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Regulation of Actin-Myosin Interaction by Conserved Periodic Sites of Tropomyosin

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Regulation of Actin-Myosin Interaction by Conserved Periodic Sites of Tropomyosin Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin Bipasha Baruaa,b,1, Donald A. Winkelmannb, Howard D. Whitec, and Sarah E. Hitchcock-DeGregoria,b aDepartment of Neuroscience and Cell Biology, bDepartment of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and cDepartment of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507 Edited by Hugh E. Huxley, Research Center, Waltham, MA, and approved September 26, 2012 (received for review July 25, 2012) Cooperative activation of actin-myosin interaction by tropomyosin actin filament, allowing strong myosin binding and force pro- (Tm) is central to regulation of contraction in muscle cells and cellular duction (11, 12). The population of thin filaments in each state is 2+ and intracellular movements in nonmuscle cells. The steric blocking modulated by the presence of Ca and myosin S1. The binding model of muscle regulation proposed 40 y ago has been sub- of myosin S1 to thin filaments shifts the equilibrium of the fila- stantiated at both the kinetic and structural levels. Even with atomic ments from the closed to open state, in which both Tm and my- fi – resolution structures of the major players, how Tm binds and is osin bind to actin with higher af nity (13 17). This cooperative activation of the thin filament by myosin from the closed to open designed for regulatory function has remained a mystery. Here we 2+ show that a set of periodically distributed evolutionarily conserved state can be produced by Tm alone, independent of Tn and Ca , surface residues of Tm is required for cooperative regulation of and is likely to be a general mechanism of regulation of acto- actomyosin. Based on our results, we propose a model of Tm on myosin by Tm in nonmuscle cells, where Tn is absent. a structure of actin-Tm-myosin in the “open” (on) state showing Previous studies showed that individual periodic repeats of Tm had varying degrees of effect on thin filament regulation, indicating potential electrostatic interactions of the residues with both actin that specific regions of Tm contribute differently to its regulatory and myosin. The sites alternate with a second set of conserved sur- function (18–21). EM and 3D reconstruction of actin-Tm filaments face residues that are important for actin binding in the inhibitory with different Tm isoforms have shown that in the absence of Tn state in the absence of myosin. The transition from the closed to and myosin, Tm occupies the blocked or closed position on the actin open states requires the sites identified here, even when troponin + fi fi + lament, depending on the Tm and actin isoform (22). This nding 2 BIOPHYSICS AND Ca is present. The evolutionarily conserved residues are impor- implies that amino acid sequence differences among isoforms can tant for actomyosin regulation, a universal function of Tm that has modulate the regulatory functions of Tm. However, the specific COMPUTATIONAL BIOLOGY a common structural basis and mechanism. residues of Tm that interact with actin in the different regulatory states as well as their effect on myosin regulation are not known. actin filament structure | cell motility | cytoskeleton | muscle contraction | We previously reported an evolutionary analysis of Tms from 26 motility assay species, ranging from cnidarians to vertebrates, and determined the conserved residues of Tm (23). Mutations at conserved surface ctin and myosin are found in almost all eukaryotic cells and positions b, c,andf of the heptad repeat of the Tm coiled-coil Aare required for diverse cellular functions, including muscle revealed that residues in the first halves of Tm’s periodic repeats or contraction, cell motility, cell adhesion, cytokinesis, and organelle periods (P) 2–6 of 7 repeats are important for actin affinity. transport (1). The actin–myosin interaction produces two types of Mutations in the second halves of P2–P6 had a relatively small ef- movements: force generation between actin filaments leading to fect on actin affinity. We hypothesize that these residues are im- contractions, such as in muscle contraction, cell motility, and cy- portant for another conserved function of Tm, myosin regulation. tokinesis; and transport of subcellular organelles and macromo- In the present study, we determined the effect of Tm mutations lecular complexes by myosin motors along actin filaments. The at conserved surface positions in the second halves of P2– P6 on actomyosin contractile apparatus is best characterized in striated myosin regulation using in vitro motility assays. Tm with mutations + muscle