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.