Is the binding of magnesium(II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance

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Abstract
Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 104 M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., and Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM .cntdot. Ca22+ (in which sites III and IV are occupied by Ca2+), CaM .cntdot. La23+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM .cntdot. Ca22+ and TR1C, moderately large Ka (2000 and 35000 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25.degree.C) were observed. For both CaM .cntdot. La23+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (Koff = 3500 and 2200 s-1, respectively). In consistency with these, the data from binding studies of Mg2+ to apo-CaM were fitted well with two strong sites (Ka = 2000 M-1, koff = 2700 s-1 at 25.degree.C) and two weak sites (Ka = 300 M-1, koff = 6600 s-1 at 25.degree.C). The results indicate that Mg2+ shows opposite site preference relative to Ca2+ and binds to sites I and II of CaM with a binding constant of ca. 2000 M-1, as compared to ca. 5 .times. 105 M-1 for Ca2+. Since the intracellular concentration of Mg2+ is higher than that of Ca2+ by a factor of ca. 104-105 at the resting state and ca. 102-103 during stimulation of cells, it is possible that sites I and II are constantly occupied by Mg2+ at the resting state and by Mg2+ or Ca2+ during stimulation. We hypothesize that sites I and II of CaM indeed be "Mg2+-Ca2+ sites" and play a structural role in some systems, in analogy to sites III and IV of skeletal muscle troponin C [Johnson, J.D., Charlton, S. C., and Potter, J. D. (1979) J. Biol. Chem. 254, 3497-3502].

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