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 Ca(2+)-free reference complex is 2.4 +/- 0.3 microM. The Ala and Arg replacements increase this to 5.4 +/- 0.4 and 6.2 +/- 0.5 microM, while the Met increases it to 26.4 +/- 2.5 microM. When Ca(2+) 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 microM, and the values for the Arg and Met variants decrease to 0.4 +/- 0.03 microM. Using mutant calmodulins, we defined the effect of Ca(2+) 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) approximately 5-fold, while it decreases the values for the variants approximately 2- to approximately 10-fold. The second step decreases the K(d) values for the all of the complexes approximately 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 approximately 15- to approximately 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 approximately 10- and approximately 15-fold and the Ala and Met variants approximately 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.
Black
D J
DJ
Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Persechini
Anthony
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D005998:Glycine / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D002147:Calmodulin; D002118:Calcium; D005998:Glycine