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Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

¹ Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA
² Division of Physical Biochemistry, National Institute for Medical Research, London NW7 1AA, United Kingdom
³ Department of Biochemistry, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1109, USA
⁴ Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA

(RECEIVED June 11, 2004; FINAL REVISION August 16, 2004; ACCEPTED August 21, 2004)

A mutation (Cam⁷) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.

Keywords: calmodulin mutant phenotype; calcium signaling in Drosophila; ryanodine receptor; calcium channels

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04928204.

Reprint requests to: Kathleen M. Beckingham, Department of Biochemistry and Cell Biology, Rice University, MS-140, P.O. Box 1892, Houston, TX 77251, USA; e-mail: kate{at}bioc.rice.edu; fax: (713) 348-5154.

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