A multipronged approach for compiling a global map of allosteric regulation in the apoptotic caspases.

One of the most promising and as yet underutilized means of regulating protein function is exploitation of allosteric sites. All caspases catalyze the same overall reaction, but they perform different biological roles and are differentially regulated. It is our hypothesis that many allosteric sites exist on various caspases and that understanding both the distinct and overlapping mechanisms by which each caspase can be allosterically controlled should ultimately enable caspase-specific inhibition. Here we describe the ongoing work and methods for compiling a comprehensive map of apoptotic caspase allostery. Central to this approach are the use of (i) the embedded record of naturally evolved allosteric sites that are sensitive to zinc-mediated inhibition, phosphorylation, and other posttranslational modifications, (ii) structural and mutagenic approaches, and (iii) novel binding sites identified by both rationally-designed and screening-derived small-molecule inhibitors.

Caspase-6 latent state stability relies on helical propensity.

Caspase-6 is an apoptotic protease that also plays important roles in neurodegenerative disorders, including Huntington's and Alzheimer's diseases. Caspase-6 is the only caspase known to form a latent state in which two extended helices block access to the active site. These helices must convert to strands for binding substrate. We probed the interconverting region and found that the absence of helix-breaking residues is more critical than a helix-bridging, hydrogen-bond network for formation of the extended conformation. In addition, our results suggest that caspase-6 must undergo a transition through a low-stability intermediate to bind the active-site ligand. Mature caspase-6 is capable of adopting a latent state not observed in any other caspase. The absence of any helix-breaking residues allows caspase-6 to adopt the extended helical conformation. When we introduced helix-breaking residues similar to those seen in caspase-3 or -7, the structure and stability of the latent state were compromised.

Substrate-induced conformational changes occur in all cleaved forms of caspase-6.

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.