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J Biol Chem. 2014 Dec 5;289(49):34267-83. doi: 10.1074/jbc.M114.598201. Epub 2014 Oct 16.

Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity.

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From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria.
Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der TU München, D-81675 Munich, Germany.
Max-Planck-Institut for Biochemistry, Proteinase Research Group, D-82152 Martinsried, Germany, and.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria,


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 Å 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 Zn(2+) concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn(2+), which located the Zn(2+) 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.


99-Loop; Autolytic Cleavage; Conformational Selection; Crystallography; Enzyme Kinetics; Kallikrein; Prostate Cancer; Serine Protease; Substrate Specificity; Zinc Inhibition

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