Abstract
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.
Highlights
Caspases are cysteine–aspartic proteases involved in the regulation of programmed cell death and a number of other biological processes
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
Hydrogen– deuterium exchange MS–mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the N-terminal domain (NTD) and the 240’s region, which serves as a general exosite for caspase-6 – specific substrate recruitment
Summary
The patch of caspase-7 consists of a highly positive tetra-lysine 38KKKK41 stretch, which helps to recruit protein substrates PARP-1 and p23 [21, 22] This exosite enables caspase-7 to cleave these substrates 30-fold more efficiently than caspase-3, despite the fact these two enzymes share the same overall specificity for peptide substrates [23, 24]. We describe the discovery of an evolutionarily conserved tri-arginine patch, 42RRR44, located within the disordered NTD of caspase-6 that serves as an exosite involved in the recruitment of protein substrates. Removal of this site does not hinder the intrinsic catalytic capability of the enzyme. This work provides new perspectives into how caspase-6 can bind, recruit, and hydrolyze protein substrates and uncovers a scaffold that may be utilized for the selective modulation of caspase-6
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