Hilfe Wegweiser Impressum Kontakt Einloggen





Structure-Function Analyses of Human Kallikrein-related Peptidase 2 Establish the 99-Loop as Master Regulator of Activity


Debela,  Mekdes
Former Research Groups, Max Planck Institute of Biochemistry, Max Planck Society;

Externe Ressourcen
Es sind keine Externen Ressourcen verfügbar
Volltexte (frei zugänglich)

J. Biol. Chem.-2014-Skala-34267-83.pdf
(beliebiger Volltext), 5MB

Ergänzendes Material (frei zugänglich)
Es sind keine frei zugänglichen Ergänzenden Materialien verfügbar

Skala, W., Utzschneider, D. T., Magdolen, V., Debela, M., Guo, S., Craik, C. S., et al. (2014). Structure-Function Analyses of Human Kallikrein-related Peptidase 2 Establish the 99-Loop as Master Regulator of Activity. JOURNAL OF BIOLOGICAL CHEMISTRY, 289(49), 34267-34283. doi:10.1074/jbc.M114.598201.

Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation. Results: Enzymatic assays and structures of KLK2 elucidate its catalytic action, especially when compared with conformations of similar proteases. Conclusion: Flexible loops around the active site of serine proteases open concertedly upon substrate binding. Significance: This mechanistic model will stimulate the design of pharmaceutical inhibitors. Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the classical KLKs 1-3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation.