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J. Biol. Chem., Vol. 280, Issue 29, 26974-26983, July 22, 2005

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Biological, Biochemical, and Kinetic Effects of Mutations of the Cardiomyopathy Loop of Dictyostelium Myosin II

IMPORTANCE OF ALA^400*

Xiong Liu, Shi Shu, Mihály Kovács¶, and Edward D. Korn||

From the Laboratory of Cell Biology and ^¶Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

The cardiomyopathy (CM)-loop of the heavy chain of class-II myosins begins with a highly conserved Arg residue (whose mutation in human -cardiac myosin II results in familial hypertrophic cardiomyopathy). The CM-loop of Dictyostelium myosin II (Arg³⁹⁷–Gln⁴⁰⁷) is essential for its biological functions and biochemical activities. We found that the CM-loop of smooth muscle myosin II substituted partially, and the CM-loop of -cardiac myosin II less well, for growth, capping of surface receptors and development, and the actin-activated MgATPase and in vitro motility activities of purified myosins. There was little correlation between the biochemical and biological activities of the two chimeras and 19 point mutants, but only the five mutants with k_cat/K_actin values equivalent to wild-type myosin supported essentially full biological function. The three point mutations of Arg³⁹⁷ equivalent to those that result in hypertrophic cardiomyopathy in humans had minimal biological effects and different biochemical effects. The A400V mutation rendered full-length wild-type myosin almost completely inactive, both in vitro and in vivo, and the reverse V400A mutation in the cardiac CM-loop chimera restored almost full activity, even though the sequence still differed from wild-type in 7 of 11 positions. Transient kinetic studies of acto-subfragment-1 (S1) showed that the chimeras and the Ala/Val, Val/Ala mutations do not affect the equilibrium or the association and dissociation rate constants for either ATP or ADP binding to acto-S1 or the rate of ATP-induced dissociation of acto-S1. We conclude that the Ala/Val, Val/Ala mutations affect the release of P_i from acto-S1·ADP·P_i. In addition, Val at position 400 substantially reduces the affinity of actin for S1 in the absence of nucleotide.

Received for publication, April 25, 2005

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^|| To whom correspondence should be addressed: Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bldg. 50, Rm. 2517–8017, Bethesda, MD 20892-8017. Tel.: 301-496-1616; Fax: 301-402-1519; E-mail: edk{at}nih.gov.

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