title: Stoichiometry of the calcineurin regulatory domain-calmodulin complex.
abstract: 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.
authors.affiliation: Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , 741 South Limestone Street, Lexington, Kentucky 40536-0509, United States.
authors.forename: Tori B
authors.initials: TB
authors.lastname: Dunlap
authors.forename: Hou-Fu
authors.initials: HF
authors.lastname: Guo
authors.forename: Erik C
authors.initials: EC
authors.lastname: Cook
authors.forename: Emily
authors.initials: E
authors.lastname: Holbrook
authors.forename: Julie
authors.initials: J
authors.lastname: Rumi-Masante
authors.forename: Terrence E
authors.initials: TE
authors.lastname: Lester
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Colbert
authors.forename: Craig W
authors.initials: CW
authors.lastname: Vander Kooi
authors.forename: Trevor P
authors.initials: TP
authors.lastname: Creamer
mesh_terms: D001483:Base Sequence; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008968:Molecular Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D017931:DNA Primers; D019703:Calcineurin