Calpain small subunit homodimerization is robust and calcium-independent

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Calpains are calcium-dependent proteases crucial for cellular processes.
• Dimerization via penta-EF-hand (PEF) domains is essential for calpain function.
• The mechanical stability and dissociation kinetics of calpain PEF domains are not well understood.

Purpose of the Study:

• To investigate the mechanical stability and dissociation mechanics of the calpain small subunit PEF (CAPNS1-PEF) homodimer.
• To determine the role of calcium ions in the mechanical properties of the CAPNS1-PEF homodimer.

Main Methods:

• Single-molecule force spectroscopy was employed to measure the forces required for homodimer dissociation.
• Experiments were conducted on an EGFP-tagged CAPNS1-PEF homodimer across various retraction speeds (0.2-10 μm/s).
• Mechanical stability was assessed in the presence and absence of calcium ions.

Main Results:

• High forces exceeding 300 pN were required to rupture the CAPNS1-PEF homodimer.
• Rupture forces and force-induced unfolding remained unchanged upon removal of calcium ions.
• These findings align with crystal structures indicating minimal calcium-induced conformational changes in the PEF domain.

Conclusions:

• The CAPNS1 homodimer exhibits significant mechanical robustness.
• The penta-EF-hand domains in the CAPNS1 homodimer act as a stable, calcium-insensitive dimerization scaffold.
• Calpain dimerization mechanics are primarily governed by structural integrity rather than calcium-dependent conformational changes.