Biomol NMR Assign DOI 10.1007/s12104-014-9543-5
ARTICLE
Solution NMR assignment of the heavy chain complex of the human cardiac myosin regulatory light chain Elena Rostkova • Mathias Gautel • Mark Pfuhl
Received: 5 November 2013 / Accepted: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The regulatory light chain (RLC) of striated and cardiac muscle myosin plays a complex role in muscle function and regulation. Together with the essential light chain it provides stability to the lever arm, which is essential for force generation. Furthermore, phosphorylation and interaction with myosin binding protein C (MyBPC) suggest an additional role in the regulation of muscle contraction. The former is of particular importance in the heart, where RLC phosphorylation appears to be correlated to the wringing motion of heart contraction. To address these questions and because of a lack of mammalian RLC structures, we initiated an NMR study of the human cardiac regulatory myosin light chain. Keywords Heart muscle Phosphorylation Myosin binding protein C Contraction Cardiomyopathy
Biological context The cardiac myosin regulatory light chain (RLC) is well positioned at the S1–S2 junction to play a key regulatory role for cardiac muscle contraction (Scruggs and Solaro 2011). In the first place, it provides—together with its neighbour, the essential light chain—stiffness to the myosin lever helix (Rayment et al. 1993). Beyond this limited mechanical role it is also able to modulate myosin head positioning through phosphorylation by myosin light chain kinase (MLCK) (Ishikawa and Kurotani 2008; Stull et al. 2011) and dephosphorylation through light chain E. Rostkova M. Gautel M. Pfuhl (&) Cardiovascular and Randall Division, King’s College London, Guy’s Campus, London SE1 1UL, UK e-mail: [email protected]
phosphatase (MLCP) (Arimura et al. 2001; Shichi et al. 2010). In smooth muscle RLC phosphorylation is the main on/off switch for contraction (Hong et al. 2011). In striated and cardiac muscle this switch is located in the troponin/ tropomyosin complex on the thin filament. In these muscle tissues the regulatory effect of the RLC is therefore much more indirect (Scruggs and Solaro 2011; Stull et al. 2011). It is currently thought that this indirect mode of regulation could relate to a repositioning of the myosin heads with the aim to increase their concentration in the vicinity of the thin filament which promotes cross bridge formation and consequently higher force development (Greenberg et al. 2009; Miller et al. 2011). Moving the S1 heads closer to the thin filament will make it more likely for them to bind and generate force (Colson et al. 2010). The precise benefit of such S1 head rearrangement is not presently known but it appears to be of particular importance for heart muscle, where a spatial gradient of phosphorylation from base to apex and from endocardium to epicardium has been reported (Davis et al. 2001, 2002). This suggests that spatially regulated structural rearrangements of the myosin heads might be important for the adaptation of force generation to the local mechanical stresses in the heart to support its characteristic wringing motion (Vandenboom and Metzger 2002) as well as to modulate hypertrophy (Huang et al. 2008). More recently, we identified the cardiac RLC as a binding partner for the C0 domain of myosin binding protein C, which is specific for its cardiac isoform MYBPC3 (Ratti et al. 2011; Schlossarek et al. 2011). Importantly, point mutations causing hypertrophic cardiomyopathy were identified in the RLC (Greenberg et al. 2010; Szczesna-Cordary et al. 2004) and recently reviewed (Kamm and Stull 2011). Consequently, the RLC in cardiac muscle is a well-positioned regulator of muscle contraction that is able to integrate several regulatory inputs to fine-
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tune local force output, thus maintaining the overall balance of contraction across the entire heart. Its importance is further supported by the identification of missense mutations causing hypertrophic cardiomyopathy. For a better understanding of such a complex role, highresolution structural information is most desirable. A few structures for regulatory light chains are available but they are limited to scallop, squid, yeast and slime mould. With the exception of chicken there are at present no available structures for other vertebrates including H. sapiens, or indeed a tissue-specific RLC isoform of a higher organism. Sequence similarities of the human cardiac light chain to those found in the PDB are low (identities range from 31 to 46 %) so that modelling is difficult. Even more importantly, the phosphorylation site in the N-terminus of the cardiac isoform is completely absent or very different in the invertebrate structures, mainly because the N-terminus is disordered in numerous structures so that its effects cannot currently be evaluated accurately in its structural context. Therefore, we began the investigation of the complex of the human cardiac regulatory light chain bound to its heavy-chain binding site.
Methods and experiments The complex was produced by co-expression of the heavychain fragment (human cardiac myosin (gene MYH7)
Fig. 1 1H-15N TROSY recorded at 298 K and 800 MHz of the 15N/2H labelled complex of the human cardiac regulatory light chain with its heavy chain binding site (residues 806–837 of human cardiac myosin)
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residues 806–837 cloned into a modified pET3a vector) with the regulatory light chain (human ventricular regulatory light chain (gene MYL3) cloned into modified pACYC9d vector) in the E. coli strain BL21* (Invitrogen) at a temperature of 18 °C over night. The complex was purified using a 5 mL HisTrap HP nickel-affinity column (GE Lifesciences) via a His6-tag on the heavy chain fragment, while the light chain was untagged. The His-tag was removed by digestion with TEV protease, upon which the His-tag, TEV protease and contaminating proteins were removed by a second round of nickel-affinity purification, leading to a protein complex with a purity [95 % and a yield of 15–20 mg/L LB, 2–3 mg/L M9 [using an improved M9 medium (Marley et al. 2001)] and about 2 mg/L M9 in D2O. NMR samples were used at concentrations from 100 to 250 lM in a buffer of 25 mM HEPES pH 7.0, 50 mM glutamic acid, 50 mM arginine, 1 mM DTT, 0.02 % NaN3 and a temperature of 298 K. Experiments for assignment were initially recorded on 15 N labelled (15N 3D NOESY and TOCSY) and 15N/13C labelled samples (HNCA, HNCO, HNCACB, HNCOCACB). However, despite a nominally modest molecular weight of 23 kDa, the triple resonance experiments came out poorly so that they had to be recorded using a triple labelled 2H/15N/13C sample. Side chain assignments were obtained from a 13C NOESY-HSQC. The limited solubility (aggregation inevitably occurred for samples [300 lM within a few hours at room temperature) suggests
Solution NMR assignment of the heavy chain complex
unspecific aggregation/low affinity self association as the most likely cause for the poor performance of the triple resonance experiments with non-deuterated samples. The limit in sample concentration significantly reduced the potentially achievable S/N which was then further reduced by the enhanced relaxation due to aggregation.
Assignments and data deposition The assignment of 1H, 15N and 13C resonances is 96 % complete for the backbone and 93 % for side chains. Most importantly, apart from residue 1 the complete N-terminus of the RLC that is missing in numerous crystal structures of regulatory light chains is seen in the NMR spectra. For a few amino acids, mainly in the light chain, we noticed the existence of a low populated (*15 %) conformer. Where possible, backbone assignments for these residues have been added to the database entry. A good impression of the overall extent of the assignment is given in the TROSY experiment in Fig. 1, which shows the good quality of the spectrum and how far it is covered by the assignment. The complete assignment has been submitted to the BioMagResBank database, accession number 19518. Acknowledgments This work was funded by a project grant from the British Heart Foundation (PG/10/65/28521) to MG and MP. NMR spectra were recorded in the Centre for Biomolecular Spectroscopy at King’s College London, at the National Institute of Medical Research and the Henry Wellcome Building NMR facility at the University of Birmingham and we would like to thank Andrew Atkinson, Geoff Kelly, Tom Frankiel, Alain Oregioni and Sara Whittaker for their help with the recording of the spectra.
References Arimura T, Suematsu N, Zhou YB et al (2001) Identification, characterization, and functional analysis of heart-specific myosin light chain phosphatase small subunit. J Biol Chem 276:6073–6082. doi:10.1074/jbc.M008566200 Colson BA, Locher MR, Bekyarova T et al (2010) Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development. J Physiol 588:981–993. doi:10.1113/jphysiol.2009.183897 Davis JS, Hassanzadeh S, Winitsky S et al (2001) The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation. Cell 107:631–641 Davis JS, Hassanzadeh S, Winitsky S et al (2002) A gradient of myosin regulatory light-chain phosphorylation across the ventricular wall supports cardiac torsion. Cold Spring Harb Symp Quant Biol 67:345–352
Greenberg MJ, Mealy TR, Watt JD et al (2009) The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation. Am J Physiol Regul Integr Comp Physiol 297:R265–R274. doi:10.1152/ajpregu.00171.2009 Greenberg MJ, Kazmierczak K, Szczesna-Cordary D, Moore JR (2010) Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry. Proc Nat Acad Sci USA 107:17403–17408. doi:10.1073/pnas.1009619107 Hong F, Haldeman BD, Jackson D et al (2011) Biochemistry of smooth muscle myosin light chain kinase. Arch Biochem Biophys 510:135–146. doi:10.1016/j.abb.2011.04.018 Huang J, Shelton JM, Richardson JA et al (2008) Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy. J Biol Chem 283:19748–19756. doi:10.1074/jbc.M802605200 Ishikawa Y, Kurotani R (2008) Cardiac myosin light chain kinase: a new player in the regulation of myosin light chain in the heart. Circ Res 102:516–518. doi:10.1161/CIRCRESAHA.108.173005 Kamm KE, Stull JT (2011) Signaling to myosin regulatory light chain in sarcomeres. J Biol Chem 286:9941–9947. doi:10.1074/jbc. R110.198697 Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75 Miller MS, Farman GP, Braddock JM et al (2011) Regulatory light chain phosphorylation and N-terminal extension increase crossbridge binding and power output in Drosophila at in vivo myofilament lattice spacing. Biophys J 100:1737–1746. doi:10. 1016/j.bpj.2011.02.028 Ratti J, Rostkova E, Gautel M, Pfuhl M (2011) Structure and interactions of myosin-binding protein C domain C0: cardiacspecific regulation of myosin at its neck? J Biol Chem 286:12650–12658. doi:10.1074/jbc.M110.156646 Rayment I, Rypniewski WR, Schmidt-Base K et al (1993) Threedimensional structure of myosin subfragment-1: a molecular motor. Science 261:50–58 Schlossarek S, Mearini G, Carrier L (2011) Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: mechanisms and therapeutic opportunities. J Mol Cell Cardiol 50:613–620. doi:10.1016/j.yjmcc.2011.01.014 Scruggs SB, Solaro RJ (2011) The significance of regulatory light chain phosphorylation in cardiac physiology. Arch Biochem Biophys 510:129–134. doi:10.1016/j.abb.2011.02.013 Shichi D, Arimura T, Ishikawa T, Kimura A (2010) Heart-specific small subunit of myosin light chain phosphatase activates rhoassociated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J Biol Chem 285:33680–33690. doi:10.1074/jbc.M110.122390 Stull JT, Kamm KE, Vandenboom R (2011) Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle. Arch Biochem Biophys 510:120–128. doi:10. 1016/j.abb.2011.01.017 Szczesna-Cordary D, Guzman G, Ng SS, Zhao J (2004) Familial hypertrophic cardiomyopathy-linked alterations in Ca2? binding of human cardiac myosin regulatory light chain affect cardiac muscle contraction. J Biol Chem 279:3535–3542 Vandenboom R, Metzger JM (2002) A ‘‘wringing’’ endorsement for myosin phosphorylation in the heart. Mol Interv 2:422–424. doi:10.1124/mi.2.7.422
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ARTICLE
Solution NMR assignment of the heavy chain complex of the human cardiac myosin regulatory light chain Elena Rostkova • Mathias Gautel • Mark Pfuhl
Received: 5 November 2013 / Accepted: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The regulatory light chain (RLC) of striated and cardiac muscle myosin plays a complex role in muscle function and regulation. Together with the essential light chain it provides stability to the lever arm, which is essential for force generation. Furthermore, phosphorylation and interaction with myosin binding protein C (MyBPC) suggest an additional role in the regulation of muscle contraction. The former is of particular importance in the heart, where RLC phosphorylation appears to be correlated to the wringing motion of heart contraction. To address these questions and because of a lack of mammalian RLC structures, we initiated an NMR study of the human cardiac regulatory myosin light chain. Keywords Heart muscle Phosphorylation Myosin binding protein C Contraction Cardiomyopathy
