TY - JOUR
T1 - Stoichiometry of the Calcineurin Regulatory Domain-Calmodulin Complex
AU - Dunlap, Tori B.
AU - Guo, Hou Fu
AU - Cook, Erik C.
AU - Holbrook, Emily
AU - Rumi-Masante, Julie
AU - Lester, Terrence E.
AU - Colbert, Christopher L.
AU - Vander Kooi, Craig W.
AU - Creamer, Trevor P.
N1 - Publisher Copyright:
© 2014 American Chemical Society.
PY - 2014/9/16
Y1 - 2014/9/16
N2 - Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.
AB - Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.
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U2 - 10.1021/bi5004734
DO - 10.1021/bi5004734
M3 - Article
C2 - 25144868
AN - SCOPUS:84908354124
SN - 0006-2960
VL - 53
SP - 5779
EP - 5790
JO - Biochemistry
JF - Biochemistry
IS - 36
ER -