Dimeric granzyme-A mediates the specific recognition and cleavage of full-length GSDMB

Gasdermin pores formed by the processed N-terminal domain of gasdermin family members induce pyroptotic cell death and are involved in multiple diseases. Gasdermin B (GSDMB) is cleaved by granzyme-A (GZMA) from killer lymphocytes, triggering pyroptosis in targeted cancer cells to enhance antitumor immunity. Previous research has indicated that the substrate specificity of GZMA is determined by interactions outside the catalytic site, but the molecular mechanism by which the exosite of GZMA recognizes GSDMB is not well understood. Here we report the cryo-EM structure of human GSDMB in complex with GZMA, revealing the specific recognition of GSDMB-CTD by GZMA. Several association interfaces between GSDMB-CTD and the GZMA monomer or the GZMA dimeric interface were uncovered, in addition to the cleavage sequence bound to the GZMA active site. These contacts function as exosites and present GSDMB substrate to the adjacent GZMA for trans-cleavage. Our results suggest that GZMA specifically recognizes and cleaves GSDMB in a GZMA dimerization interface-dependent manner, implying that a similar mechanism may be applicable to other physiological substrates of granzymes. Cytotoxic lymphocytes-derived granzyme-A (GZMA) cleaves the pore-forming protein GSDMB to trigger pyroptotic killing in target cells. Here, the authors show that the homodimer GZMA provides dual exosites and active sites, allowing specific recognition and process of GSDMB in a trans mode.