Electrostatic interactions of domain III stabilize the inactive conformation of mu-calpain.
The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human mu-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive micro-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.
Fernández-Montalván
Amaury
A
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Ludwig-Maximilians-Universität München, Nussbaumstr. 20, D-80336 München, Germany.
Assfalg-Machleidt
Irmgard
I
Pfeiler
Dietmar
D
Fritz
Hans
H
Jochum
Marianne
M
Machleidt
Werner
W
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013379:Substrate Specificity
D010455:Peptides; D002154:Calpain; C449002:mu-calpain; D002118:Calcium