Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+ Ions

Anita Alexa, Zoltán Bozóky, Attila Farkas, Peter Tompa, Peter Friedrich

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a Kd of 3400 μM. Phospholipids lower this value to 220 μM. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mM for Calpain B and from 250 to 7 μM for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this "extended transducer" mechanism is presented.

Original languageEnglish
Pages (from-to)20118-20126
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number19
DOIs
Publication statusPublished - May 7 2004

Fingerprint

Calpain
Transducers
Chemical activation
Ions
Mutagenesis
Enzyme activity
Enzymes
Schematic diagrams
Site-Directed Mutagenesis
Rats
Phospholipids
Binding Sites
Mutation
Molecules
m-calpain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+ Ions. / Alexa, Anita; Bozóky, Zoltán; Farkas, Attila; Tompa, Peter; Friedrich, Peter.

In: Journal of Biological Chemistry, Vol. 279, No. 19, 07.05.2004, p. 20118-20126.

Research output: Contribution to journalArticle

Alexa, Anita ; Bozóky, Zoltán ; Farkas, Attila ; Tompa, Peter ; Friedrich, Peter. / Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+ Ions. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 19. pp. 20118-20126.
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