Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain.
The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.
Hosfield
C M
CM
Department of Biochemistry, Queen's University and The Protein Engineering Network of Centres of Excellence, Kingston, Ontario K7L 3N6, Canada.
Moldoveanu
T
T
Davies
P L
PL
Elce
J S
JS
Jia
Z
Z
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D012492:Salts / Q000737:chemistry; D012694:Serine / Q000737:chemistry
D020033:Protein Isoforms; D012492:Salts; D018698:Glutamic Acid; D012694:Serine; D002154:Calpain; D003546:Cysteine Endopeptidases; D008239:Lysine; D002118:Calcium