Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
Abstract
:1. Introduction
2. Results
2.1. Design of Tpm1.1 and Tpm3.12 Peptides
2.2. Structural Stability of Tpm1.164–154 and Tpm3.1265–155
2.3. Disruptive Effects of Missense Mutations on Stability of Tpm1.164–154 and Tpm3.1265–155
2.4. R91P Mutation Increased Disorder in Tpm3.1265–155
2.5. Analysis of Three-Dimensional Structures of the Tpm Fragments Using Molecular Dynamics Simulations
3. Discussion
3.1. Differences in Stability of Tpm1.1 and Tpm3.12 Middle Regions
3.2. Effects of Disease-Causing Mutations on Structural Transitions of Tpm1.1 and Tpm3.12 Middle Regions
4. Materials and Methods
4.1. Construction and Cloning of TPM1 and TPM3 cDNA Fragments
4.2. Site-Directed Mutagenesis
4.3. Peptide Expression and Purification
4.4. Peptide Concentration Determination
4.5. Circular Dichroism Measurements
4.6. Molecular Dynamics Simulations
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
- Hitchcock-DeGregori, S.E.; Barua, B. Tropomyosin Structure, Function, and Interactions: A Dynamic Regulator. Fibrous Proteins Struct. Mech. 2017, 82, 253–284. [Google Scholar] [CrossRef]
- Gordon, A.M.; Homsher, E.; Regnier, M. Regulation of contraction in striated muscle. Physiol. Rev. 2000, 80, 853–924. [Google Scholar] [CrossRef]
- Geeves, M.A.; Hitchcock-DeGregori, S.E.; Gunning, P.W. A systematic nomenclature for mammalian tropomyosin isoforms. J. Muscle Res. Cell Motil. 2014, 36, 147–153. [Google Scholar] [CrossRef] [Green Version]
- Moraczewska, J. Thin filament dysfunctions caused by mutations in tropomyosin Tpm3.12 and Tpm1.1. J. Muscle Res. Cell. Motil. 2020, 41, 39–53. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Matyushenko, A.M.; Shchepkin, D.V.; Kopylova, G.V.; Bershitsky, S.Y.; Levitsky, D.I. Unique functional properties of slow skeletal muscle tropomyosin. Biochimie 2020, 174, 1–8. [Google Scholar] [CrossRef] [PubMed]
- Robaszkiewicz, K.; Sliwinska, M.; Moraczewska, J. Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin. Int. J. Mol. Sci. 2020, 21, 4285. [Google Scholar] [CrossRef]
- Hitchcock-DeGregori, S.E. Tropomyosin: Function follows structure. Adv. Exp. Med. Biol. 2008, 644, 60–72. [Google Scholar]
- Barua, B. Periodicities designed in the tropomyosin sequence and structure define its functions. Bioarchitecture 2013, 3, 51–56. [Google Scholar] [CrossRef] [Green Version]
- Barua, B.; Pamula, M.C.; Hitchcock-DeGregori, S.E. Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin. Proc. Natl. Acad. Sci. USA 2011, 108, 10150–10155. [Google Scholar] [CrossRef] [Green Version]
- Li, X.E.; Tobacman, L.S.; Mun, J.Y.; Craig, R.; Fischer, S.; Lehman, W. Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry. Biophys. J. 2011, 100, 1005–1013. [Google Scholar] [CrossRef] [Green Version]
- Hitchcock-DeGregori, S.E.; Song, Y.; Greenfield, N.J. Functions of tropomyosin’s periodic repeats. Biochemistry 2002, 41, 15036–15044. [Google Scholar] [CrossRef]
- Singh, A.; Hitchcock-DeGregori, S.E. Tropomyosin’s periods are quasi-equivalent for actin binding but have specific regulatory functions. Biochemistry 2007, 46, 14917–14927. [Google Scholar] [CrossRef]
- Behrmann, E.; Muller, M.; Penczek, P.A.; Mannherz, H.G.; Manstein, D.J.; Raunser, S. Structure of the rigor actin-tropomyosin-myosin complex. Cell 2012, 150, 327–338. [Google Scholar] [CrossRef] [Green Version]
- Doran, M.H.; Pavadai, E.; Rynkiewicz, M.J.; Walklate, J.; Bullitt, E.; Moore, J.R.; Regnier, M.; Geeves, M.A.; Lehman, W. Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments. Biophys. J. 2020, 119, 821–830. [Google Scholar] [CrossRef]
- Barua, B.; Winkelmann, D.A.; White, H.D.; Hitchcock-DeGregori, S.E. Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin. Proc. Natl. Acad. Sci. USA 2012, 109, 18425–18430. [Google Scholar] [CrossRef] [Green Version]