contraction, which is regulated in a Ca2 -dependent man- in P3– P6 showed a large inhibition of filament velocities compared ner by the proteins, tropomyosin (Tm) and troponin (Tn). with WT Tm. The period 3 and period 6 mutants inhibited filament Tm is a universal regulator of the actin cytoskeleton. It is a two- velocities, even in the presence of N-ethylmaleimide (NEM)-S1 or 2+ chained, α-helical coiled-coil actin binding protein that associates Tn + Ca . These results, supported by kinetic and fluorescent end-to-end to form a continuous strand along both sides of actin probe studies, indicate that the mutated residues in second halves filaments and regulates the functions and stability of actin fila- of P3– P6 are required for shifting the equilibrium of actin-Tm ments (2, 3). Tm regulates the interaction of actin filaments with filaments from closed to open state and may be binding sites for actin binding proteins, including myosin, Tn, ADF-cofilin, Arp2/3, actin or myosin in the open or activated state. A molecular model formin, and tropomodulin. Tm can activate or inhibit actomyosin, for interacting sites of Tm with actin and myosin in the open reg- depending on the myosin and Tm isoforms (4–7). The Tm-de- ulatory state is presented to give additional insights into this uni- pendent regulation is widespread, having been reported in fission versal regulatory mechanism. yeast and mammalian muscle and nonmuscle systems. The regu- lation of actomyosin by Tm may be viewed as universal, although Results there is little understanding of the mechanism or structural basis, The striated muscle αTm constructs used in this study have an the focus of the present work. Ala-Ser extension at the N terminus and have three to four In striated muscle, regulation of myosin binding to the thin filament (actin-Tm-Tn) is described by a three-state model, where the three kinetic states are thought to correspond to three Author contributions: B.B., D.A.W., H.D.W., and S.E.H.-D. designed research; B.B. and positions of Tm on the actin filament (8–10). The three states are H.D.W. performed research; B.B. contributed new reagents/analytic tools; B.B. and D.A.W. defined as: (i) “blocked” or “off” state, in the presence of Tn and made the models and movie; B.B., D.A.W., H.D.W., and S.E.H.-D. analyzed data; and B.B. and + absence of Ca2 , where Tm blocks the interaction of myosin with S.E.H.-D. wrote the paper. + actin; (ii) “closed” state, in the presence of Ca2 and Tn, where The authors declare no conflict of interest. Tm shifts azimuthally on the actin filament to partially expose This article is a PNAS Direct Submission. the myosin binding site and allow weak myosin binding; and (iii) 1To whom correspondence should be addressed. E-mail: [email protected]. “open” or “on” state, in the presence of myosin heads (sub- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fragment 1 or S1), where Tm azimuthally shifts further on the 1073/pnas.1212754109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212754109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Tm mutations at evolutionarily conserved surface residues (23). (A) The rat striated αTm se- quence showing conserved b, c, and f residues that were mutated to Ala in P2–P6. Each mutant has three to four mutations within the first-half (red) or second-half (blue) of each period. (B) Tm mutations shown in the 7 Å striated muscle αTm structure (1C1G) (43). Mutations in the first-half of periods 2– 6 are in red, and mutations in the second-half are in blue. The periodic repeats do not correspond to the half-turns of the coiled-coil in the 7 Å structure because there are five and three-quarter half-turns of the supercoil per molecule, not seven (43, 44). That is, the half-turns do not correspond to the seven actins along the length of one Tm molecule. conserved residues mutated to Ala in the second halves of 2A). The inhibition by A-Tm(WT) is consistent with previous periods P2–P6 (blue in Fig. 1). A first-half mutant, P3-1-EE, was reports (24, 25) and with kinetics and EM structural evidence that used as a control for the effect of mutations in the first half of the Tm is predominantly in the closed state in the absence of Tn (11, periods (red in Fig. 1). This mutant was selected because it had the 22). The velocities of A-Tm(mutant) filaments, P2-2-EKD and P3- smallest effect on actin affinity (23) compared with other first-half 1-EE, were not significantly different from A-Tm(WT) (P > 0.05). mutants, to separate the effects of Tm mutations on myosin reg- The velocities of all other A-Tm(mutant) filaments were inhibited ulation from the effect on regulation caused by weaker actin af- by ∼40–80% relative to A-Tm(WT) (P < 0.05) (Fig. 2A and Table finity of Tms in the absence of myosin.
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