CA2-CHANGES IN A CALMODULIN DOMAIN( BINDING AND CONFORMATIONAL)

Calcium activation of the C-terminal domain of calmodulin was studied using H-1 and N-15 NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emph asized by the striking effects resulting from a mutation of this gluta mic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop TV. The study involves determination of Ca2+ binding constants, assignment s, and structural characterizations of the ape, (Ca2+)(1), and (Ca2+)( 2) states of the E104Q mutant and comparisons to the wild-type protien and the E140Q mutant [Evenas et al. (1997) Biochemistry 36, 3448-3457 ]. NMR titration data show sequential Ca2+ binding in the E104Q mutant . The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both muta nts, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-ter minal EF-hands. Spectral characteristics show that the (Ca2+)(1) and ( Ca2+)(2) states of the E104Q mutant undergo global exchange on a 10-10 0 mu s time scale between conformations seemingly similar to the close d and open structures of this domain in wild-type calmodulin, parallel ing earlier observations for the (Ca2+)(2) state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are requir ed for stabilization of the open conformation in the (Ca2+)(2) state. To verify that the NOE constraints cannot be fulfilled in a single str ucture, solution structures of the (Ca2+)(2) state of the E104Q mutant are calculated. Within the ensemble of structures the precision is go od. However, the clearly dynamic nature of the state, a large number o f violated distance restraints, ill-defined secondary structural eleme nts, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)(2) state of th e E104Q mutant but rather represents the distance-averaged structure o f at least two distinct different conformations.