Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis

• Published in: The Journal of biological chemistry Vol. 294; no. 1; pp. 71 - 88
• Main Authors: MacPherson, Derek J., Mills, Caitlyn L., Ondrechen, Mary Jo, Hardy, Jeanne A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.01.2019; American Society for Biochemistry and Molecular Biology
• Subjects: allosteric regulation; Amino Acid Substitution; apoptosis; Arginine - chemistry; Arginine - genetics; Arginine - metabolism; cancer; Caspase 6 - chemistry; Caspase 6 - genetics; Caspase 6 - metabolism; caspase-6; cysteine protease; Humans; hydrogen–deuterium exchange; Hydrolysis; mass spectrometry; Mutation, Missense; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Neoplasms - enzymology; Neoplasms - genetics; Neoplasms - pathology; neurodegeneration; Neurodegenerative Diseases - enzymology; Neurodegenerative Diseases - genetics; Neurodegenerative Diseases - pathology; Protein Domains; protein dynamic; Protein Structure and Folding; substrate recognition; substrate specificity; tri-arginine exosite; cysteine protease; caspase-6; hydrogen–deuterium exchange; substrate specificity; tri-arginine exosite; neurodegeneration; apoptosis; mass spectrometry; cancer; allosteric regulation; substrate recognition; protein dynamic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.005914

Caspases are cysteine–aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23–45, and core of the caspase-6 structure. We observed that substitutions of the tri-arginine patch Arg-42–Arg-44 or the R44K cancer-associated mutation in caspase-6 markedly alter its rates of protein substrate hydrolysis. Notably, turnover of protein substrates but not of short peptide substrates was affected by these exosite alterations, underscoring the importance of this region for protein substrate recruitment. Hydrogen–deuterium exchange MS–mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the NTD and the 240's region (containing residues 236–246), which serves as a general exosite for caspase-6–specific substrate recruitment. In summary, we have identified an exosite on caspase-6 that is critical for protein substrate recognition and turnover and therefore highly relevant for diseases such as cancer in which caspase-6–mediated apoptosis is often disrupted, and in neurodegeneration in which caspase-6 plays a central role.