Structural Insight Into The Nisin Leader Peptidase NisP

Nisin is a natural antibiotic produced by Lactococcus lactis. It belongs to the Group A lantibiotics. In mature nisin molecule, there are five lanthionine rings, which confers good stability on nisin even at high temperature up to100℃.Nisin effectively kills a broad range of gram-positive microorganisms including several pathogenic bacteria. For it’s nontoxic to mammalians and humans, nisin has been widely used as food preservative for a long history.Nisin is synthesized in a precursor form.To become a mature lantibiotic, the nisin precursor needs to undergo several modification steps, including dehydration, ring formation, transport across the membrane and cleavage of the leader peptide. Unmodified or modified nisin precursor with the leader peptide attached shows no activity. NisP, which belongs to the subtilase superfamily of serine proteases, functions as the leader peptidase in nisin leader peptide cleavage.Disruption of the nisP gene in LAC71results in the production of inactive nisin with the leader peptide attached.In this study, mature NisP protein was crystallized and the high-resolution structure was resolved using single wavelength anomalous dispersion method. The NisP structure shows great similarity to other members of the subtilase family. However, in the structure we solved, a short peptide from the C-terminal part of the NisP protein was bonded. It located at the surface groove of the protein, and the last Arg647residue was right at the catalytic triad of the protein, indicating the peptide was possibly from the autocleavage activity of NisP. With the synthesized peptide corresponding to the cleavage site, enzyme activity assay was performed. The results showed that NisP is capable of getting its C-teminus autocleaved. Through western blot and mass spectrum the C-terminal autocleavage activity of NisP was confirmed. By mutating the cleavage site residues Arg647and Ser648to two prolines, the C-termius of NisP was not cleaved, indicating the Arg647/Ser648was the right and unique autocleavage site of NisP. In addition, the C-terminal cleavage site is on the N-terminal of the LPXTG cell wall binding motif, the auto-cleavage should lead to the release of NisP from the cell wall.To explore the physiology function of the autocleavage activity, bacterial inhibition assays were performed both underin vivo and in vitro conditions. The results showed that cleavage of the C-terminus did not affect the activity of NisP in nisin maturation. However, remaining the NisP C-terminus uncleaved, growth rate of the Lactococcus lactis expressing NisP protein was lowered, indicating the significance of the NisP C-terminal autocleavage activity.Meanwhile, previous studies reported that many members of the subtilase family got calcium ions bond in their structures. And putative calcium binding sites in NisP were predicted. However, no calcium ion was found in the NisP structure solved. The predicted calcium ion chelating residues distribute linearly and did not form a good metal ion binding system. Also, effect of calcium ions on NisP activity was exanmined. Distinguishied form several other members of the subtilase family, NisP’s activity was not dependent on the calcium ions.In conclusion, the study found an unusual C-terminal autocleavage activity of NisP and the activity is of significant biological function. In addition, the NisP structure demonstrates how NisP recognizes the consensus sequence before the cutting site, and provides clues for the study of substrate specificity of NisP.