Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.
We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides.
Vetter
S W
SW
Institute of Biochemistry, ETH-Zentrum, Zürich, Switzerland. svetter@scripps.edu
Leclerc
E
E
D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008962:Models, Theoretical; D009479:Neuropeptides; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010455:Peptides; D020033:Protein Isoforms; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D012694:Serine; D001120:Arginine