The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation.
Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein.
O'Neil
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E.I. du Pont de Nemours and Company, Central Research and Development Department, Wilmington, Delaware 19880-0328.
DeGrado
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D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin; D002417:Cattle; D003196:Computer Graphics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000096:biosynthesis; D010649:Phenylalanine / Q000031:analogs & derivatives*; D010782:Photolysis; D011487:Protein Conformation; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D002147:Calmodulin; D010649:Phenylalanine; C053764:benzoylphenylalanine; D009219:Myosin-Light-Chain Kinase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase