Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif.
The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an alpha-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an alpha-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed.
Yuan
T
T
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Vogel
H J
HJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D019657:Solanaceae / Q000201:enzymology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002147:Calmodulin; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; D002118:Calcium