Modifying Mg2+ binding and exchange with the N-terminal of calmodulin.
To follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substituted Phe in position 19, which immediately precedes the first Ca2+/Mg2+ binding loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues in chelating positions, two of which form a native Z-acid pair. We then generated seven additional N-terminal CaM mutants to examine the role of chelating acidic residues in Mg2+ binding and exchange with the first EF-hand of CaM. A CaM mutant with acidic residues in all of the chelating positions exhibited Mg2+ affinity similar to that of W-Z. Only CaM mutants that had a Z-acid pair were able to bind Mg2+ with physiologically relevant affinities. Removal of the Z-acid pair from the first EF-hand produced a dramatic 58-fold decrease in its Mg2+ affinity. Additionally, removal of the Z-acid pair led to a 1.8-fold increase in the rate of Mg2+ dissociation. Addition of an X- or Y-acid pair could not restore the high Mg2+ binding lost with removal of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of CaM supports high Mg2+ binding primarily by increasing the rate of Mg2+ association.
Tikunova
S B
SB
Department of Molecular and Cellular Biochemistry, and Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210, USA. tikunova.1@osu.edu
Black
D J
DJ
Johnson
J D
JD
Davis
J P
JP
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002147:Calmodulin / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010649:Phenylalanine; D014364:Tryptophan; D008274:Magnesium