The 2.2-Å Crystal Structure of Human Pro-granzyme K Reveals a Rigid Zymogen with Unusual Features

Abstract

Granzyme K (GzmK) belongs to a family of trypsin-like serine proteases localized in electron dense cytoplasmic granules of activated natural killer and cytotoxic T-cells. Like the related granzymes A and B, GzmK can trigger DNA fragmentation and is involved in apoptosis. We expressed the Ser 195 3 Ala variant of human proGzmK in Escherichia coli, crystallized it, and determined its 2.2-Å x-ray crystal structure. Pro-GzmK possesses a surprisingly rigid structure, which is most similar to activated serine proteases, in particular complement factor D, and not their proforms. The N-terminal peptide Met 14 -Ile 17 projects freely into solution and can be readily approached by cathepsin C, the natural convertase of pro-granzymes. The pre-shaped S1 pocket is occupied by the ion paired residues Lys 188B -Asp 194 and is hence not available for proper substrate binding. The Ser 214 -Cys 220 segment, which normally provides a template for substrate binding, bulges out of the active site and is distorted. With analogy to complement factor D, we suggest that this strand will maintain its nonproductive conformation in mature GzmK, mainly due to the unusual residues Gly 215 , Glu 219 , and Val 94 . We hypothesize that GzmK is proteolytically active only toward specific, as yet unidentified substrates, which upon approach transiently induce a functional activesite conformation.

Granzyme F induces a novel death pathway characterized by Bid-independent cytochrome c release without caspase activation

“…Recombinant GzmF was expressed in E. coli and refolded from inclusion bodies as previously described. 19 The inactive counterpart, Ser195AlaGzmF (S-AGzmF), was generated by a point mutation of the active site, Ser 195, to Ala. The Gzms were purified with nickel chelation columns, followed by cation exchange chromatography.…”

“…Both proteins were expressed and refolded from Rosetta (DE3) (Novagen, Darmstadt, Germany) inclusion bodies as described. 19 After thoroughly washing and refolding for 3 days, proteins were harvested and activated by enterokinase treatment. The enterokinase was removed and GzmF was purified on a Hitrap ion exchange column (Amersham Biosciences, Pittsburgh, PA, USA).…”

Identification and characterization of common carp (Cyprinus carpio L.) granzyme A/K, a cytotoxic cell granule-associated serine protease

“…GzmK, like GzmA and GzmB, is synthesized as a zymogen precursor, containing a signal and a short propeptide preceding the mature polypeptide chain [15]. GzmK and GzmA differ from GzmB by their substrate specificity [5]. The study showed that GzmA-deficient CTLs still contain 20% tryptase activity and their cytolytic activity is just slightly reduced [16], suggesting that GzmK might be a potent Gzm to rescue the GzmA activity.…”

“…Gzms belongs to a family of serine proteases (SPs) localized in the electron dense cytoplasmic granules of activated NK and Tc cells [5]. Gzm loci have been placed into three groups by their chromosomal location and structural similarities.…”

“…GzmK was originally discovered in granules of human lymphokine-stimulated killer cells as a second tryptase among all the Gzms [5]. In evolutionary terms, GzmK and GzmA are the only two tryptases among all the Gzms, and are the most closely related Gzms.…”