- Landis, C.; Back, N.; Homsher, E.; Tobacman, L.S. Effects of tropomyosin internal deletions on thin filament function. J. Biol. Chem. 1999, 274, 31279–31285. [Google Scholar] [CrossRef] [Green Version]
- Landis, C.A.; Bobkova, A.; Homsher, E.; Tobacman, L.S. The active state of the thin filament is destabilized by an internal deletion in tropomyosin. J. Biol. Chem. 1997, 272, 14051–14056. [Google Scholar] [CrossRef] [Green Version]
- Singh, A.; Hitchcock-DeGregori, S.E. Dual requirement for flexibility and specificity for binding of the coiled-coil tropomyosin to its target, actin. Structure 2006, 14, 43–50. [Google Scholar] [CrossRef] [Green Version]
- Singh, A.; Hitchcock-Degregori, S.E. A peek into tropomyosin binding and unfolding on the actin filament. PLoS ONE 2009, 4, e6336. [Google Scholar] [CrossRef] [Green Version]
- Hitchcock-DeGregori, S.E.; An, Y. Integral repeats and a continuous coiled coil are required for binding of striated muscle tropomyosin to the regulated actin filament. J. Biol. Chem. 1996, 271, 3600–3603. [Google Scholar] [CrossRef] [Green Version]
- Kawai, M.; Lu, X.; Hitchcock-Degregori, S.E.; Stanton, K.J.; Wandling, M.W. Tropomyosin period 3 is essential for enhancement of isometric tension in thin filament-reconstituted bovine myocardium. J. Biophys. 2009, 2009, 380967. [Google Scholar] [CrossRef] [Green Version]
- Oguchi, Y.; Ishizuka, J.; Hitchcock-DeGregori, S.E.; Ishiwata, S.; Kawai, M. The role of tropomyosin domains in cooperative activation of the actin-myosin interaction. J. Mol. Biol. 2011, 414, 667–680. [Google Scholar] [CrossRef] [Green Version]
- Marttila, M.; Lehtokari, V.L.; Marston, S.; Nyman, T.A.; Barnerias, C.; Beggs, A.H.; Bertini, E.; Ceyhan-Birsoy, O.; Cintas, P.; Gerard, M.; et al. Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies. Hum. Mutat. 2014, 35, 779–790. [Google Scholar] [CrossRef] [Green Version]
- Redwood, C.; Robinson, P. Alpha-tropomyosin mutations in inherited cardiomyopathies. J. Muscle Res. Cell. Motil. 2013, 34, 285–294. [Google Scholar] [CrossRef]
- Karibe, A.; Tobacman, L.S.; Strand, J.; Butters, C.; Back, N.; Bachinski, L.L.; Arai, A.E.; Ortiz, A.; Roberts, R.; Homsher, E.; et al. Hypertrophic cardiomyopathy caused by a novel alpha-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis. Circulation 2001, 103, 65–71. [Google Scholar] [CrossRef] [Green Version]
- Hershberger, R.E.; Norton, N.; Morales, A.; Li, D.; Siegfried, J.D.; Gonzalez-Quintana, J. Coding sequence rare variants identified in MYBPC3, MYH6, TPM1, TNNC1, and TNNI3 from 312 patients with familial or idiopathic dilated cardiomyopathy. Circ. Cardiovasc. Genet. 2010, 3, 155–161. [Google Scholar] [CrossRef] [Green Version]
- Lawlor, M.W.; Dechene, E.T.; Roumm, E.; Geggel, A.S.; Moghadaszadeh, B.; Beggs, A.H. Mutations of tropomyosin 3 (TPM3) are common and associated with type 1 myofiber hypotrophy in congenital fiber type disproportion. Hum. Mutat. 2010, 31, 176–183. [Google Scholar] [CrossRef] [Green Version]
- Shapovalov, M.V.; Dunbrack, R.L., Jr. A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions. Structure 2011, 19, 844–858. [Google Scholar] [CrossRef] [Green Version]
- Matyushenko, A.M.; Kleymenov, S.Y.; Susorov, D.S.; Levitsky, D.I. Thermal unfolding of homodimers and heterodimers of different skeletal-muscle isoforms of tropomyosin. Biophys. Chem. 2018, 243, 1–7. [Google Scholar] [CrossRef]
- Gonchar, A.D.; Kopylova, G.V.; Kochurova, A.M.; Berg, V.Y.; Shchepkin, D.V.; Koubasova, N.A.; Tsaturyan, A.K.; Kleymenov, S.Y.; Matyushenko, A.M.; Levitsky, D.I. Effects of myopathy-causing mutations R91P and R245G in the TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin. Biochem. Biophys. Res. Commun. 2021, 534, 8–13. [Google Scholar] [CrossRef]
