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Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis
T Moldoveanu1, C M Hosfield, Z Jia
1Department of Biochemistry and the Protein Engineering Network of Centres of Excellence, Queen's University, Kingston, Ont. K7L 3N6, Canada.
Biochimica Et Biophysica Acta
|May 9, 2001
Summary
Calcium ions (Ca2+) induce conformational changes in m-calpain, a thiol protease. Proteolysis studies reveal Ca2+ enhances domain III accessibility and protects the active site, supporting a model of calpain activation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- m-Calpain is a calcium-dependent thiol protease.
- Understanding its structure and activation mechanism is crucial for various cellular processes.
Purpose of the Study:
- To map protease-resistant domains in m-calpain.
- To investigate calcium-induced conformational changes in m-calpain.
Main Methods:
- Partial proteolysis using trypsin and chymotrypsin on an inactive calpain mutant (Cys105Ser).
- N-terminal sequencing of major proteolytic products.
- Comparative analysis of digestion in the presence and absence of Ca(2+).
Main Results:
- Proteolysis primarily affected the large subunit (domains I-IV), with limited impact on the small subunit (domain VI).
- Hydrolysis sites were identified in the N-terminal anchor peptide, near domain II, and within domain III.
- Domain III showed increased mobility and accessibility upon Ca(2+) binding.
- A Ca(2+)-sensitive site within the active site cleft was protected by Ca(2+).
Conclusions:
- Ca(2+) binding significantly alters m-calpain conformation, increasing domain III accessibility.
- Ca(2+) promotes the formation of the active site by bringing domains I and II closer together.
- These findings support a model for Ca(2+)-mediated activation of calpain.