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Variations at the semiconserved glycine in the IQ domain consensus sequence have a major impact on Ca-dependent switching in calmodulin-IQ domain complexes

Biochemistry, ISSN: 0006-2960, Vol: 49, Issue: 1, Page: 78-83
2010
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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 for the Ca-free reference complex is 2.4 ± 0.3 μM. The Ala and Arg replacements increase this to 5.4 ± 0.4 and 6.2 ± 0.5 μM, while the Met increases it to 26.4 ± 2.5 μM. When Ca is bound to both calmodulin lobes, the K for the reference complex is not significantly affected, but the K for the Ala variant decreases to 0.9 ± 0.04 μM, and the values for the Arg and Met variants decrease to 0.4 ± 0.03 μM. Using mutant calmodulins, we defined the effect of Ca 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 ∼5-fold, while it decreases the values for the variants ∼2- to ∼10-fold. The second step decreases the K values for the all of the complexes ∼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 values for the reference complex and Met and Ala variants ∼15- to ∼200-fold but does not affect the value for the Arg variant. The second step decreases the K values for the reference and Arg variant ∼10- and ∼15-fold and the Ala and Met variants ∼2000-fold. Thus, both steps in the N→C order are 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. © 2009 American Chemical Society.

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