Variations at the Semiconserved Glycine in the IQ Domain Consensus Sequence Have a Major Impact on Ca2+-Dependent Switching in Calmodulin-IQ Domain Complexes

We have replaced the semiconserved Gly in the IQ domain consensus sequence with Ala, Arg, or Met in a reference sequence and determined how this affects its complexes with calmodulin. The K-d for the Ca2+-free reference complex is 2.4 +/- 0.3 mu M. The Ala and Arg replacements increase tills to 5.4 +/- 0.4 and 6.2 +/- 0.5 mu M, while the Met increases it to 26.4 +/- 2.5 mu M. When Ca2+ is bound to both calmodulin lobes, the K-d for the reference complex is not significantly affected, but the K-d for the Ala variant decreases to 0.9 +/- 0.04 mu M, and the values for the Arg and Met variants decrease to 0.4 +/- 0.03 mu M. Using mutant calmodulin, we defined the effect of Ca2+ binding to each lobe, with the C-terminal preceding the N-terminal (C -> N) or vice versa (N -> C). In the C -> N order the first step increases the reference K-d similar to 5-fold, while it decreases the values for the variants similar to 2- to similar to 10-fold. The second step decreases the K-d values for the all of the complexes similar to 5-fold, suggesting that the N-terminal lobe does not interact with the semiconserved position after the first step. In the N -> C order the first step increases the K-d values for the reference complex and Met and Ala variants similar to 15- to similar to 200-fold but does not affect the value for the Arg variant. The second step decreases the K-d values for the reference and Arg variant similar to 10- and similar to 15-fold and the Ala and Met variants similar to 2000-fold. Thus, both steps in the N -> C order arc sensitive to variations at the semiconserved position, while only the first is in the C -> N order. Due to energy coupling, this order is followed under equilibrium conditions.