The ubiquitous Ca2+-regulatory protein calmodulin activates target enzymes
as a response to submicromolar Ca2+ increases in a background of millimolar
Mg2+. Th, potential influence of Mg2+/ Ca2+ competition is especially intr
iguing for the N-terminal domain of the protein which possesses the sites w
ith the lowest Ca2+ specificity. The interdependence of Ca2+ and Mg2+ bindi
ng in the N-terminal domain of calmodulin was therefore studied using Ca-43
NMR, H-1-N-15 NMR, and fluorescent Ca2+ chelator techniques. The apparent
affinity for Ca2+ was found to be significantly decreased at physiological
Mg2+ levels. At Ca2+ concentrations of an activated cell the (Ca2+)(2) stat
e of the N-terminal domain is therefore only weakly populated, indicating t
hat for this domain Ca2+ binding is intimately associated with binding of t
arget molecules. The data are in good agreement with a two-site model in wh
ich each site can bind either Ca2+ or Mg2+. The Mg2+-Ca2+ binding interacti
on is slightly positively allosteric, resulting in a significantly populate
d (Mg2+)(1)(Ca2+)(1) state. The Ca2+ off-rate from this state is determined
to be at least one order of magnitude faster than from the (Ca2+)(2) state
. These two findings indicate that the (Mg2+)(1)(Ca2+)(1) state is structur
ally and/or dynamically different from the (Ca2+)(2) state. The Ca-43 quadr
upolar coupling constant and the H-1 and N-15 chemical shifts of the (Mg2+)
,(Ca2+), State were calculated from titration data. The values of both para
meters suggest that the (Mg2+)(1)(Ca2+)(1) State has a conformation more si
milar to the "closed" apo and (Mg2+)(2) states than to the "open" (Ca2+)(2)
state.