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| Title | Human calpain-3 and its structural plasticity: Dissociation of a homohexamer into dimers on binding titin. |
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| Journal, issue, pages | J Biol Chem, Vol. 301, Issue 2, Page 108133, Year 2025 |
| Publish date | Dec 24, 2024 |
 Authors | Qilu Ye / Amy Henrickson / Borries Demeler / Vitor Hugo Balasco Serrão / Peter L Davies /   |
| PubMed Abstract | Calpain-3 is an intracellular Ca-dependent cysteine protease abundant in skeletal muscle. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These ...Calpain-3 is an intracellular Ca-dependent cysteine protease abundant in skeletal muscle. 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 multidomain protein that includes three 40+-residue sequences (NS, IS1, and IS2). The latter sequences are unique to this calpain isoform and are hypersensitive to proteolysis. To investigate the whole enzyme structure and how mutations might affect its activity, we produced the proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in Escherichia coli. During size-exclusion chromatography, this calpain-3 was consistently eluted as a much larger 0.5-MDa complex rather than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS, Blue Native PAGE, and AUC, made the complex amenable to single-particle cryo-EM analysis. From two data sets, we obtained a 3.85-Å reconstruction map that shows the complex is a trimer of calpain-3 dimers with six penta-EF-hand domains at its core. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When this 37-kDa region of titin was co-expressed with calpain-3, the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to several 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. |
 External links | J Biol Chem / PubMed:39725035 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 3.85 Å |
| Structure data |  EMDB-43646: Human calpain-3 forms a homohexamer |
| Source |
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Homo sapiens (human)