The Journal of Cell Biology, Vol 134, 895-909, Copyright © 1996 by The Rockefeller University Press

ARTICLES

Glycine 699 is pivotal for the motor activity of skeletal muscle myosin

F Kinose, SX Wang, US Kidambi, CL Moncman and DA Winkelmann
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

• Lowey, S., Lesko, L. M., Rovner, A. S., Hodges, A. R., White, S. L., Low, R. B., Rincon, M., Gulick, J., Robbins, J. (2008). Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an {alpha}- or {beta}-Myosin Heavy Chain Backbone. J. Biol. Chem. 283: 20579-20589 [Abstract] [Full Text]  
• Miller, B. M., Trybus, K. M. (2008). Functional Effects of Nemaline Myopathy Mutations on Human Skeletal {alpha}-Actin. J. Biol. Chem. 283: 19379-19388 [Abstract] [Full Text]  
• Matsson, H., Eason, J., Bookwalter, C. S., Klar, J., Gustavsson, P., Sunnegardh, J., Enell, H., Jonzon, A., Vikkula, M., Gutierrez, I., Granados-Riveron, J., Pope, M., Bu'Lock, F., Cox, J., Robinson, T. E, Song, F., Brook, D. J, Marston, S., Trybus, K. M., Dahl, N. (2008). Alpha-cardiac actin mutations produce atrial septal defects. Hum Mol Genet 17: 256-265 [Abstract] [Full Text]  
• Ajtai, K., Garamszegi, S. P., Watanabe, S., Ikebe, M., Burghardt, T. P. (2004). The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction. J. Biol. Chem. 279: 23415-23421 [Abstract] [Full Text]  
• Srikakulam, R., Winkelmann, D. A. (2004). Chaperone-mediated folding and assembly of myosin in striated muscle. J. Cell Sci. 117: 641-652 [Abstract] [Full Text]  
• Wang, Q., Moncman, C. L., Winkelmann, D. A. (2003). Mutations in the motor domain modulate myosin activity and myofibril organization. J. Cell Sci. 116: 4227-4238 [Abstract] [Full Text]  
• Himmel, D. M., Gourinath, S., Reshetnikova, L., Shen, Y., Szent-Gyorgyi, A. G., Cohen, C. (2002). Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor. Proc. Natl. Acad. Sci. USA 99: 12645-12650 [Abstract] [Full Text]  
• Chow, D., Srikakulam, R., Chen, Y., Winkelmann, D. A. (2002). Folding of the Striated Muscle Myosin Motor Domain. J. Biol. Chem. 277: 36799-36807 [Abstract] [Full Text]  
• Hettmann, C., Herm, A., Geiter, A., Frank, B., Schwarz, E., Soldati, T., Soldati, D. (2000). A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization. Mol. Biol. Cell 11: 1385-1400 [Abstract] [Full Text]  
• Gillespie, P. G., Gillespie, S. K. H., Mercer, J. A., Shah, K., Shokat, K. M. (1999). Engineering of the Myosin-Ibeta Nucleotide-binding Pocket to Create Selective Sensitivity to N6-modified ADP Analogs. J. Biol. Chem. 274: 31373-31381 [Abstract] [Full Text]  
• Hiratsuka, T. (1999). ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence. J. Biol. Chem. 274: 29156-29163 [Abstract] [Full Text]  
• Srikakulam, R., Winkelmann, D. A. (1999). Myosin II Folding Is Mediated by a Molecular Chaperonin. J. Biol. Chem. 274: 27265-27273 [Abstract] [Full Text]  
• Xiao, M., Li, H., Snyder, G. E., Cooke, R., Yount, R. G., Selvin, P. R. (1998). Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin. Proc. Natl. Acad. Sci. USA 95: 15309-15314 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents