Molecular Mechanism of Latency and Substrate Specificity in Pappalysin-like Metallopeptidases

Objectives: Bacterial proteases contribute to periodontitis development. Tannerella forsythia (Tf) is the sole periodontopathogen secreting protease mirolysin, a M43B-metalloprotease family member with human pappalysins. M43B proteases share unique specificity for Xaa-Arg and Xaa-Lys (P1-P1’) peptide bonds and are produced as inactive zymogens. Therefore, this project’s aims were to i) determine molecular basis of mechanism of latency and substrate specificity in M43B proteases and ii) check if mirolysin is responsible for Tf resistance to LL-37, a crucial human oral cavity antimicrobial peptide.
Methods: Differential scanning fluorimetry (DSF) was performed using Tycho NT.6. Activation of promirolysins: wild-type (wt) and point mutants: C23A and C23L, was analyzed by SDS-PAGE and measurement of proteolytic activity employing FTC-Casein as substrate. Crystal structure of mature mirolysin bound with product of hydrolysis was solved and analyzed. LL-37’s bactericidal effect on Tf: wt and mirolysin deletion mutant (delta mir) was evaluated using colony reduction assay.
Results: Point mutation of Cys²³ into both Ala and Leu significantly decreased promirolysin’s inflection temperature. DSF findings were corroborated by studies of activation in trans and in cis showing that promirolysin point mutants: C23A and C23L were processed more quickly than the wt protein. Crystal structure of mirolysin with product of the hydrolysis revealed that mirolysin S1’ specificity pocket is narrow but deep and D²⁸⁹, conserved among M43B proteases, forms its negatively-charged bottom. Such properties of S1’ pocket is ideal for accommodating both Lys and Arg. Tf delta mir, contrary to wt bacteria, was not resistant to the antimicrobial activity of LL-37. Phenotype of knock-out strain was restored by supplementation of bacteria with recombinant mirolysin.
Conclusions: Latency of mirolysin and other M43B proteases is exerted by cysteine-switch. Properties of S1’ subsite of substrate-binding pocket explain the unique substrate specificity of M43B enzymes. Mirolysin is responsible for Tf resistance to LL-37.