An examination of glutamic acid in the -X chelating position of the helix-loop-helix calcium binding motif.
Poor calcium affinity was exhibited in helix-loop-helix calcium binding motifs with X-axis acid pairs containing aspartic acid in the -X chelating position. In order to increase interaction of the -X chelating residue with the cation, helix-loop-helix calcium binding motifs were synthesized containing three and four acid residues in chelating positions, with a glutamic acid replacing aspartic acid in the -X chelating position. The glutamate-containing motif gave an unexpected 6-fold decrease in cation affinity for the three-acid residue loop motif (KCa = 524 microM vs KCa = 3140 microM) and a 46-fold decrease for the four-acid residue loop motif (KCa = 42.1 microM vs KCa 1950 microM). To improve calcium binding of the glutamate-containing motifs, peptides were synthesized keeping glutamate in the -X position and inserting serine in the +Z position to provide a hydrogen-bonded system stabilizing the glutamate interaction with the cation. The serine residue further reduced calcium affinity in both the three-acid residue loop (KCa = 19.6 mM) and the four-acid residue loop (KCa = 2806 microM). These results indicate that glutamate and serine residues in the -X and +Z positions, respectively, can be detrimental to calcium binding. However, in natural calcium binding proteins, glutamate in the -X chelating position can confer high affinity for calcium in helix-loop-helix calcium binding motifs, but this may be dependent on the environment created by as yet undetermined factors.
Procyshyn
R M
RM
Faculty of Pharmaceutical Sciences, Division of Pharmaceutical Chemistry, University of British Columbia, Vancouver, Canada.
Reid
R E
RE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002614:Chelating Agents; D002942:Circular Dichroism; D005971:Glutamates; D018698:Glutamic Acid; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002614:Chelating Agents; D005971:Glutamates; D010455:Peptides; D018698:Glutamic Acid; D002118:Calcium