Abstract
Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro function. The Met8Arg mutation reduces tropomyosin affinity for regulated actin 30- to 100-fold. Ca(2+)-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca(2+) is reduced. The poor activation may reflect weakened actin affinity or reduced effectiveness in switching the thin filament to the open, force-producing state. The presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impair end-to-end association between TMs on the thin filament. In muscle, the mutation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end of the filament. The mutation may also reduce force generation during activation.
Full Text
The Full Text of this article is available as a PDF (106.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cassell M., Tobacman L. S. Opposite effects of myosin subfragment 1 on binding of cardiac troponin and tropomyosin to the thin filament. J Biol Chem. 1996 May 31;271(22):12867–12872. doi: 10.1074/jbc.271.22.12867. [DOI] [PubMed] [Google Scholar]
- Cho Y. J., Hitchcock-DeGregori S. E. Relationship between alternatively spliced exons and functional domains in tropomyosin. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10153–10157. doi: 10.1073/pnas.88.22.10153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clayton L., Reinach F. C., Chumbley G. M., MacLeod A. R. Organization of the hTMnm gene. Implications for the evolution of muscle and non-muscle tropomyosins. J Mol Biol. 1988 Jun 5;201(3):507–515. doi: 10.1016/0022-2836(88)90633-x. [DOI] [PubMed] [Google Scholar]
- Eaton B. L. Tropomyosin binding to F-actin induced by myosin heads. Science. 1976 Jun 25;192(4246):1337–1339. doi: 10.1126/science.131972. [DOI] [PubMed] [Google Scholar]
- Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
- Fowler V. M., Sussmann M. A., Miller P. G., Flucher B. E., Daniels M. P. Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle. J Cell Biol. 1993 Jan;120(2):411–420. doi: 10.1083/jcb.120.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenfield N. J., Hitchcock-DeGregori S. E. The stability of tropomyosin, a two-stranded coiled-coil protein, is primarily a function of the hydrophobicity of residues at the helix-helix interface. Biochemistry. 1995 Dec 26;34(51):16797–16805. doi: 10.1021/bi00051a030. [DOI] [PubMed] [Google Scholar]
- Greenfield N. J., Montelione G. T., Farid R. S., Hitchcock-DeGregori S. E. The structure of the N-terminus of striated muscle alpha-tropomyosin in a chimeric peptide: nuclear magnetic resonance structure and circular dichroism studies. Biochemistry. 1998 May 26;37(21):7834–7843. doi: 10.1021/bi973167m. [DOI] [PubMed] [Google Scholar]
- Greenfield N. J., Stafford W. F., Hitchcock-DeGregori S. E. The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin. Protein Sci. 1994 Mar;3(3):402–410. doi: 10.1002/pro.5560030304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hammell R. L., Hitchcock-DeGregori S. E. Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon. J Biol Chem. 1996 Feb 23;271(8):4236–4242. doi: 10.1074/jbc.271.8.4236. [DOI] [PubMed] [Google Scholar]
- Hammell R. L., Hitchcock-DeGregori S. E. The sequence of the alternatively spliced sixth exon of alpha-tropomyosin is critical for cooperative actin binding but not for interaction with troponin. J Biol Chem. 1997 Sep 5;272(36):22409–22416. doi: 10.1074/jbc.272.36.22409. [DOI] [PubMed] [Google Scholar]
- Heald R. W., Hitchcock-DeGregori S. E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin. J Biol Chem. 1988 Apr 15;263(11):5254–5259. [PubMed] [Google Scholar]
- Hitchcock-De Gregori S. E., Mandala S., Sachs G. A. Changes in actin lysine reactivities during polymerization detected using a competitive labeling method. J Biol Chem. 1982 Nov 10;257(21):12573–12580. [PubMed] [Google Scholar]
- Hitchcock-DeGregori S. E., Heald R. W. Altered actin and troponin binding of amino-terminal variants of chicken striated muscle alpha-tropomyosin expressed in Escherichia coli. J Biol Chem. 1987 Jul 15;262(20):9730–9735. [PubMed] [Google Scholar]
- Hitchcock S. E. Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin. Eur J Biochem. 1975 Mar 17;52(2):255–263. doi: 10.1111/j.1432-1033.1975.tb03993.x. [DOI] [PubMed] [Google Scholar]
- Jockusch B. M., Veldman H., Griffiths G. W., van Oost B. A., Jennekens F. G. Immunofluorescence microscopy of a myopathy. alpha-Actinin is a major constituent of nemaline rods. Exp Cell Res. 1980 Jun;127(2):409–420. doi: 10.1016/0014-4827(80)90445-0. [DOI] [PubMed] [Google Scholar]
- Koradi R., Billeter M., Wüthrich K. MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph. 1996 Feb;14(1):51-5, 29-32. doi: 10.1016/0263-7855(96)00009-4. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Laing N. G. Inherited disorders of sarcomeric proteins. Curr Opin Neurol. 1999 Oct;12(5):513–518. doi: 10.1097/00019052-199910000-00004. [DOI] [PubMed] [Google Scholar]
- Laing N. G., Wilton S. D., Akkari P. A., Dorosz S., Boundy K., Kneebone C., Blumbergs P., White S., Watkins H., Love D. R. A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nat Genet. 1995 Jan;9(1):75–79. doi: 10.1038/ng0195-75. [DOI] [PubMed] [Google Scholar]
- Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
- Lehrer S. S., Geeves M. A. The muscle thin filament as a classical cooperative/allosteric regulatory system. J Mol Biol. 1998 Apr 17;277(5):1081–1089. doi: 10.1006/jmbi.1998.1654. [DOI] [PubMed] [Google Scholar]
- Lehrer S. S., Golitsina N. L., Geeves M. A. Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions. Biochemistry. 1997 Nov 4;36(44):13449–13454. doi: 10.1021/bi971568w. [DOI] [PubMed] [Google Scholar]
- Lehrer S. S. Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3377–3381. doi: 10.1073/pnas.72.9.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lehrer S. S. The regulatory switch of the muscle thin filament: Ca2+ or myosin heads? J Muscle Res Cell Motil. 1994 Jun;15(3):232–236. doi: 10.1007/BF00123476. [DOI] [PubMed] [Google Scholar]
- Lumb K. J., Carr C. M., Kim P. S. Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. Biochemistry. 1994 Jun 14;33(23):7361–7367. doi: 10.1021/bi00189a042. [DOI] [PubMed] [Google Scholar]
- MacLeod A. R., Gooding C. Human hTM alpha gene: expression in muscle and nonmuscle tissue. Mol Cell Biol. 1988 Jan;8(1):433–440. doi: 10.1128/mcb.8.1.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Margossian S. S., Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982;85(Pt B):55–71. doi: 10.1016/0076-6879(82)85009-x. [DOI] [PubMed] [Google Scholar]
- McKillop D. F., Geeves M. A. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J. 1993 Aug;65(2):693–701. doi: 10.1016/S0006-3495(93)81110-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michele D. E., Albayya F. P., Metzger J. M. A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production. J Clin Invest. 1999 Dec;104(11):1575–1581. doi: 10.1172/JCI7842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michele D. E., Albayya F. P., Metzger J. M. Thin filament protein dynamics in fully differentiated adult cardiac myocytes: toward a model of sarcomere maintenance. J Cell Biol. 1999 Jun 28;145(7):1483–1495. doi: 10.1083/jcb.145.7.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moraczewska J., Nicholson-Flynn K., Hitchcock-DeGregori S. E. The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state. Biochemistry. 1999 Nov 30;38(48):15885–15892. doi: 10.1021/bi991816j. [DOI] [PubMed] [Google Scholar]
- Morris E. P., Nneji G., Squire J. M. The three-dimensional structure of the nemaline rod Z-band. J Cell Biol. 1990 Dec;111(6 Pt 2):2961–2978. doi: 10.1083/jcb.111.6.2961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mukherjea P., Tong L., Seidman J. G., Seidman C. E., Hitchcock-DeGregori S. E. Altered regulatory function of two familial hypertrophic cardiomyopathy troponin T mutants. Biochemistry. 1999 Oct 5;38(40):13296–13301. doi: 10.1021/bi9906120. [DOI] [PubMed] [Google Scholar]
- North K. N., Laing N. G., Wallgren-Pettersson C. Nemaline myopathy: current concepts. The ENMC International Consortium and Nemaline Myopathy. J Med Genet. 1997 Sep;34(9):705–713. doi: 10.1136/jmg.34.9.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nowak K. J., Wattanasirichaigoon D., Goebel H. H., Wilce M., Pelin K., Donner K., Jacob R. L., Hübner C., Oexle K., Anderson J. R. Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet. 1999 Oct;23(2):208–212. doi: 10.1038/13837. [DOI] [PubMed] [Google Scholar]
