Crystal structure of calpain‐3 penta‐EF‐hand (PEF) domain – a homodimerized PEF family member with calcium bound at the fifth EF‐hand

Abstract

Calpains are Ca 2+ dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca 2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3 0 s PEF domain and its crystal structure in the presence of Ca 2+ , which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca 2+ bound at the EF5-hand used for homodimer association. Three of the four Ca 2+ -binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca 2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…This protease shows significant sequence homology with both human µ-(54%) and m-calpain (51%) large subunits (Sorimachi et al, 1989). A recent study analyzed the crystal structure of calpain 3 in the presence of Ca 2+ , confirming the ability of the PEF domain to form a stable homodimer (Partha et al, 2014). However, differently from other calpains, the PEF domain of calpain 3 contains a Ca 2+ ion bound at the fifth EF-hand, which plays a role in homodimer association (Herasse et al, 1999;Partha et al, 2014).…”

Calcium and Proteases

“…To date, 15 calpain-like genes have been described for mammals, 4 in Drosophila, 12 in Caenorhabditis elegans, and 1 in plants (Sorimachi and Suzuki 2001). With the exception of the well-studied calpains, m-calpain, m-calpain, and calpastatin, all other calpain isoforms have only been identified based on gene homology and therefore have not been purified or characterized in the protein form; however, a recent crystal structure demonstrating the penta-EF-hand domain of calpain 3 was published by the Davies laboratory (Partha and Partha, 2014).…”

The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation

“…However, a similar heterodimer of CAPN3 with CAPNS1 has not been detected, which may also suggest that the carboxyl-terminal region of CAPN3 and other CAPNs plays different roles in these related proteases. In line with such difference, the EF5-hand of CAPN3 is unique in that it is a binding site for Ca 2+ as well as an interacting site for homodimer formation of the PEF(L) domain (33). It was also shown that a CAPN3 mutant completely lacking IS2 and PEF(L) (CAPN3: S581X), expresses protease activity comparable to that of FL-CAPN3 (35).…”

“…Studies focusing on homodimerization of CAPN3 have revealed the unique feature in the C-terminal PEF(L) domain (32,33). With respect to the pathological mechanisms of Significance CAPN3/p94/calpain-3, a calpain protease family member, has uniquely rapid and exhaustive autolytic activity.…”