Effects of KCL on Calmodulin Mutants Defective in Ion Channel Regulation

Abstract

Small ankyrin-1 (sAnk1, Ank1.5) is a splice variant of the ANK1 gene that binds to the large modular protein, obscurin A, with nanomolar affinity, a reaction that may help to organize the sarcoplasmic reticulum in striated muscle. A subset of lysine and arginine residues in the 2 ankyrin repeats of sAnk1 interact specifically with 4 glutamate residues in a stretch of 30 amino acids of obscurin to mediate binding. Homology modeling and molecular dynamics simulations have revealed a ''hot spot'' of 4 hydrophobic residues exposed on the surface of the ankyrin repeat domain of sAnk1. We used site-directed mutagenesis of bacterially expressed fusion proteins, followed by blot overlays and surface plasmon resonance assays, to study the contribution of these 4 residues, V70, F71, I102 and I103, to binding to the 30-mer of obscurin. Alanine mutations of each of these four residues inhibited binding to residues 6316-6345 of obscurin (Obsc 6316-6345 ). In contrast, V70A and I102A mutations had no effect on binding to a second sAnk1 binding site on obscurin, located within residues 6231-6260 (Obsc 6231-6260 ). Using the same methods, we mutated the 5 hydrophobic residues present in Obsc 6316-6345 to alanine and identified V6328, I6332, and V6334 as critical for proper binding. Our results suggest that hydrophobic interactions as well as electrostatic interactions are important for the binding of sAnk1 to Obsc 6316-6345 , consistent with studies of the complexes formed by other ankyrin repeat proteins with their ligands. Hydrophobic interactions are likely to contribute to the difference in affinity of sAnk1 for Obsc 6316-6345 and Obsc 6231-6260 , and for the dominant role played by the more C-terminal sequence in binding. Supported by grant R01-AR056330 from the NIH to RJB and training grants T32 GM08181 (to RJB) and T32 AR07592 (to M. Schneider).