Journal of Molecular Biology
Volume 384, Issue 4, 26 December 2008, Pages 780-797
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Three-Dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

https://doi.org/10.1016/j.jmb.2008.10.013Get rights and content

Abstract

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen–hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305–Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.

Introduction

Striated muscle is formed by two sets of overlapping filaments, the thick myosin-containing filaments and the thin actin-containing filaments. During contraction, the two sets actively slide past each other, shortening the sarcomere. The myosin heads are helically ordered on the backbone of relaxed thick filaments. When filaments are activated, the heads detach from the backbone and become disordered. Sliding force is produced when these heads cyclically attach to and pull on the thin filaments.1

Contraction of most muscles is regulated via Ca2+-controlled molecular switches located on either or both sets of filaments.2 While the mechanism of actin-linked regulation by troponin and tropomyosin is well understood,3, 4 our knowledge on the mechanism of myosin-linked regulation is less complete.5, 6 It can occur by direct Ca2+ binding to the essential light chains (ELCs; see below)5 (scallop striated muscle) or by regulatory light chain (RLC) phosphorylation (invertebrate striated muscle7, 8 and vertebrate smooth muscle9). While RLC phosphorylation occurs in many muscles, its functional importance varies. In the case of vertebrate striated muscle, for example, phosphorylation appears to modulate contraction, but it is not essential for activity.6

Myosin contains two heavy chains and two pairs of light chains (the ELC and the RLC).10, 11 One RLC and one ELC are noncovalently bound to the heavy-chain α-helix in the lever arm of each myosin head,10 stabilizing it and forming the regulatory domain.11, 12 The RLC has two domains9 connected by a “linker helix” and an N-terminal fragment (NTF) or extension; all are important for regulation. The NTF varies in length, depending on species, and includes phosphorylation sites. While the NTF is absent from S1 crystal structures,10 its structure has emerged from EPR of smooth muscle myosin;13 revealing that it acts as a distinct phosphorylation domain, changing from solvent inaccessible and weakly helical when unphosphorylated to solvent accessible with helical order and increased rotational mobility when Ser19 is phosphorylated. Electron microscopy (EM) of chicken smooth muscle myosin 14, 15, 16, 17 revealed that, in the switched-off state (dephosphorylated), the two heads establish an asymmetric “interacting-head” structure in which actin binding activity of one head (“blocked”) is sterically blocked by binding of its actin binding interface to the converter domain of the other head. Actin binding activity of this head is not blocked (it is therefore called the “free” head), while its ATPase activity is inhibited, by prevention of converter movements needed for phosphate release. In addition to interaction between the two motor domains, there is also an interaction between the blocked-head motor domain and the free-head ELC. This interacting-head structure, deduced from two-dimensional crystals14, 15, 16 and single-molecule17 studies, has also been shown to be present in native thick filaments of tarantula striated muscle.18 It is therefore not an artifact of myosin isolation. In the filament, three additional interactions are seen:18 one intramolecular interaction between the blocked head and its subfragment 2 (S2); subsequently also seen in single-molecule studies17 and two intermolecular interactions between the blocked-head SH3 domain and the S2 from its axially adjacent neighbor as well as between the blocked-head ELC and the axially neighboring free-head motor domain. The interacting-head motif has now been observed in other isolated myosin molecules (scallop,19 tarantula, Limulus, and mouse skeletal and cardiac muscles20) and in thick filaments from mouse cardiac muscle,21 Limulus and scallop striated muscle,22 and scorpion striated muscles (Sanchez et al., unpublished results), supporting the concept18, 23, 24 that this motif is highly conserved and underlies the relaxed state of thick filaments in both smooth and striated muscles over a wide range of species.

When regulated, thick filaments are activated by phosphorylation of their RLCs, the interaction between the heads is broken,14, 16 and the heads are released from the filament surface, becoming disordered.8, 25, 26 Breakage of the interaction could occur through a disorder-to-order transition of the phosphorylation domain,13 apparently by establishing an Arg16–Ser19 salt bridge,27 a sequence known to be essential for regulation by Ser19 phosphorylation.28

Our goal in this study was to elucidate the mechanism by which phosphorylation-induced changes in the RLC activate the thick filament by releasing the heads from each other and from the filament surface so that they can interact with the thin filament. To achieve this, we calculated a new three-dimensional map of tarantula filaments that better defines the RLC region, revealing two new interactions with functional significance. We propose a mechanism to explain how RLC phosphorylation weakens these inhibitory head interactions, releasing them so they can interact with the thin filament.

Section snippets

Three-dimensional reconstruction of frozen–hydrated tarantula thick filaments

We carried out a new three-dimensional reconstruction of tarantula filaments by the iterative helical real-space reconstruction (IHRSR) technique, using an initial reference viewed at 0° or tilted up to ± 12°, to account for possible filament tilt in the images (see Materials and Methods).29, 30, 31 The reconstruction was more detailed, clearly showing two new interactions in addition to those seen previously18 (see Introduction)—an intramolecular density (“a” in Fig. 1) between the motor domain

The three-dimensional reconstruction

The three-dimensional map together with flexible atomic fitting reveals a new intramolecular interaction between the motor domain of the blocked head and its S2 (“a” in Fig. 1) and a new intermolecular interaction between the blocked-head RLC and the free-head motor domain (“b” in Fig. 1), in addition to the interactions previously described16, 17, 18, 45 (Fig. 1). The map shows specific density protrusions that match well with the fitted atomic structure (Fig. 5b). The interaction between the

Sequencing

Total RNA was isolated from the flexor metatarsus longus striated muscle of the legs of the tarantula A. avicularia in the presence of Trizol® (Invitrogen) to clone the RLC gene (rlcAa). An initial cDNA was obtained using oligo d(T) and an internal degenerate primer, designed against the uniquely conserved region of published RLCs. Nucleotide sequence analysis of the 315-bp fragment amplified allowed the design of two gene-specific primers over the carboxy-terminus (GSP2:

Note added in proof

It has come to our attention, after this paper was submitted, that a paper published by Espinoza-Fonseca, L. M., Kast, D. & Thomas, D. D. (2008). J. Am. Chem. Soc. 130, 12208–12209. proposed that entropically balanced disorder-order transitions are a common theme in phosphorylation-induced conformational shifts involved in cell signaling. cf., Fig. 7.

