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 kD a) can activate some 40 distinct proteins in a calcium-dependent manne r. The majority of the CaM-binding domain regions of the target protei ns are basic and hydrophobic in nature, are devoid of multiple negativ ely charged residues, and have a propensity to form an alpha-helix. Th e CaM-binding domain in the C-terminal region of petunia glutamate dec arboxylase (PGD) is atypical because it contains five negatively charg ed residues. Therefore, we chose to study the binding of calcium-CaM t o a as-residue synthetic peptide encompassing the C-terminaI region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titrat ion studies showed that a single unique complex of calcium-CaM with tw o PGD peptides is formed. The formation of a 1:2 protein-peptide compl ex is unusual; normally, calcium-CaM forms 1:1 complexes with the majo rity of its target proteins. Circular dichroism spectroscopy showed th at the bound PGD peptides have an alpha-helical structure. NMR studies of biosynthetically [methyl-C-13]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 lo bes of CaM. These results predict that binding of calcium-CaM to PGD w ill give rise to dimerization of the protein, which may be necessary f or activation. Possible models for the structure of the protein-peptid e complex, such as a dimeric peptide structure, are discussed.