Battle for the EF-hands: magnesium-calcium interference in calmodulin.
The ubiquitous Ca(2+)-regulatory protein calmodulin activates target enzymes as a response to submicromolar Ca(2+) increases in a background of millimolar Mg(2+). The potential influence of Mg(2+)/Ca(2+) competition is especially intriguing for the N-terminal domain of the protein which possesses the sites with the lowest Ca(2+) specificity. The interdependence of Ca(2+) and Mg(2+) binding in the N-terminal domain of calmodulin was therefore studied using (43)Ca NMR, (1)H-(15)N NMR, and fluorescent Ca(2+) chelator techniques. The apparent affinity for Ca(2+) was found to be significantly decreased at physiological Mg(2+) levels. At Ca(2+) concentrations of an activated cell the (Ca(2+))(2) state of the N-terminal domain is therefore only weakly populated, indicating that for this domain Ca(2+) binding is intimately associated with binding of target molecules. The data are in good agreement with a two-site model in which each site can bind either Ca(2+) or Mg(2+). The Mg(2+)-Ca(2+) binding interaction is slightly positively allosteric, resulting in a significantly populated (Mg(2+))(1)(Ca(2+))(1) state. The Ca(2+) off-rate from this state is determined to be at least one order of magnitude faster than from the (Ca(2+))(2) state. These two findings indicate that the (Mg(2+))(1)(Ca(2+))(1) state is structurally and/or dynamically different from the (Ca(2+))(2) state. The (43)Ca quadrupolar coupling constant and the (1)H and (15)N chemical shifts of the (Mg(2+))(1)(Ca(2+))(1) state were calculated from titration data. The values of both parameters suggest that the (Mg(2+))(1)(Ca(2+))(1) state has a conformation more similar to the "closed" apo and (Mg(2+))(2) states than to the "open" (Ca(2+))(2) state.
Malmendal
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A
Physical Chemistry 2, Lund University, P.O. Box 124, S-221 00 Lund, Sweden. anders@scripps.edu
Linse
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S
Evenäs
J
J
Forsén
S
S
Drakenberg
T
T
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
D002147:Calmodulin; D002614:Chelating Agents; D011994:Recombinant Proteins; D004533:Egtazic Acid; D008274:Magnesium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium