Flexibility Analysis and Structure Comparison of Two Crystal Forms of Calcium-Free Human m-Calpain

Abstract

The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papainrelated catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and fulllength human apomcalpain revealed the domain arrangement and explained the inactivity of mcalpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that mcalpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papainlike catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of mcalpain, we have determined and refined the human fulllength apomcalpain structure of a second crystal form to 3.15 å resolution. Here we present this new structure, compare it with our first structure now rerefined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calciuminduced activation process.