Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106.

Calcium-dependent conformational states of calmodulin (CaM) were probed by thrombin to determine quantitative differences in the susceptibility of two bonds: Arg37-Ser38 (R37-S38, near site I in the N-terminal domain) and Arg106-His107 (R106-H107, near site III in the C-terminal domain). Quantitative thrombin footprinting of a discontinuous equilibrium calcium titration of wild-type calmodulin showed that the R37-S38 bond of the apoprotein was cleaved at a barely detectable level while the R106-H107 bond was maximally susceptible. Calcium binding to sites III and IV monotonically protected R106-H107 from proteolysis; concomitantly, the susceptibility of R37-S38 increased. However, calcium binding to sites I and II protected R37-S38 from cleavage, yielding a peaked biphasic profile composed of equal and opposite transitions. Both bonds were fully protected when calmodulin was saturated with calcium. Susceptibility profiles resolved from the fractional abundance of primary cleavage products (peptides 1-37, 38-148, 1-106, 107-148) were interpreted as directly reflecting calcium-induced conformational changes in whole calmodulin; free energies of calcium binding and cooperativity were estimated. Secondary cleavage was never observed; both R37 and R106 were sites of thrombinolysis in whole calmodulin only. In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monotonically. Thus, the biphasic character of cleavage of R37 in helix B was not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patterns of susceptibility indicated that partially saturated wild-type calmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.