Structural characterization of Ca2+/CaM in complex with the phosphorylase kinase PhK5 peptide.
Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (alphabetagammadelta)4. An intrinsic calmodulin (CaM, the delta subunit) binds directly to the gamma protein kinase chain. The interaction site of CaM on gamma has been localized to a C-terminal extension of the kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical 'compact' CaM complex.
Cook
Atlanta G
AG
Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, UK.
Johnson
Louise N
LN
McDonnell
James M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D021122:Protein Subunits; D010764:Phosphorylase Kinase; D002118:Calcium