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

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


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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cations, Divalent
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Kallikreins / chemistry*
  • Kallikreins / genetics
  • Kallikreins / metabolism
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Structure-Activity Relationship
  • Zinc / chemistry*
  • Zinc / metabolism


  • Cations, Divalent
  • Recombinant Proteins
  • KLK2 protein, human
  • Kallikreins
  • Zinc

Associated data

  • PDB/1ao5
  • PDB/1ce5
  • PDB/1gvl
  • PDB/1gvz
  • PDB/1hia
  • PDB/1l2e
  • PDB/1npm
  • PDB/1sgf
  • PDB/1spj
  • PDB/1ton
  • PDB/1yph
  • PDB/2bdh
  • PDB/2kai
  • PDB/2pka
  • PDB/2psx
  • PDB/2qxg
  • PDB/2zch
  • PDB/3bsq
  • PDB/3qum
  • PDB/3vfe
  • PDB/4nfe
  • PDB/4nff