Biological context The cardiac myosin regulatory light chain (RLC) is well positioned at the S1–S2 junction to play a key regulatory role for cardiac muscle contraction (Scruggs and Solaro 2011). In the first place, it provides—together with its neighbour, the essential light chain—stiffness to the myosin lever helix (Rayment et al. 1993). Beyond this limited mechanical role it is also able to modulate myosin head positioning through phosphorylation by myosin light chain kinase (MLCK) (Ishikawa and Kurotani 2008; Stull et al. 2011) and dephosphorylation through light chain E. Rostkova M. Gautel M. Pfuhl (&) Cardiovascular and Randall Division, King’s College London, Guy’s Campus, London SE1 1UL, UK e-mail: [email protected]
phosphatase (MLCP) (Arimura et al. 2001; Shichi et al. 2010). In smooth muscle RLC phosphorylation is the main on/off switch for contraction (Hong et al. 2011). In striated and cardiac muscle this switch is located in the troponin/ tropomyosin complex on the thin filament. In these muscle tissues the regulatory effect of the RLC is therefore much more indirect (Scruggs and Solaro 2011; Stull et al. 2011). It is currently thought that this indirect mode of regulation could relate to a repositioning of the myosin heads with the aim to increase their concentration in the vicinity of the thin filament which promotes cross bridge formation and consequently higher force development (Greenberg et al. 2009; Miller et al. 2011). Moving the S1 heads closer to the thin filament will make it more likely for them to bind and generate force (Colson et al. 2010). The precise benefit of such S1 head rearrangement is not presently known but it appears to be of particular importance for heart muscle, where a spatial gradient of phosphorylation from base to apex and from endocardium to epicardium has been reported (Davis et al. 2001, 2002). This suggests that spatially regulated structural rearrangements of the myosin heads might be important for the adaptation of force generation to the local mechanical stresses in the heart to support its characteristic wringing motion (Vandenboom and Metzger 2002) as well as to modulate hypertrophy (Huang et al. 2008). More recently, we identified the cardiac RLC as a binding partner for the C0 domain of myosin binding protein C, which is specific for its cardiac isoform MYBPC3 (Ratti et al. 2011; Schlossarek et al. 2011). Importantly, point mutations causing hypertrophic cardiomyopathy were identified in the RLC (Greenberg et al. 2010; Szczesna-Cordary et al. 2004) and recently reviewed (Kamm and Stull 2011). Consequently, the RLC in cardiac muscle is a well-positioned regulator of muscle contraction that is able to integrate several regulatory inputs to fine-
123
E. Rostkova et al.
tune local force output, thus maintaining the overall balance of contraction across the entire heart. Its importance is further supported by the identification of missense mutations causing hypertrophic cardiomyopathy. For a better understanding of such a complex role, highresolution structural information is most desirable. A few structures for regulatory light chains are available but they are limited to scallop, squid, yeast and slime mould. With the exception of chicken there are at present no available structures for other vertebrates including H. sapiens, or indeed a tissue-specific RLC isoform of a higher organism. Sequence similarities of the human cardiac light chain to those found in the PDB are low (identities range from 31 to 46 %) so that modelling is difficult. Even more importantly, the phosphorylation site in the N-terminus of the cardiac isoform is completely absent or very different in the invertebrate structures, mainly because the N-terminus is disordered in numerous structures so that its effects cannot currently be evaluated accurately in its structural context. Therefore, we began the investigation of the complex of the human cardiac regulatory light chain bound to its heavy-chain binding site.
Methods and experiments The complex was produced by co-expression of the heavychain fragment (human cardiac myosin (gene MYH7)
Fig. 1 1H-15N TROSY recorded at 298 K and 800 MHz of the 15N/2H labelled complex of the human cardiac regulatory light chain with its heavy chain binding site (residues 806–837 of human cardiac myosin)
123
residues 806–837 cloned into a modified pET3a vector) with the regulatory light chain (human ventricular regulatory light chain (gene MYL3) cloned into modified pACYC9d vector) in the E. coli strain BL21* (Invitrogen) at a temperature of 18 °C over night. The complex was purified using a 5 mL HisTrap HP nickel-affinity column (GE Lifesciences) via a His6-tag on the heavy chain fragment, while the light chain was untagged. The His-tag was removed by digestion with TEV protease, upon which the His-tag, TEV protease and contaminating proteins were removed by a second round of nickel-affinity purification, leading to a protein complex with a purity [95 % and a yield of 15–20 mg/L LB, 2–3 mg/L M9 [using an improved M9 medium (Marley et al. 2001)] and about 2 mg/L M9 in D2O. NMR samples were used at concentrations from 100 to 250 lM in a buffer of 25 mM HEPES pH 7.0, 50 mM glutamic acid, 50 mM arginine, 1 mM DTT, 0.02 % NaN3 and a temperature of 298 K. Experiments for assignment were initially recorded on 15 N labelled (15N 3D NOESY and TOCSY) and 15N/13C labelled samples (HNCA, HNCO, HNCACB, HNCOCACB). However, despite a nominally modest molecular weight of 23 kDa, the triple resonance experiments came out poorly so that they had to be recorded using a triple labelled 2H/15N/13C sample. Side chain assignments were obtained from a 13C NOESY-HSQC. The limited solubility (aggregation inevitably occurred for samples [300 lM within a few hours at room temperature) suggests
Solution NMR assignment of the heavy chain complex
unspecific aggregation/low affinity self association as the most likely cause for the poor performance of the triple resonance experiments with non-deuterated samples. The limit in sample concentration significantly reduced the potentially achievable S/N which was then further reduced by the enhanced relaxation due to aggregation.
Assignments and data deposition The assignment of 1H, 15N and 13C resonances is 96 % complete for the backbone and 93 % for side chains. Most importantly, apart from residue 1 the complete N-terminus of the RLC that is missing in numerous crystal structures of regulatory light chains is seen in the NMR spectra. For a few amino acids, mainly in the light chain, we noticed the existence of a low populated (*15 %) conformer. Where possible, backbone assignments for these residues have been added to the database entry. A good impression of the overall extent of the assignment is given in the TROSY experiment in Fig. 1, which shows the good quality of the spectrum and how far it is covered by the assignment. The complete assignment has been submitted to the BioMagResBank database, accession number 19518. Acknowledgments This work was funded by a project grant from the British Heart Foundation (PG/10/65/28521) to MG and MP. NMR spectra were recorded in the Centre for Biomolecular Spectroscopy at King’s College London, at the National Institute of Medical Research and the Henry Wellcome Building NMR facility at the University of Birmingham and we would like to thank Andrew Atkinson, Geoff Kelly, Tom Frankiel, Alain Oregioni and Sara Whittaker for their help with the recording of the spectra.
