Premium
Crystal structure of recombinant human tissue kallikrein at 2.0 Å resolution
Author(s) -
Katz Bradley A.,
Liu Beishan,
Barnes Micael,
Springman Eric B.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070405
Subject(s) - kallikrein , proteases , trypsin , chemistry , biochemistry , serine , pichia pastoris , serine protease , binding site , recombinant dna , microbiology and biotechnology , protease , enzyme , biology , gene
Human tissue kallikrein, a trypsin‐like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris , purified, and crystallized. The crystal structure at 2.0 Å resolution is described and compared with that of porcine kallikrein and of other trypsin‐like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27 :157‐162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis ‐Pro 219. The replacement Tyr 228 → Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between Oδ1 Asp189 and Oγ ser226 . These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin‐like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg‐Ser and a Met‐Lys peptide bond in its natural protein substrate, kininogen. A Zn +2 ‐dependent, small‐molecule competitive inhibitor of kallikrein (K i = 3.3 μM) has been identified and the bound structure modeled to guide drug design.