Phosphorylation Regulates Assembly of the Caspase-6 Substrate-Binding Groove

Abstract

Summary Caspases, a family of apoptotic proteases, are increasingly recognized as being extensively phosphorylated, usually leading to inactivation. To date, no structural mechanism for phosphorylation-based caspase inactivation is available, although this information may be key to achieving caspase-specific inhibition. Caspase-6 has recently been implicated in neurodegenerative conditions including Huntington's and Alzheimer's diseases. A full understanding of caspase-6 regulation is crucial to caspase-6-specific inhibition. Caspase-6 is phosphorylated by ARK5 kinase at serine 257 leading to suppression of cell death via caspase-6 inhibition. Our structure of the fully inactive phosphomimetic S257D reveals that phosphorylation results in a steric clash with P201 in the L2′ loop. Removal of the proline side chain alleviates the clash resulting in nearly wild-type activity levels. This phosphomimetic-mediated steric clash causes misalignment of the substrate-binding groove, preventing substrate binding. Substrate-binding loop misalignment appears to be a widely used regulatory strategy among caspases and may present a new paradigm for caspase-specific control.

Structural snapshots reveal distinct mechanisms of procaspase-3 and -7 activation

“…Rather, this suggests that the primary purpose of cleavage is to generate the loop bundle, which functions as an allosteric activator of the mature enzyme. This observation is supported by studies showing that mutations within the loop bundle can alter catalytic activity without altering the structure of the active enzyme, and by the observation that phosphorylation of the loop bundle can regulate activity in C6 (25,30,31).…”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…Among the apoptotic caspases, caspase-6 is uniquely resistant to X-linked inhibitor of apoptosis protein-mediated inhibition at either the active site or the dimer interface (22), underscoring the fact that regulation of caspase-6 is distinctive. To date, only caspase-6 has been reported to be inactivated by phosphorylation at Ser-257 by ARK5 kinase (2), which allosterically prevents the proper conformation of one of the substratebinding loops (23). The understanding of the unique features of such regulatory pathways improves our ability to utilize caspase-6 as a therapeutic target.…”

Palmitoylation of caspase-6 by HIP14 regulates its activation

“…To further determine how palmitoylation of CASP6 might affect its processing and activation (Figure 4a), we utilized computational molecular modelling based on the published crystal structures of CASP6. 24,25,[49][50][51][52][53][54][55] Generation of the model of CASP6 required the construction of coordinates of loop 2 (L2), which forms the substrate-binding groove (active site) and is not resolved in any of the several crystal structures available, presumably owing to high flexibility. The structure of CASP6 has a six-stranded β-sheet flanked by five α-helices and two small β-strands.…”

“…Crystal structure studies of the phospho-mimetic S257D suggested that phosphorylation of S257 inactivates CASP6 through a steric clash with the proline side chain of residue P201 in the L2' loop, 49 causing reorganization of the L2 loop into an inactive position. 59 Indeed, removal of the clashing proline side chain of P201 (P201G) reversed the S257 phosphorylation-mediated inhibition of CASP6 activity, suggesting that steric hindrance was the underlying mechanism of inhibition.…”