been implicated in bacterial virulence, can effectively induce an immune response and thus are good antigen candidates for MD vaccine design. Based on such reason, a numbers of OMPs have been identified and studied, including NMB0315, a putative peptidase.NMB0315is an OMP identified in Neisseria meningitidis as belonging to serogroup B. Although few studies have been dedicated to the uncharacterized protein NMB0315, sequence similarities implicate that it is a lysostaphin-type metallopeptidase belonging to the M23peptidase family. Proteins from this family are all zinc-dependent peptidase, believed to have a hydrolytic specificity on glycine-glycine peptide bond. They are involved in the degradation of cell wall peptidoglycan during cell growth and separation in Gram-positive bacteria, and are also related to bacterial virulence in some Gram-negative bacteria. Over the past few decades, many members of the M23metallopeptidase family have been identified, and fine structure analysis has been performed for some. Although they all share a conserved zinc containing active site, the diversity of the overall structure confused us about their functionality in different kinds of bacteria. Moreover, it has been reported that some of lysostaphin-type peptidases were synthesized as proenzymes and thus are inactive against the preferred substrates, such as polyglycine peptides.Here, we expressed, purified and crystallized the protein of NMB0315, solved its structure based on X-ray crystallography. The overall structure of NMBO315contains three well-separated domains, with a putative catalytic domain from its carboxyl-terminus. Moreover, a conserved active site was observed in its structure, and as predicted, the metal atom from the active site was proved to be Zn2+originally. Intriguingly, the full-length NMB0315was found in an auto-inhibition state and the kind of inhibition is different from other family members. Our studies improved the understanding of catalytic mechanism of M23metallopeptidase and provided structural basis for exploring the physiological function of NMB0315in N. meningitides.Moreover, The CC1+SOAR domain of the stromal interaction molecule1(STIM1) was expressed and purified, and some artifical reconstructions of the protein were applied to simulate the activated state of STIM1. These works provide enlightening guidance for the structural study of STIM1activation process in the future.