Myosin Flexibility: Structural Domains and Collective Vibrations Isabelle Navizet, 1,2 Richard Lavery, 2 and Robert L. Jernigan 1 * 1 Molecular Structure Section, Laboratory of Experimental and Computational Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5677 2 Laboratoire de Biochimie The ´orique, CNRS UPR 9080, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France ABSTRACT The movement of the myosin mo- tor along an actin filament involves a directed con- formational change within the cross-bridge formed between the protein and the filament. Despite the structural data that has been obtained on this sys- tem, little is known of the mechanics of this confor- mational change. We have used existing crystallo- graphic structures of three conformations of the myosin head, containing the motor domain and the lever arm, for structural comparisons and mechani- cal studies with a coarse-grained elastic network model. The results enable us to define structurally conserved domains within the protein and to better understand myosin flexibility. Notably they point to the role of the light chains in rigidifying the lever arm and to changes in flexibility as a consequence of nucleotide binding. Proteins 2004;54:384 –393. © 2003 Wiley-Liss, Inc. Key words: motor proteins; Gaussian network model; structural blocks; B-factors INTRODUCTION Myosin is an enzyme that converts the chemical energy resulting from the hydrolysis of ATP into directed mechani- cal movement along an actin filament. The actomyosin system is involved in numerous cell processes including vesicle trafficking, determinant partitioning, cell motility, neurosensory function, and muscle contraction. 1 Although considerable crystallographic data have been gathered on this system, 2–6 many questions concerning the molecular mechanisms underlying myosin mobility remain unan- swered. Myosin II, so-called conventional myosin, forms fila- ments and constitutes large assemblies of noncooperative motors within muscular tissues. It is an important mem- ber of a diverse family of myosin motor proteins. 7 Various mechanisms have been proposed for myosin movement. The majority of biophysicists explain muscle contraction by the movement of the myosin lever arm, 8 but other evidence has pointed to a biased Brownian ratchet mecha- nism and to the possibility of multiple myosin steps per ATP-driven cycle. 9 It may however be possible to reconcile these apparently conflicting viewpoints. 10 A part of the mechanism proposed by Houdusse et al. 10 based on insights from X-ray structures, cryo-electron microscopy, and kinetic studies is presented in Figure 1. The strong binding of myosin to actin (rigor state) weakens with ATP binding. This conformation is termed the near rigor state. The detached state, where myosin releases the actin filament may prevent a reverse power stroke and increase the lifetime of the prehydrolysis state. After hydrolysis of ATP in the myosin motor, phosphate binding stabilizes the so-called transition state until actin binds. This is followed by force generation and ADP release returning the system to its rigor state. In the present article, we use theoretical methods to study myosin II, in an attempt to better understand the mechanics of its conformational changes. Because the myosin head is a large system (1147 amino acids, 130 kDa) and, moreover, undergoes large conformational changes, it is not easy to use conventional all-atom molecular mechan- ics or dynamics methods. We have thus chosen to study the problem with an anisotropic network model 11,12 and also via a rigid block decomposition method. Both of these methods are coarse-grained and only use a single point, C, to represent each amino acid residue. The anisotropic network model provides data on the large-scale collective modes of vibration by converting the protein structure into a set of coupled springs between neighboring residues and carrying out a normal mode style analysis. It has been shown to provide data in excellent agreement with more refined all-atom approaches and with crystallographic temperature factors. 12–17 The rigid block decomposition method is based on a comparison of inter-C distances between two structures of the same protein and the identification of blocks based on virtually constant inter- residue distances. Together, these methods enable us to identify the rigid and flexible domains within the myosin structure and highlight the respective roles of the light chains and of nucleotide binding. Grant sponsor: Foundation for Advanced Education in the Sciences to I.N. Grant sponsor: National Institutes of Health to I.N. R.L. Jernigan’s present address is Baker Center for Bioinformatics and Biological Statistics, Iowa State University, 123 Office and Lab Building, Ames, IA 50011-3020. *Correspondence to: Robert L. Jernigan, E-mail: jernigan@iastate.edu Received 20 December 2002; Revised 3 March 2003; Accepted 25 March 2003 PROTEINS: Structure, Function, and Bioinformatics 54:384 –393 (2004) © 2003 WILEY-LISS, INC.