Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association.
A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca(2+) affinity and the number of paired, negatively charged Ca(2+) chelating residues in the N-terminal Ca(2+)-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and -x (X-axis), +y and -y (Y-axis), and +z and -z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca(2+) affinities. The maximal ranges of the increases observed in the N-terminal Ca(2+) affinity were approximately 8-8.5-fold for site I, approximately 4.5-5-fold for site II, and approximately 11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca(2+) affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca(2+)-binding sites reduced the N-terminal Ca(2+) affinity. The increases in Ca(2+) affinity observed were caused by an increase in the Ca(2+) association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca(2+) dissociation rates.
Black
D J
DJ
Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210, USA. black.215@osu.edu
Tikunova
S B
SB
Johnson
J D
JD
Davis
J P
JP
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D010446:Peptide Fragments; D010649:Phenylalanine; D014364:Tryptophan; D002118:Calcium