The Myosin Relay Helix to Converter Interface Remains Intact throughout the Actomyosin ATPase Cycle*

Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850–15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559–571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a “cysteine-light” myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683–694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.

[1]  M. Yoshida,et al.  GroEL Locked in a Closed Conformation by an Interdomain Cross-link Can Bind ATP and Polypeptide but Cannot Process Further Reaction Steps* , 1996, The Journal of Biological Chemistry.

[2]  D A Winkelmann,et al.  Three-dimensional structure of myosin subfragment-1: a molecular motor. , 1993, Science.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  J. Eccleston,et al.  Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. , 1991, Biochemistry.

[5]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[6]  J. Spudich,et al.  Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. , 1987, Science.

[7]  A. Muhlrad,et al.  Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes. , 1992, Biochemistry.

[8]  E. Taylor,et al.  Transient state phosphate production in the hydrolysis of nucleoside triphosphates by myosin. , 1970, Biochemistry.

[9]  D. Koshland,et al.  Global flexibility in a sensory receptor: a site-directed cross-linking approach. , 1987, Science.

[10]  J. Spudich,et al.  Phenotypically selected mutations in myosin's actin binding domain demonstrate intermolecular contacts important for motor function. , 1997, Biochemistry.

[11]  Roberto Dominguez,et al.  Crystal Structure of a Vertebrate Smooth Muscle Myosin Motor Domain and Its Complex with the Essential Light Chain Visualization of the Pre–Power Stroke State , 1998, Cell.

[12]  J. Falke,et al.  Thermal motions of surface alpha-helices in the D-galactose chemosensory receptor. Detection by disulfide trapping. , 1992, Journal of molecular biology.

[13]  M. Geeves,et al.  The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin. , 1985, The Biochemical journal.

[14]  E. Taylor,et al.  Mechanism of adenosine triphosphate hydrolysis by actomyosin. , 1971, Biochemistry.

[15]  H M Holden,et al.  X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. , 1995, Biochemistry.

[16]  Ivan Rayment,et al.  X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. , 1996 .

[17]  A. Houdusse,et al.  Atomic Structure of Scallop Myosin Subfragment S1 Complexed with MgADP A Novel Conformation of the Myosin Head , 1999, Cell.

[18]  J. Spudich,et al.  Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates. , 1998, Biochemistry.

[19]  Y. Goldman,et al.  Wag the Tail: Structural Dynamics of Actomyosin , 1998, Cell.

[20]  D A Knecht,et al.  Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. , 1987, Science.

[21]  Ivan Rayment,et al.  The Structural Basis of the Myosin ATPase Activity* , 1996, The Journal of Biological Chemistry.

[22]  Zygmunt Gryczynski,et al.  A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin , 2000, Cell.

[23]  J. Spudich,et al.  Molecular genetic tools for study of the cytoskeleton in Dictyostelium. , 1991, Methods in enzymology.