Distinguishing between calpain heterodimerization and homodimerization

Abstract

The two main mammalian calpains, 1 and 2, are heterodimers of a large 80 kDa and a small 28 kDa subunit that together bind multiple calcium ions during enzyme activation. The main contact between the two subunits of these intracellular cysteine proteases is through a pairing of the fifth EF‐hand of their C‐terminal penta‐EF‐hand (PEF) domains. From modeling studies and observation of crystal structures, it is not obvious why these calpains form heterodimers with the small subunit rather than homodimers of the large subunit, as suggested for calpain 3 (p94). Therefore, we have used a differential tagging system to determine which of the other PEF domain‐containing calpains form heterodimers and which form homodimers. His6‐tagged PEF domains of calpains 1, 3, 9 and 13 were coexpressed with the PEF domain of the small subunit that had been tagged with an antifreeze protein. As predicted, the PEF domain of calpain 1 heterodimerized and that of calpain 3 formed a homodimer. The PEF domain of digestive tract‐specific calpain 9 heterodimerized with the small subunit, and that of calpain 13, prevalent in lung and testis, was mainly found as a homodimer with a small amount of heterodimer. These results indicate whether recombinant production of a particular calpain requires coexpression of the small subunit, and whether this calpain is likely to be active in a small subunit knockout mouse. Furthermore, as the endogenous inhibitor calpastatin binds to PEF domains on the large and small subunit, it is less likely that the homodimeric calpains 3 and 13 with two active sites will bind or be silenced by calpastatin.

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

“…As the PEF domain of calpain‐3 has an unpaired EF‐hand, it was natural to suspect that this could act as the homodimer interface. Our previous studies on the isolated calpain‐3 PEF domain expressed as a recombinant protein showed that it formed a stable homodimer and did not form a heterodimer when coexpressed in E. coli with the calpain small subunit . The present crystal structure confirms the ability of the calpain‐3 PEF domain to form a stable homodimer, and provides direct structural support for the homodimerization of the full‐length enzyme.…”

“…To further investigate the nature of this homodimerization, we have solved the crystal structure of the calpain‐3 PEF domain dimer in the presence of Ca 2+ . The structure supports computer models of the calpain‐3 dimer with two active sites at opposite ends of the molecule . Unexpectedly, the PEF domain binds Ca 2+ at EF5, used for dimerization, and is the only member of the calpain family known to do so.…”

A New Insight into the Role of Calpains in Post-mortem Meat Tenderization in Domestic Animals: A review

“…i) CAPN3 is mainly expressed in skeletal muscle (Sorimachi et al, 1989), yet, during development, it also occurs in lens, liver, brain and cardiac muscle (Poussard et al, 1996; Fougerousse et al, 1998; Ma et al, 1998; Konig et al, 2003). ii) CAPN3 is likely to function as a homodimer due to its lacking a small subunit (28 kDa) (Blanchard et al, 1996; Blanchard et al, 1997; Kinbara et al, 1998; Ravulapalli et al, 2005; Ravulapalli et al, 2009). At the same time, CAPN3 has some unique domains distinguishing it from the ubiquitous CAPNs, including its NH2-terminal domain I (contains 20 to 30 additional amino acids) and two “insertion sequences” which contain 62 and 77 amino acids at the COOH-terminal regions of domain II and III, called IS1 and IS2, respectively (Goll et al, 2003).…”

Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation

“…Evidence for this association came initially from the absence of small subunit in calpain-3 preparations (12) and from the apparent molecular weight of the enzyme (~180,000 Da) being twice that expected for the monomer (94 000 Da) when autolysis was suppressed by mutation of the catalytic Cys to Ser (13). Subsequently, we showed direct evidence of homodimerization of the calpain-3 PEF domain (14,15) and have subsequently solved the crystal structure of the Ca 2+ -bound PEF homodimer (16). Other structural differences from the large subunit of calpain-1 and -2 are the presence of two insertion sequences (IS) that are not seen in any of the other calpain isoforms.…”