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Human calpain-3 and its structural plasticity: dissociation of a homohexamer into dimers on binding titin
Open accessPreprint — published as 10.1016/j.jbc.2024.108133 · Paper page
Abstract
Calpain-3 is an intracellular Ca 2+ -dependent cysteine protease abundant 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 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 produce the 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 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.
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Highly accurate protein structure prediction with AlphaFold
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Metadata
- Authors
- Ye, Q., Henrickson, A., Demeler, B., Serrão, V. H. B., & Davies, P. L.
- Affiliations
- Queen's University (CA)
- University of Lethbridge (CA)
- University of California, Santa Cruz (US)
- Corresponding author
- Davies, Peter L.
- Venue
- Journal of Biological Chemistry
- Publisher
- openRxiv
- Published
- 2024-03-03
- Volume
- 301
- Issue
- 2
- Pages
- 108133-108133
- ISSN
- 0021-9258 (ISSN-L)
- PMC
- PMC11795594
- OpenAlex
- W4405756850
- Cited by
- 4
- References
- 1
- Citations by year
- 2026: 2 · 2025: 2
- FWCI
- 0.90
- Type
- journal article
- Language
- en
- Status
- Retracted
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- PubMed Central (submittedVersion, cc-by-nd)
- Funders
- National Science Foundation (TG-MCB070039N, 1829070, U24GM129547, 1-P30-GM124166-01A1)
- U.S. Department of Energy (DMR-1829070)
- Empire State Development Corporation
- Muscular Dystrophy Association (MDA577340)
- University of California, Santa Cruz (SCR_021755)
- Oregon Health and Science University
- University of Lethbridge
- Canada Research Chairs
- Queen's University
- University of Toronto (U24GM129547)
- National Institutes of Health (S10OD02509, 1s10od025091-01, TG-MCB070039N, GM124166)
- Office of Science
- University of California, San Francisco
- Oregon Institute of Occupational Health Sciences, Oregon Health and Science University
- Canadian Institutes of Health Research (148422_1, 14842_2)
- Natural Sciences and Engineering Research Council of Canada (rgpin-2019-05637)
- National Institute of General Medical Sciences (1-P30-GM124166, 1‐P30‐GM124166‐01A1, GM‐124166, U24GM129547, DMR-1829070, R01GM120600, P30GM124166, R01GM129325)
- National Institute of Allergy and Infectious Diseases
- Division of Materials Research (DMR-1829070, 1829070)
- Biological and Environmental Research
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Calpain Protease Function and Regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- CAPN3, titin, limb-girdle muscular dystrophy, sarcomere
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