Acknowledgements

This work was supported in part by the National Institutes of Health through grant R01GM62968, Alfred P. Sloan Foundation (BR-4297), and Human Frontier Science Program (RGP0026/2003; to W.W.); the National Institutes of Health through grant AR34711 (to R.C.); and FONACIT, Venezuela, and the Howard Hughes Medical Institute, USA (to R.P.). We thank Dr. Neal Epstein, Dr. Ulf Lunberg, Dr. Jose Reinaldo Guerrero, Reicy Brito, MSc, and Sol Patiño, MSc, for discussions; Dr. Kenneth Taylor for

References (88)

  • PomfretA.J. et al.

    Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases

    J. Struct. Biol.

    (2007)
  • CombetC. et al.

    NPS@: network protein sequence analysis

    Trends Biochem. Sci.

    (2000)
  • BystroffC. et al.

    HMMSTR: a hidden Markov model for local sequence–structure correlations in proteins

    J. Mol. Biol.

    (2000)
  • KnellerD.G. et al.

    Improvements in protein secondary structure prediction by an enhanced neural network

    J. Mol. Biol.

    (1990)
  • KempB.E. et al.

    Protein kinase recognition sequence motifs

    Trends Biochem. Sci.

    (1990)
  • WriggersW. et al.

    Modeling tricks and fitting techniques for multiresolution structures

    Structure

    (2001)
  • DominguezR. 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

    Cell

    (1998)
  • YangY. et al.

    Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor

    Structure

    (2007)
  • HoudusseA. et al.

    Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head

    Cell

    (1999)
  • CrowtherR.A. et al.

    Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle

    J. Mol. Biol.

    (1985)
  • BrownJ.H. et al.

    An unstable head–rod junction may promote folding into the compact off-state conformation of regulated myosins

    J. Mol. Biol.

    (2008)
  • ZoghbiM.E. et al.

    Helical order in tarantula thick filaments requires the “closed” conformation of the myosin head

    J. Mol. Biol.

    (2004)
  • ZhaoF.Q. et al.

    Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

    Biophys. J.

    (2008)
  • Geisterfer-LowranceA.A. et al.

    A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation

    Cell

    (1990)
  • DeckerB. et al.

    Periodically arranged interactions within the myosin filament backbone revealed by mechanical unzipping

    J. Mol. Biol.

    (2008)
  • IkebeM.

    Regulation of the function of mammalian myosin and its conformational change

    Biochem. Biophys. Res. Commun.

    (2008)
  • BaranyK. et al.

    Phosphorylation–dephosphorylation of the 18,000-dalton light chain of myosin during the contraction–relaxation cycle of frog muscle

    J. Biol. Chem.

    (1979)
  • EgelmanE.H.

    The iterative helical real space reconstruction method: surmounting the problems posed by real polymers

    J. Struct. Biol.

    (2007)
  • EgelmanE.H.

    Single-particle reconstruction from EM images of helical filaments

    Curr. Opin. Struct. Biol.

    (2007)
  • FrankJ. et al.

    SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields

    J. Struct. Biol.

    (1996)
  • PadrónR. et al.

    Three-dimensional reconstruction of thick filaments from rapidly frozen, freeze-substituted tarantula muscle

    J. Struct. Biol.

    (1995)
  • PadrónR. et al.

    Towards an atomic model of the thick filaments of muscle

    J. Mol. Biol.

    (1998)
  • OfferG. et al.

    A new model for the surface arrangement of myosin molecules in tarantula thick filaments

    J. Mol. Biol.

    (2000)
  • BauerC.B. et al.

    X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain

    J. Biol. Chem.

    (2000)
  • ChaconP. et al.

    Multi-resolution contour-based fitting of macromolecular structures

    J. Mol. Biol.

    (2002)
  • WriggersW. et al.

    Topology representing neural networks reconcile biomolecular shape, structure, and dynamics

    Neurocomputing

    (2004)
  • OpalkaN. et al.

    Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase

    Cell

    (2003)
  • HumphreyW. et al.

    VMD: visual molecular dynamics

    J. Mol. Graphics

    (1996)
  • CraigR. et al.

    Molecular structure of the sarcomere

  • LehmanW. et al.

    Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom

    J. Gen. Physiol.

    (1975)
  • Szent-GyorgyiA.G.

    Regulation by myosin: how calcium regulates some myosins, past and present

    Adv. Exp. Med. Biol.

    (2007)
  • SweeneyH.L. et al.

    Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function

    Am. J. Physiol.

    (1993)
  • CraigR. et al.

    Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    J. Cell Biol.

    (1987)
  • ChackoS. et al.

    Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation

    Proc. Natl Acad. Sci. USA

    (1977)
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    Present address: W. Wriggers, D. E. Shaw Research, 39th Floor, 120 West Forty-Fifth Street, New York, NY 10036, USA.

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