- O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
- Pelin K., Hilpelä P., Donner K., Sewry C., Akkari P. A., Wilton S. D., Wattanasirichaigoon D., Bang M. L., Centner T., Hanefeld F. Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2305–2310. doi: 10.1073/pnas.96.5.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pittenger M. F., Kazzaz J. A., Helfman D. M. Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol. 1994 Feb;6(1):96–104. doi: 10.1016/0955-0674(94)90122-8. [DOI] [PubMed] [Google Scholar]
- Potter J. D. Preparation of troponin and its subunits. Methods Enzymol. 1982;85(Pt B):241–263. doi: 10.1016/0076-6879(82)85024-6. [DOI] [PubMed] [Google Scholar]
- Reinach F. C., MacLeod A. R. Tissue-specific expression of the human tropomyosin gene involved in the generation of the trk oncogene. Nature. 1986 Aug 14;322(6080):648–650. doi: 10.1038/322648a0. [DOI] [PubMed] [Google Scholar]
- Ruiz-Opazo N., Nadal-Ginard B. Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. J Biol Chem. 1987 Apr 5;262(10):4755–4765. [PubMed] [Google Scholar]
- SHY G. M., ENGEL W. K., SOMERS J. E., WANKO T. NEMALINE MYOPATHY. A NEW CONGENITAL MYOPATHY. Brain. 1963 Dec;86:793–810. doi: 10.1093/brain/86.4.793. [DOI] [PubMed] [Google Scholar]
- Salviati G., Betto R., Danieli Betto D., Zeviani M. Myofibrillar-protein isoforms and sarcoplasmic-reticulum Ca2+-transport activity of single human muscle fibres. Biochem J. 1984 Nov 15;224(1):215–225. doi: 10.1042/bj2240215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiaffino S., Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev. 1996 Apr;76(2):371–423. doi: 10.1152/physrev.1996.76.2.371. [DOI] [PubMed] [Google Scholar]
- Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
- Tan P., Briner J., Boltshauser E., Davis M. R., Wilton S. D., North K., Wallgren-Pettersson C., Laing N. G. Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy. Neuromuscul Disord. 1999 Dec;9(8):573–579. doi: 10.1016/s0960-8966(99)00053-x. [DOI] [PubMed] [Google Scholar]
- Tobacman L. S. Thin filament-mediated regulation of cardiac contraction. Annu Rev Physiol. 1996;58:447–481. doi: 10.1146/annurev.ph.58.030196.002311. [DOI] [PubMed] [Google Scholar]
- Urbancikova M., Hitchcock-DeGregori S. E. Requirement of amino-terminal modification for striated muscle alpha-tropomyosin function. J Biol Chem. 1994 Sep 30;269(39):24310–24315. [PubMed] [Google Scholar]
- Wallgren-Pettersson C., Jasani B., Newman G. R., Morris G. E., Jones S., Singhrao S., Clarke A., Virtanen I., Holmberg C., Rapola J. Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy. Neuromuscul Disord. 1995 Mar;5(2):93–104. doi: 10.1016/0960-8966(94)00035-8. [DOI] [PubMed] [Google Scholar]
- Weber A., Pennise C. R., Babcock G. G., Fowler V. M. Tropomodulin caps the pointed ends of actin filaments. J Cell Biol. 1994 Dec;127(6 Pt 1):1627–1635. doi: 10.1083/jcb.127.6.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber A., Pennise C. R., Fowler V. M. Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP.P(i)-actin to ADP-actin at all pointed filament ends. J Biol Chem. 1999 Dec 3;274(49):34637–34645. doi: 10.1074/jbc.274.49.34637. [DOI] [PubMed] [Google Scholar]
- Whitby F. G., Phillips G. N., Jr Crystal structure of tropomyosin at 7 Angstroms resolution. Proteins. 2000 Jan 1;38(1):49–59. [PubMed] [Google Scholar]
- White H. D. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol. 1982;85(Pt B):698–708. doi: 10.1016/0076-6879(82)85057-x. [DOI] [PubMed] [Google Scholar]
- Yamaguchi M., Robson R. M., Stromer M. H., Dahl D. S., Oda T. Actin filaments form the backbone of nemaline myopathy rods. Nature. 1978 Jan 19;271(5642):265–267. doi: 10.1038/271265a0. [DOI] [PubMed] [Google Scholar]
- Yamaguchi M., Robson R. M., Stromer M. H., Dahl D. S., Oda T. Nemaline myopathy rod bodies. Structure and composition. J Neurol Sci. 1982 Oct;56(1):35–56. doi: 10.1016/0022-510x(82)90059-4. [DOI] [PubMed] [Google Scholar]