pubs.acs.org/Biochemistry Published on Web 05/01/2009 r 2009 American Chemical Society Biochemistry 2009, 48, 5263–5275 5263 DOI: 10.1021/bi900584q The Myosin C-Loop Is an Allosteric Actin Contact Sensor in Actomyosin † Katalin Ajtai, ‡ Miriam F. Halstead, ‡ Mikl  os Nyitrai, ^ Alan R. Penheiter, § Ye Zheng, ‡ and Thomas P. Burghardt* ,‡, ) ‡ Biochemistry and Molecular Biology and § Molecular Medicine Program and ) Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and ^ Department of Biophysics, University of P ecs, P ecs, Hungary Received April 6, 2009; Revised Manuscript Received April 30, 2009 ABSTRACT: Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V max . In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop. Myosin is an actin-dependent molecular motor driving sarco- meric shortening and muscle contraction by transducing ATP hydrolysis free energy into directed protein movement (1). ATP hydrolysis is coupled to a series of conformational changes in myosin, which result in a cycle of attachment to the actin filament, strain development, protein translation, actin release, and reattachment (2). Myosin conformation change associated with strain development and protein translation, termed the power stroke, is shown in crystal structures by a lever arm rotation through ∼70° (3, 4). Myosin heavy chain (MHC) 1 is a linear molecule consisting of an N-terminal globular head domain called subfragment 1 (S1) and a C-terminal tail responsible for cargo binding or dimeriza- tion. Elliptically shaped S1 (∼130 A ˚  70 A ˚ ) contains subdo- mains referring to proteolytic fragments (25, 50, and 20 kDa) (5) or functional domains (6). Functional domains retain their structure, but move relatively, during transduction. Transduction starts in the active site with ATP hydrolysis. A long R-helical switch 2 helix transmits linear force originating from the active site to a converter domain converting linear force into torque to rotate the lever arm. The lever arm is another long R-helix stabilized by bound myosin light chains. The lever arm amplifies displacement and impels myosin relative to actin (7, 8). Multiple peptides within the 50K portion of S1 constitute the actin binding site. Protein-protein contacts in actomyosin were identified by experimental structural studies combined with simulated docking of the myosin and actin crystal structures (9- 13), via detection of changes in actin binding strength, actin- activated myosin ATPase, and in vitro motility caused by the mutation of small peptide segments or individual residues in myosin (14-16). Primary hydrophobic actin contacts are helical segments, amino acids 529-558 2 and 647-659, on S1, while the † This work was supported by NIH-NIAMS Grant R01AR049277, Mayo Clinic, and by Hungarian Science Foundation OTKA Grant K60968 to M.Ny. *To whom correspondence should be addressed. Telephone: (507) 284-8120. Fax: (507) 284-9349. E-mail: burghardt@mayo.edu. 1 Abbreviations: βS1, β-cardiac myosin subfragment 1; C-loop, loop 4 of amino acids 362-376; cCl, cardiac C-loop smooth muscle myosin chimera; Dc, Dictyostelium discoideum; ΔG, ΔH, and ΔS, change in free energy, enthalpy, and entropy, respectively; DOF, degree of freedom; ELC, essential light chain; HCTm, human cardiac tropomyosin; HMM, heavy meromyosin; mATP and mADP, fluorescent nucleotides mant- ATP and mant-ADP, respectively; MHC, myosin heavy chain; MLCK, myosin light chain kinase; MCMD, Monte Carlo molecular dynamics; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethanesul- fonyl fluoride; Rh, rhodamine; RLC, regulatory light chain; S1, myosin subfragment 1; sCl, skeletal C-loop smooth muscle myosin chimera; SKTm, rabbit skeletal tropomyosin; Tm, tropomyosin; U50a or U50b, myosin skeletal sequence of amino acids 145-361 or 362-462, respec- tively; W510, nucleotide sensitive tryptophan; WT, wild-type isoform. 2 All myosin residue numbering uses the skeletal sequence unless noted otherwise.