Disruption of interdomain interactions via partial calcium occupancy of calmodulin.
Binding of calcium to CaM exposes clefts in both N- and C-domains to promote their cooperative association with a diverse array of target proteins, functioning to relay the calcium signal regulating cellular metabolism. To clarify relationships between the calcium-dependent activation of individual domains and interdomain structural transitions associated with productive binding to target proteins, we have utilized three engineered CaM mutants that were covalently labeled with N-(1-pyrene) maleimide at introduced cysteines in the C- and N-domains, i.e., T110C (PyC-CaM), T34C (PyN-CaM), and T34C/T110C (Py2-CaM). These sites were designed to detect known conformers of CaM such that upon association with classical CaM-binding sequences, the pyrenes in Py2-CaM are brought close together, resulting in excimer formation. Complementary measurements of calcium-dependent enhancements of monomer fluorescence of PyC-CaM and PyN-CaM permit a determination of the calcium-dependent activation of individual domains and indicate the sequential calcium occupancy of the C- and N-terminal domains, with full saturation at 7.0 and 300 microM calcium, respectively. Substantial amounts of excimer formation are observed for apo-CaM prior to peptide association, indicating that interdomain interactions occur in solution. Calcium binding results in a large and highly cooperative reduction in the level of excimer formation; its calcium dependence coincides with the occupancy of C-terminal sites. These results indicate that interdomain interactions between the opposing domains of CaM occur in solution and that the occupancy of C-terminal calcium binding sites is necessary for the structural coupling between the opposing domains associated with the stabilization of the interdomain linker to enhance target protein binding.
Boschek
Curt B
CB
Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Squier
Thomas C
TC
Bigelow
Diana J
DJ
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D008301:Maleimides; C003944:N-(3-pyrene)maleimide; D002118:Calcium