References Arimura T, Suematsu N, Zhou YB et al (2001) Identification, characterization, and functional analysis of heart-specific myosin light chain phosphatase small subunit. J Biol Chem 276:6073–6082. doi:10.1074/jbc.M008566200 Colson BA, Locher MR, Bekyarova T et al (2010) Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development. J Physiol 588:981–993. doi:10.1113/jphysiol.2009.183897 Davis JS, Hassanzadeh S, Winitsky S et al (2001) The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation. Cell 107:631–641 Davis JS, Hassanzadeh S, Winitsky S et al (2002) A gradient of myosin regulatory light-chain phosphorylation across the ventricular wall supports cardiac torsion. Cold Spring Harb Symp Quant Biol 67:345–352
Greenberg MJ, Mealy TR, Watt JD et al (2009) The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation. Am J Physiol Regul Integr Comp Physiol 297:R265–R274. doi:10.1152/ajpregu.00171.2009 Greenberg MJ, Kazmierczak K, Szczesna-Cordary D, Moore JR (2010) Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry. Proc Nat Acad Sci USA 107:17403–17408. doi:10.1073/pnas.1009619107 Hong F, Haldeman BD, Jackson D et al (2011) Biochemistry of smooth muscle myosin light chain kinase. Arch Biochem Biophys 510:135–146. doi:10.1016/j.abb.2011.04.018 Huang J, Shelton JM, Richardson JA et al (2008) Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy. J Biol Chem 283:19748–19756. doi:10.1074/jbc.M802605200 Ishikawa Y, Kurotani R (2008) Cardiac myosin light chain kinase: a new player in the regulation of myosin light chain in the heart. Circ Res 102:516–518. doi:10.1161/CIRCRESAHA.108.173005 Kamm KE, Stull JT (2011) Signaling to myosin regulatory light chain in sarcomeres. J Biol Chem 286:9941–9947. doi:10.1074/jbc. R110.198697 Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75 Miller MS, Farman GP, Braddock JM et al (2011) Regulatory light chain phosphorylation and N-terminal extension increase crossbridge binding and power output in Drosophila at in vivo myofilament lattice spacing. Biophys J 100:1737–1746. doi:10. 1016/j.bpj.2011.02.028 Ratti J, Rostkova E, Gautel M, Pfuhl M (2011) Structure and interactions of myosin-binding protein C domain C0: cardiacspecific regulation of myosin at its neck? J Biol Chem 286:12650–12658. doi:10.1074/jbc.M110.156646 Rayment I, Rypniewski WR, Schmidt-Base K et al (1993) Threedimensional structure of myosin subfragment-1: a molecular motor. Science 261:50–58 Schlossarek S, Mearini G, Carrier L (2011) Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: mechanisms and therapeutic opportunities. J Mol Cell Cardiol 50:613–620. doi:10.1016/j.yjmcc.2011.01.014 Scruggs SB, Solaro RJ (2011) The significance of regulatory light chain phosphorylation in cardiac physiology. Arch Biochem Biophys 510:129–134. doi:10.1016/j.abb.2011.02.013 Shichi D, Arimura T, Ishikawa T, Kimura A (2010) Heart-specific small subunit of myosin light chain phosphatase activates rhoassociated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J Biol Chem 285:33680–33690. doi:10.1074/jbc.M110.122390 Stull JT, Kamm KE, Vandenboom R (2011) Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle. Arch Biochem Biophys 510:120–128. doi:10. 1016/j.abb.2011.01.017 Szczesna-Cordary D, Guzman G, Ng SS, Zhao J (2004) Familial hypertrophic cardiomyopathy-linked alterations in Ca2? binding of human cardiac myosin regulatory light chain affect cardiac muscle contraction. J Biol Chem 279:3535–3542 Vandenboom R, Metzger JM (2002) A ‘‘wringing’’ endorsement for myosin phosphorylation in the heart. Mol Interv 2:422–424. doi:10.1124/mi.2.7.422
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