Calcium-Induced Refolding of the Calmodulin V136G Mutant Studied by NMR Spectroscopy:  Evidence for Interaction between the Two Globular Domains

The Ca2+ titration of the 15N-labeled mutant V136G calmodulin has been monitored using 1H−15N HSQC NMR spectra. Up to a [Ca2+]/[CaM] ratio of 2, the Ca2+ ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca2+. Surprisingly, the Ca2+-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca2+]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca2+ binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca2+-bound N-domain appear as two signals during the Ca2+ titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca2+ affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca2+-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.