CALCIUM-INDUCED INTERACTIONS OF CALMODULIN DOMAINS REVEALED BY QUANTITATIVE THROMBIN FOOTPRINTING OF ARG37 AND ARG106

Calcium-dependent conformational states of calmodulin (CaM) were probe d by thrombin to determine quantitative differences in the susceptibil ity 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 equilib rium 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 site s III and IV monotonically protected ii ty of R37-S38 increased, Howev er, calcium R106-H107 from proteolysis; concomitantly, the susceptibil ity of R37-S38 increased. However, calcium binding to sites I and II p rotected R37-S38 from cleavage, yielding a peaked biphasic profile com posed of equal and opposite transitions. Both bonds were fully protect ed 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 re flecting calcium-induced conformational changes in whole calmodulin; f ree energies of calcium binding and cooperativity were estimated, Seco ndary cleavage was never observed; both R37 and R106 were sites of thr ombinolysis in whole calmodulin only, In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monoto nically, Thus, the biphasic character of cleavage of R37 in helix B wa s not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patt erns of susceptibility indicated that partially saturated wild-type ca lmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.