CALPAIN-3 STRUCTURE AND ITS ROLE IN LIMB-GIRDLE MUSCULAR DYSTROPHY R1

講演要旨

Calpain-3 (P94) is an intracellular Ca2+-dependent cysteine protease produced in skeletal muscle. Its physiological role in the sarcomere is thought to include removing damaged muscle proteins after exercise. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb girdle muscles. These mutations, of which there are over 500 in humans, are spread all along this 94-kDa multi-domain protein. Three 40+-residue sequences (NS, IS1 and IS2) are unique to this calpain isoform and are sensitive to proteolysis. We have previously published the crystal structures of the two protease core domains with IS1 present as a largely unstructured loop, and the penta-EF-hand domain from the C terminus that forms a tight homodimer. To investigate the whole enzyme structure, we are producing a proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in E. coli. During size-exclusion chromatography this calpain-3 eluted as a much larger 480-kDa complex than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS and Blue Native PAGE, encouraged us to solve the multimer structure by single particle cryo-EM. From two data sets we have a 3.5-Å volume map under refinement that shows the complex is a trimer of calpain-3 dimers. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When a 37-kDa section of titin from this region was co-expressed in E. coli with calpain-3 the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to four copies of the titin fragment. We suggest that newly synthesized calpain-3 is kept as an inactive hexamer until it binds the N2A region of titin in the sarcomere, whereupon it dissociates into functional dimers. This seminar is dedicated to the memory of Professor Hiroyuki Sorimachi.