A muscle-specific calpain, CAPN3, forms a homotrimer

Area of Science:

• Muscle physiology
• Protease function
• Molecular complex formation

Background:

• Calpain-3 (CAPN3) is crucial for skeletal muscle function, with mutations causing limb girdle muscular dystrophy type 2A.
• Previous crystallographic data suggested CAPN3 functions as a homodimeric protease.
• Understanding CAPN3's native complex formation is key to elucidating its physiological roles.

Purpose of the Study:

• To investigate the native oligomeric state of Calpain-3 (CAPN3).
• To determine the domains responsible for CAPN3 complex formation.
• To clarify the functional quaternary structure of CAPN3.

Main Methods:

• Blue native polyacrylamide gel electrophoresis (BN-PAGE) to assess native molecular weight.
• Cross-linking experiments to stabilize protein complexes.
• Sequential immunoprecipitation to confirm protein interactions.
• Domain deletion analysis to identify key structural elements.

Main Results:

• Native and recombinant CAPN3 exhibited a molecular weight exceeding 240 kDa, inconsistent with a homodimer.
• Further analysis confirmed that CAPN3 forms a homotrimer.
• Trimer formation was dependent on the penta-EF-hand (PEF) domain, but not the NS, IS1, or IS2 insertion sequences.
• The PEF domain alone formed a homodimer, but its trimerization capability was enhanced by the adjacent CBSW domain.

Conclusions:

• Calpain-3 (CAPN3) predominantly exists as a homotrimer in solution.
• The penta-EF-hand (PEF) domain is essential for CAPN3 trimerization.
• The N-terminal CBSW domain influences the trimer-forming property of the PEF domain.
• These findings refine our understanding of CAPN3 structure-function relationships in muscle.