title: Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity.
abstract: 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.
authors.affiliation: From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Skala
authors.affiliation: Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der TU München, D-81675 Munich, Germany.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Utzschneider
authors.affiliation: Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der TU München, D-81675 Munich, Germany.
authors.forename: Viktor
authors.initials: V
authors.lastname: Magdolen
authors.affiliation: Max-Planck-Institut for Biochemistry, Proteinase Research Group, D-82152 Martinsried, Germany, and.
authors.forename: Mekdes
authors.initials: M
authors.lastname: Debela
authors.affiliation: From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria.
authors.forename: Shihui
authors.initials: S
authors.lastname: Guo
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.affiliation: From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria.
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
authors.affiliation: From the Division of Structural Biology, Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria, peter.goettig@sbg.ac.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Goettig
mesh_terms: D000595:Amino Acid Sequence; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007610:Kallikreins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; C000595667:KLK2 protein, human; D007610:Kallikreins; D015032:Zinc
keywords: 99-Loop; Autolytic Cleavage; Conformational Selection; Crystallography; Enzyme Kinetics; Kallikrein; Prostate Cancer; Serine Protease; Substrate Specificity; Zinc Inhibition