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 Ser195 → Ala variant of human pro-GzmK 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 Met14-Ile17 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 Lys188B-Asp194 and is hence not available for proper substrate binding. The Ser214-Cys220 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 Gly215, Glu219, and Val94. We hypothesize that GzmK is proteolytically active only toward specific, as yet unidentified substrates, which upon approach transiently induce a functional activesite conformation.