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Cited by 4Calpain Protease Function and RegulationMuscle Physiology and DisordersProtein Structure and Dynamics

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Human calpain-3 and its structural plasticity: Dissociation of a homohexamer into dimers on binding titin

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Ye, Qilu, Henrickson, Amy, Demeler, Borries, Balasco Serrão, Vitor Hugo, Davies, Peter L.·2025·Journal of Biological Chemistry
DOI 10.1016/j.jbc.2024.108133PMC PMC11795594

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Abstract

-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.

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Metadata

Authors
Ye, Q., Henrickson, A., Demeler, B., Balasco Serrão, V. H., & Davies, P. L.
Affiliations
  • Queen's University (CA)
  • University of Lethbridge (CA)
Corresponding author
Davies, Peter L.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2025-02
Volume
301
Issue
2
Pages
108133
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/j.jbc.2024.108133
PMC
PMC11795594
OpenAlex
W4405756850
Cited by
4
References
2
Citations by year
2026: 2 · 2025: 2
FWCI
0.90
Type
journal article
Language
en
Status
Retracted
Update notices
erratum (2026-08-01)
Open access
Licence
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 (DMR-1829070, TG-MCB070039N, 1-P30-GM124166-01A1)
  • Muscular Dystrophy Association (MDA577340)
  • University of California, Santa Cruz (SCR_021755)
  • Natural Sciences and Engineering Research Council of Canada (DG-RGPIN-2019-05637)
  • National Institutes of Health (R01-GM129325, S10OD02509, U24GM129547)
  • University of Toronto (U24GM129547)
  • Queen's University
  • Biological and Environmental Research
  • National Institute of Allergy and Infectious Diseases
  • Canada Research Chairs
  • Office of Science
  • Oregon Institute of Occupational Health Sciences, Oregon Health and Science University
  • National Institute of General Medical Sciences (1R01GM120600)
  • Canadian Institutes of Health 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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