Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin.
The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.
Ohki
S
S
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon.
Ikura
M
M
Zhang
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D010727:Phosphoric Diester Hydrolases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D008274:Magnesium; D002118:Calcium