- Matyushenko, A.M.; Artemova, N.V.; Sluchanko, N.N.; Levitsky, D.I. Effects of two stabilizing substitutions, D137L and G126R, in the middle part of alpha-tropomyosin on the domain structure of its molecule. Biophys. Chem. 2015, 196, 77–85. [Google Scholar] [CrossRef]
- Moraczewska, J.; Robaszkiewicz, K.; Śliwinska, M.; Czajkowska, M.; Ly, T.; Kostyukova, A.; Wen, H.; Zheng, W. Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding. FEBS J. 2019, 286, 1877–1893. [Google Scholar] [CrossRef]
- Letai, A.; Coulombe, P.A.; Fuchs, E. Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations. J. Cell Biol. 1992, 116, 1181–1195. [Google Scholar] [CrossRef] [Green Version]
- Ly, T.; Krieger, I.; Tolkatchev, D.; Krone, C.; Moural, T.; Samatey, F.A.; Kang, C.; Kostyukova, A.S. Structural destabilization of tropomyosin induced by the cardiomyopathy-linked mutation R21H. Protein Sci. 2018, 27, 498–508. [Google Scholar] [CrossRef]
- Demers, J.P.; Mittermaier, A. Binding mechanism of an SH3 domain studied by NMR and ITC. J. Am. Chem. Soc. 2009, 131, 4355–4367. [Google Scholar] [CrossRef]
- Xue, Y.; Yuwen, T.; Zhu, F.; Skrynnikov, N.R. Role of electrostatic interactions in binding of peptides and intrinsically disordered proteins to their folded targets. 1. NMR and MD characterization of the complex between the c-Crk N-SH3 domain and the peptide Sos. Biochemistry 2014, 53, 6473–6495. [Google Scholar] [CrossRef]
- Potekhin, S.A.; Privalov, P.L. Co-operative blocks in tropomyosin. J. Mol. Biol. 1982, 159, 519–535. [Google Scholar] [CrossRef]
- Itzhaki, R.F.; Gill, D.M. A Micro-Biuret Method for Estimating Proteins. Anal. Biochem. 1964, 9, 401–410. [Google Scholar] [CrossRef]
- Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612. [Google Scholar] [CrossRef] [Green Version]
- Jorgensen, W.L.; Chandrasekhar, J.; Madura, J.D.; Impey, R.W.; Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926–935. [Google Scholar] [CrossRef]
- Lee, T.S.; Cerutti, D.S.; Mermelstein, D.; Lin, C.; LeGrand, S.; Giese, T.J.; Roitberg, A.; Case, D.A.; Walker, R.C.; York, D.M. GPU-Accelerated Molecular Dynamics and Free Energy Methods in Amber18: Performance Enhancements and New Features. J. Chem. Inf. Model. 2018, 58, 2043–2050. [Google Scholar] [CrossRef]
- Maier, J.A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K.E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696–3713. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Tpm | Melting Temperature (Tm), °C | |
---|---|---|
No DTT | DTT | |
Tpm1.164–154 | 46.1 ± 0.2 | 23.7 ± 0.7 |
Tpm1.164–154I92T | 28.2 ± 0.1 | 5.6 ± 0.5 |
Tpm1.164–154V95A | 42.2 ± 0.5 | 21.7 ± 0.3 |
Tpm 3.1265–155 | 60.9 ± 0.2 | 45.8 ± 0.9 |
Tpm 3.1265–155R91C | 51.5 ± 0.7 | 31.3 ± 0.2 |
Tpm3.1265–155R91P | 28.9 ± 0.1 | ND |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Kuruba, B.; Kaczmarek, M.; Kęsik-Brodacka, M.; Fojutowska, M.; Śliwinska, M.; Kostyukova, A.S.; Moraczewska, J. Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin. Molecules 2021, 26, 6980. https://doi.org/10.3390/molecules26226980
Kuruba B, Kaczmarek M, Kęsik-Brodacka M, Fojutowska M, Śliwinska M, Kostyukova AS, Moraczewska J. Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin. Molecules. 2021; 26(22):6980. https://doi.org/10.3390/molecules26226980
Chicago/Turabian StyleKuruba, Balaganesh, Marta Kaczmarek, Małgorzata Kęsik-Brodacka, Magdalena Fojutowska, Małgorzata Śliwinska, Alla S. Kostyukova, and Joanna Moraczewska. 2021. "Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin" Molecules 26, no. 22: 6980. https://doi.org/10.3390/molecules26226980