Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.
Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].
Yang
S A
SA
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Building 37, Room 4E28, Bethesda, Maryland 20892, USA.
Klee
C B
CB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D002614:Chelating Agents; D010446:Peptide Fragments; D004492:Edetic Acid; D019703:Calcineurin; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium