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• W2016296503 abstract "Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction. Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction. Muscle contraction occurs as the result of many sarcomeric proteins interacting with one another in a precise manner. The main interacting system is formed by myosin and actin, with proteins such as troponin and tropomyosin being essential for regulation. Other proteins have in recent years become established as additional regulatory proteins in muscle contraction, one of them being myosin-binding protein C (MyBP-C) 4The abbreviations used are: MyBP-C, myosin-binding protein C; MyBS, binding site on myosin for the regulatory light chain; RLC, regulatory light chain; ELC, essential light chain; DSC, differential scanning calorimetry; ITC, isothermal titration calorimetry; HCM, hypertrophic cardiomyopathy; r.m.s.d., root mean square displacement; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum correlation. (1Oakley C.E. Hambly B.D. Curmi P.M. Brown L.J. Cell Res. 2004; 14: 95-110Crossref PubMed Scopus (48) Google Scholar, 2Winegrad S. Circ. Res. 2000; 86: 6-7Crossref PubMed Scopus (42) Google Scholar, 3Winegrad S. Adv. Exp. Med. Biol. 2003; 538: 31-41Crossref PubMed Google Scholar), a multidomain protein formed of immunoglobulin I (IgI) and fibronectin type III domains, similarly to titin (Fig. 1). Three isoforms exist of MyBP-C, for fast skeletal, slow skeletal, and cardiac muscle (4Bennett P.M. Fürst D.O. Gautel M. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 203-234Crossref PubMed Google Scholar). The cardiac form presents some unique features such as the long insertion in the CD loop of domain C5 (5Gautel M. Zuffardi O. Freiburg A. Labeit S. EMBO J. 1995; 14: 1952-1960Crossref PubMed Scopus (330) Google Scholar, 6Idowu S.M. Gautel M. Perkins S.J. Pfuhl M. J. Mol. Biol. 2003; 329: 745-761Crossref PubMed Scopus (34) Google Scholar), the insertion of two extra phosphorylation sites in the MyBP-C motif, located at the N terminus between domains C1 and C2, and an extra domain at its N terminus (C0) (5Gautel M. Zuffardi O. Freiburg A. Labeit S. EMBO J. 1995; 14: 1952-1960Crossref PubMed Scopus (330) Google Scholar). MyBP-C was suggested to function as a tether that holds on to myosin heads close to the S1-S2 neck region through its N terminus (7Weisberg A. Winegrad S. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 8999-9003Crossref PubMed Scopus (149) Google Scholar, 8McClellan G. Kulikovskaya I. Winegrad S. Biophys. J. 2001; 81: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9Levine R. Weisberg A. Kulikovskaya I. McClellan G. Winegrad S. Biophys. J. 2001; 81: 1070-1082Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Gruen M. Prinz H. Gautel M. FEBS Lett. 1999; 453: 254-259Crossref PubMed Scopus (161) Google Scholar) in a phosphorylation-regulated manner involving the MyBP-C motif (10Gruen M. Prinz H. Gautel M. FEBS Lett. 1999; 453: 254-259Crossref PubMed Scopus (161) Google Scholar, 11Kunst G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circ. Res. 2000; 86: 51-58Crossref PubMed Scopus (181) Google Scholar). The observation that short N-terminal fragments of MyBP-C, too short to perform a tethering role, are also able to influence S1 activity hinted at an additional, more direct way in which MyBP-C could regulate muscle contraction (11Kunst G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circ. Res. 2000; 86: 51-58Crossref PubMed Scopus (181) Google Scholar, 12Herron T.J. Rostkova E. Kunst G. Chaturvedi R. Gautel M. Kentish J.C. Circ. Res. 2006; 98: 1290-1298Crossref PubMed Scopus (76) Google Scholar, 13Harris S.P. Rostkova E. Gautel M. Moss R.L. Circ. Res. 2004; 95: 930-936Crossref PubMed Scopus (64) Google Scholar). Recently, we positioned the binding site for MyBP-C domain C1 right next to the S1-S2 hinge in immediate proximity to the regulatory light chain (14Ababou A. Rostkova E. Mistry S. Le Masurier C. Gautel M. Pfuhl M. J. Mol. Biol. 2008; 384: 615-630Crossref PubMed Scopus (74) Google Scholar). As domain C2 is located further C-terminal on S2 (15Ababou A. Gautel M. Pfuhl M. J. Biol. Chem. 2007; 282: 9204-9215Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), it would be plausible to look for an interaction site for the most N-terminal domain of MyBP-C further N-terminally on myosin. An interaction between the cardiac isoform of MyBP-C and the regulatory light chain (RLC) of myosin has been proposed as early as 1985 (16Margossian S.S. J. Biol. Chem. 1985; 260: 13747-13754Abstract Full Text PDF PubMed Google Scholar). Most likely, such an interaction might take place via the cardiac-specific domain C0. This would be entirely in agreement with results obtained with short, N-terminal MyBP-C fragments because these contained domain C0 (11Kunst G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circ. Res. 2000; 86: 51-58Crossref PubMed Scopus (181) Google Scholar, 12Herron T.J. Rostkova E. Kunst G. Chaturvedi R. Gautel M. Kentish J.C. Circ. Res. 2006; 98: 1290-1298Crossref PubMed Scopus (76) Google Scholar, 13Harris S.P. Rostkova E. Gautel M. Moss R.L. Circ. Res. 2004; 95: 930-936Crossref PubMed Scopus (64) Google Scholar). The assumption would be that these fragments somehow influence myosin activity via the RLC. The RLC is positioned in the neck region of myosin and, together with the essential light chain (ELC), stabilizes the 8.5-nm-long end of the lever helix by wrapping around it (17Rayment I. Rypniewski W.R. Schmidt-Bäse K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1877) Google Scholar, 18Xie X. Harrison D.H. Schlichting I. Sweet R.M. Kalabokis V.N. Szent-Györgyi A.G. Cohen C. Nature. 1994; 368: 306-312Crossref PubMed Scopus (266) Google Scholar) in the region spanning residues 808 and 842 (Fig. 1). The RLC seems to be of great importance for both myosin structure and function. Selective removal of the RLC causes a change in the structure of the cardiac myosin molecule (19Margossian S.S. Slayter H.S. J. Muscle Res. Cell. Motil. 1987; 8: 437-447Crossref PubMed Scopus (17) Google Scholar), leading to myosin disorder (9Levine R. Weisberg A. Kulikovskaya I. McClellan G. Winegrad S. Biophys. J. 2001; 81: 1070-1082Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and, importantly, weakens binding to MyBP-C (16Margossian S.S. J. Biol. Chem. 1985; 260: 13747-13754Abstract Full Text PDF PubMed Google Scholar). The RLC is also phosphorylated at its N terminus upon adrenergic stimulation (20Takashima S. Circ. J. 2009; 73: 208-213Crossref PubMed Scopus (70) Google Scholar), although the relevance of this in cardiac muscle, in contrast to e.g. smooth muscle, is not completely understood (21Scruggs S.B. Hinken A.C. Thawornkaiwong A. Robbins J. Walker L.A. de Tombe P.P. Geenen D.L. Buttrick P.M. Solaro R.J. J. Biol. Chem. 2009; 284: 5097-5106Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 22Davis J.S. Hassanzadeh S. Winitsky S. Lin H. Satorius C. Vemuri R. Aletras A.H. Wen H. Epstein N.D. Cell. 2001; 107: 631-641Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 23Colson B.A. Locher M.R. Bekyarova T. Patel J.R. Fitzsimons D.P. Irving T.C. Moss R.L. J. Physiol. 2010; 588: 981-993Crossref PubMed Scopus (124) Google Scholar). The regulatory light chain is a member of the superfamily of EF-hand Ca2+-binding protein and is also linked to hypertrophic cardiomyopathy (HCM), with currently seven known mutations that are linked to hereditary cardiac disease (UniProtKB/Swiss-Prot accession number P10916). Several different genes exist for RLC encoding numerous isoforms, among others, for cardiac and skeletal muscle, similar to MyBP-C. In the present work, our focus has been determining the structure of the cardiac-specific domain C0 and studying its interaction with the S1 fragment of myosin and more precisely with the regulatory light chain that associates with it. MyBP-C and RLC, together with other sarcomeric proteins, are both linked to HCM, a genetic disorder leading to cardiac dysfunction that can manifest itself through arrhythmias, heart failure, and sudden cardiac death, especially in the young. Domain C0 shows three missense mutations linked to HCM in human (Sarcomere Protein Gene Mutation Database and UniProtKB/Swiss-Prot accession number Q14896) and one in Maine Coon cats (24Meurs K.M. Sanchez X. David R.M. Bowles N.E. Towbin J.A. Reiser P.J. Kittleson J.A. Munro M.J. Dryburgh K. Macdonald K.A. Kittleson M.D. Hum. Mol. Genet. 2005; 14: 3587-3593Crossref PubMed Scopus (183) Google Scholar), making it one of the domains presenting the fewest HCM mutations, surprisingly given its unique cardiac nature. In this work, we study the structure and dynamics of domain C0 and show that it binds to the RLC. We map the binding site and show the effect upon the interaction by some HCM-related mutations. Our biophysical results are backed up by immunofluorescence studies showing C0 co-localizing by itself in the A-band, consistent with an interaction around the myosin heads. Domain C0 (residues 1–95 of human cardiac MyBP-C) was cloned in vector pET8c and expressed and purified as described previously for domain C5 (6Idowu S.M. Gautel M. Perkins S.J. Pfuhl M. J. Mol. Biol. 2003; 329: 745-761Crossref PubMed Scopus (34) Google Scholar). Isotope-enriched samples were produced by expression in M9 minimal medium supplemented with uniformly 13C-enriched glucose (CIL) and/or [15N]ammonium chloride (CIL) as unique sources of carbon and nitrogen, respectively. The four mutants produced for the mutagenesis studies were cloned in vector pLEICS-03 (available from the Protex laboratory at the University of Leicester) and expressed in LB medium as wild type. The miniHMM heavy chain construct contains residues 806–963 of the cardiac β-myosin heavy chain (NM_000257). The fragment contains the RLC binding site (residues 806–835) and the N-terminal part of the coiled-coil myosin rod, S2Δ (848–963) (10Gruen M. Prinz H. Gautel M. FEBS Lett. 1999; 453: 254-259Crossref PubMed Scopus (161) Google Scholar). The myosin fragment was cloned into a modified pET8c vector with ampicillin resistance containing a His6 tag and tobacco etch virus protease cleavage site. This fragment was impossible to express on its own, probably due to its instability caused by the very likely unfolded RLC binding site. In contrast, the S2Δ fragment alone was easily expressed in Escherichia coli with a high yield as described previously (25Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (201) Google Scholar). To stabilize the RLC binding site, we co-expressed the miniHMM myosin heavy chain fragment with cardiac RLC (GI_4557774) cloned into the pACYC vector with kanamycin resistance. The two plasmids were co-transformed into BL21 [DE3] RIL-competent cells and grown on plates and media containing both antibiotics. Protein was purified using polyhistidine binding nickel chelate resin (HiTrap) by standard procedures. The purified protein was cleaved by tobacco etch virus protease, and tag, uncleaved protein, and tobacco etch virus protease were removed by one pass over a nickel HiTrap. The same procedure was used for cloning and expression of the RLC binding site cloned in pET8c and co-expressed with the RLC. Different samples were prepared to perform the NMR experiments: [13C/15N C0] = 1.4 mm, [15N C0] = 670 μm, [C0Ar] = 1.7 mm in 50 mm phosphate buffer at pH 7 containing 50 mm NaCl, 2 mm DTT, and 0.01% NaN3. The first, doubly labeled sample was used to record the triple resonance experiments, HNCaCb, HN(CO)CaCb, HN(Ca)CO, HNCO, HN(CaCb)HaHb, HN(CaCbCO)HaHb (26Clore G.M. Gronenborn A.M. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 29-63Crossref PubMed Scopus (130) Google Scholar, 27Grzesiek S. Bax A. J. Am. Chem. Soc. 1992; 114: 6291-6293Crossref Scopus (931) Google Scholar, 28Wang A.C. Lodi P.J. Qin J. Vuister G.W. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1994; 105: 196-198Crossref Scopus (43) Google Scholar), and the 13C-specific experiments such as 1H/13C HSQC, 1H/13C HCCH TOCSY (29Bax A. Clore M. Driscoll P.C. Gronenborn A.M. Ikura M. Kay L.E. J. Magn. Reson. 1990; 87: 620Google Scholar), and 1H/13C NOESY-HSQC (30Majumdar A. Zuiderweg E.P. J. Magn. Reson. 1993; 102: 242Crossref Scopus (54) Google Scholar); from the 15N-enriched sample were obtained 1H/15N HSQC, 1H/15N TOCSY-HSQC, and 1H/15N NOESY-HSQC. An unlabeled sample was used to record the spectra relative to the aromatic side chains 1H/1H TOCSY and 1H/1H nuclear Overhauser enhancement spectroscopy (NOESY). The NMR samples were concentrated in Vivaspin 20 concentrators with 3-kDa molecular mass cutoff (Sartorius) and transferred to a clean NMR tube (Shigemi). The 13C/15N C0 and C0Ar samples were frozen and lyophilized overnight to eliminate the water and then resuspended in high purity D2O (Sigma). All NMR spectra were obtained using in-house modified pulse sequences based on the standard pulse sequences provided by Bruker. They were collected on Bruker Avance spectrometers at 600 MHz, with and without cryoprobe, or 800 MHz with cryoprobe at 303 K. NMR spectra were processed with Topspin and analyzed with CCPNMR Analysis software. Sequence-specific assignments were deposited in the Biological Magnetic Resonance Bank (BMRB) with accession code 5679. Relaxation analysis was performed by measuring 15N R1, R2, and 1H-15N heteronuclear NOE experiments (31Barbato G. Ikura M. Kay L.E. Pastor R.W. Bax A. Biochemistry. 1992; 31: 5269-5278Crossref PubMed Scopus (893) Google Scholar) on a 15N-labeled sample. R1 was measured with delays of 16, 48, 96, 192, 288, 384, 512, 704, 880, 1120, and 1440 ms; R2 was measured with delays of 5, 10, 15, 20, 31, 41, 61, 82, 102, 133, 154 ms. The heteronuclear NOEs were measured with a proton saturation period of 3 s. Relaxation rates were extracted from the time series by exponential fit using customized macros in the program Mathematica (Wolfram Research). Relaxation rates were initially used to obtain the rotational correlation time from R1/R2. using the value of τc obtained, and a Lipari-Szabo analysis (32Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3409) Google Scholar, 33Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4559-4570Crossref Scopus (1877) Google Scholar, 34Clore G.M. Szabo A. Bax A. Kay L.E. Driscoll P.C. Gronenborn A.M. J. Am. Chem. Soc. 1990; 112: 4989-4991Crossref Scopus (972) Google Scholar) was performed for all individual residues. Interaction studies were carried out by measuring 1H-15N HSQC spectra (35Gronenborn A.M. Bax A. Wingfield P.T. Clore G.M. FEBS Lett. 1989; 243: 93-98Crossref PubMed Scopus (181) Google Scholar) of 50 μm 15N-labeled C0 without and with binding partner, RLC, RLC-MyBS, or miniHMM at a concentration of 200 μm. The experiment was repeated for G5R, R35W, and K87E mutants of C0 under identical conditions. Spectra were recorded at 800 MHz and a temperature of 298 K using standard NMR buffer. For analysis, 1H and 15N chemical shift perturbations were combined according to Δδ = |Δδ(1H)| + 0.15 × |Δδ(15N)| (36Mulder F.A. Schipper D. Bott R. Boelens R. J. Mol. Biol. 1999; 292: 111-123Crossref PubMed Scopus (224) Google Scholar) and plotted against the sequence. The weighting used in this study is different from that used in previous work on interactions of domains of MyBP-C (14Ababou A. Rostkova E. Mistry S. Le Masurier C. Gautel M. Pfuhl M. J. Mol. Biol. 2008; 384: 615-630Crossref PubMed Scopus (74) Google Scholar, 15Ababou A. Gautel M. Pfuhl M. J. Biol. Chem. 2007; 282: 9204-9215Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) so that the chemical shift perturbations appear relatively smaller. The automatic assignment of the peaks picked manually in the three-dimensional 1H/13C NOESY HSQC experiment was carried out using the CANDID protocol (37Herrmann T. Güntert P. Wüthrich K. J. Biomol. NMR. 2002; 24: 171-189Crossref PubMed Scopus (430) Google Scholar), as part of the software CYANA 2.1 (38Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2558) Google Scholar). Out of the total 2460 peaks, 88% were assigned (2170), whereas just 292 (12%) were left unassigned at the end of the procedure; all these unassigned peaks were the product of artifacts in the NMR experiments and were all checked manually at the end of the automatic assignment procedure. The final family of structures for domain C0 was obtained using 1360 structural constraints derived from experimental NMR data, including 441 sequential (i, i + 1), 85 medium range (i, i ≤ 4), and 628 long range (i, i ≥ 5) upper distance limits, 180 backbone torsion angle constraints (90 ϕ and 90 ψ) produced by the program TALOS (39Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar), and 26 hydrogen bond constraints found in regions with defined secondary structure. Following the final round of CYANA calculation, 81 converged structures were obtained from 100 random starting structures. The converged structures contain no distance or van der Waals violation grater than 0.5 Å and no dihedral angle violations grater than 5° (Table 1).TABLE 1Structural statistics for domain C0Input constraintsDihedral angle constraints180NOE-derived distances1142Hydrogen bond constraints26Structure statisticsBackbone r.m.s.d.0.4 ÅHeavy atom r.m.s.d.Residues in core region of Ramachandran plot83.70%Residues in allowed region of Ramachandran plot100.00%Average/maximal violation of NOE constraints0.013/0.2 ÅAverage/maximal violation of dihedral constraints0.223/4° Open table in a new tab Investigation of thermally induced protein denaturation was performed using a VP-DSC differential scanning calorimeter (MicroCal). Protein samples were in 25 mm MES, pH 7.0, 100 mm NaCl, and 10 mm β-mercaptoethanol and were heated from 10 to 80 °C with constant rate 1 °C/min. Protein concentrations were 80 μm for C0 and C1, 65 μm for S2Δ, 70 μm for RLC, and 65 μm for miniHMM (summarized molecular weights of extended S2Δ and RLC were used for calculation of molar concentration of miniHMM). To obtain information on the reversibility of thermally induced denaturation, samples were heated at the same rate immediately after cooling down after the first denaturation. As some of investigated proteins were unfolding reversibly, we could not use traces obtained from the second denaturation to correct for instrument background. In our experiments, calorimetric traces were therefore corrected by subtracting the scans of the buffer in both cells of the calorimeter. Data were analyzed using Origin 7 software (OriginLab Corp.). Cardiac MyBP-C C0 was cloned into an HA-tagged mammalian expression vector as described previously (40Lange S. Himmel M. Auerbach D. Agarkova I. Hayess K. Fürst D.O. Perriard J.C. Ehler E. J. Mol. Biol. 2005; 345: 289-298Crossref PubMed Scopus (62) Google Scholar). The constructs were transfected into neonatal rat ventricular myocytes using standard procedures, and cells were maintained as described (41Auerbach D. Bantle S. Keller S. Hinderling V. Leu M. Ehler E. Perriard J.C. Mol. Biol. Cell. 1999; 10: 1297-1308Crossref PubMed Scopus (63) Google Scholar). The transfected cells were fixed with 4% paraformaldehyde after 2–3 days in culture, and stained with anti-HA tag antibody (clone 3F10, Roche Applied Science), anti-myosin heavy chain (clone A4.1025, a gift from S. Hughes), and anti α-actinin (Sigma) or Alexa Fluor 633 phalloidin (Molecular Probes) for visualization of F-actin. The specimens were analyzed by confocal microscopy using a Zeiss LSM-510 Meta microscope under 63× magnification and 2–5× zoom. The molecular structure of domain C0 was determined using three-dimensional 15N- and 13C-resolved NOESY spectra supplemented by dihedral restraints obtained from chemical shift analysis and hydrogen bond constraints. The NMR data are available from the Biological Magnetic Resonance Bank (accession code 5679), whereas the atomic coordinates were deposited in the Protein Data Bank (2K1M). The good quality of the structure is shown by the high degree of agreement between the 20 structures shown in Fig. 2A, which shows 20 of the 81 converged structures as result of the structure calculation carried out with the program CYANA. The average backbone r.m.s.d. is 0.4 Å for the structured portion with the only exception of the N terminus of the domain, which also corresponds to the N terminus of the whole cardiac isoform of MyBP-C, which is completely unstructured (Figs. 2A and Fig. 3).FIGURE 3Dynamics of domain C0. Order parameters S2 from the Lipari-Szabo analysis of 15N relaxation data are shown in the top panel, and average r.m.s.d. values from the top 20 structures in the ensemble are shown in the bottom panel. Both are plotted against the sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm the highly disordered nature of the N terminus, the dynamic properties of C0 were studied by 15N relaxation experiments at T = 298 K. The relaxation results were analyzed using the Lipari-Szabo approach (32Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4546-4559Crossref Scopus (3409) Google Scholar, 33Lipari G. Szabo A. J. Am. Chem. Soc. 1982; 104: 4559-4570Crossref Scopus (1877) Google Scholar). The rotation correlation time (τc) for the domain was determined based on the ratios of the two relaxation times T1 and T2, giving a value of 5.81 ± 0.16 ns, in good agreement with the expected value of about 5 ns for a protein of 10 kDa, according to a simple calculation using the Stokes-Einstein equation. The main result of the Lipari-Szabo analysis is the information about the local rigidity of the molecule, expressed by the order parameter S2 values determined for each residue (Fig. 3). The plot of S2 against residue number shows a uniformly rigid structure with S2 values of 0.8–0.9, typical for a well structured protein. In contrast, the N terminus is unstructured with S2 values dropping to around 0.2 for the first 7–8 amino acids, in agreement with the r.m.s.d. values (Fig. 3), which are significantly increased for residues 1–9 when compared with the rest of the protein. Based on the results obtained for domains C1 and C2 (14Ababou A. Rostkova E. Mistry S. Le Masurier C. Gautel M. Pfuhl M. J. Mol. Biol. 2008; 384: 615-630Crossref PubMed Scopus (74) Google Scholar, 15Ababou A. Gautel M. Pfuhl M. J. Biol. Chem. 2007; 282: 9204-9215Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and in accordance with previous suggestions (16Margossian S.S. J. Biol. Chem. 1985; 260: 13747-13754Abstract Full Text PDF PubMed Google Scholar), it was hypothesized that domain C0 could interact, among others, with the RLC. The interaction was investigated in vitro using differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and NMR spectroscopy, using both RLC bound to its myosin binding site as well as miniHMM and S2Δ as a negative control (Fig. 1). DSC was used to analyze thermally induced denaturation of domain C0, fragments of myosin, and their complexes (as control, domain C1 was also investigated in exactly the same way; supplemental Fig. S1). Thermal denaturation curves of C0 with miniHMM and S2Δ are shown in Fig. 4, A and B, respectively. In each, the denaturation of C0 alone (red), the myosin fragment alone (green), and their mixture (black) are superimposed. Denaturation of C0 alone is well reproducible with a single well defined peak at a temperature of 57 °C, almost completely reversible (supplemental Fig. S1). Thermal denaturation of miniHMM in Fig. 4A shows a sharp peak with a maximum at 50 °C and a broad, less thermostable shoulder with a maximum approximately at 38 °C. The denaturation of S2Δ in Fig. 4B has only a single peak around 34 °C, suggesting that the low temperature transition belongs to the coiled-coil portion, whereas the higher temperature transition corresponds to the RLC bound to the myosin binding site. The small increase in melting temperature from S2Δ to miniHMM is likely to be caused by the interaction of the myosin fragment on the RLC. Unfolding of the coiled-coil domain was partially reversible (supplemental Fig. S1) in good agreement with our own data (42Holtzer M.E. Holtzer A. Biopolymers. 1990; 30: 985-993Crossref PubMed Scopus (16) Google Scholar, 43Levitsky D.I. Rostkova E.V. Orlov V.N. Nikolaeva O.P. Moiseeva L.N. Teplova M.V. Gusev N.B. Eur. J. Biochem. 2000; 267: 1869-1877Crossref PubMed Scopus (35) Google Scholar). The experimental denaturation curves of the mixtures of C0 with miniHMM (Fig. 4A) and S2Δ (Fig. 4B) are markedly different; in the mixture with S2Δ, the denaturation curve is virtually identical to the addition of the two individual curves. The high temperature denaturation peak, corresponding to C0, is slightly shifted by 2 °C to a lower melting temperature, possibly indicating a weak, unspecific interaction. The DSC profile of the denaturation of the mixture of miniHMM and C0 is more complex. At 51.5 °C, a sharp exothermic drop brings the curve to baseline, and then it returns back and forms a shoulder of the main peak. Such an exothermic peak is a very rare effect and normally reflects either fast aggregation (43Levitsky D.I. Rostkova E.V. Orlov V.N. Nikolaeva O.P. Moiseeva L.N. Teplova M.V. Gusev N.B. Eur. J. Biochem. 2000; 267: 1869-1877Crossref PubMed Scopus (35) Google Scholar, 44Merabet E.K. Walker M.C. Yuen H.K. Sikorski J.A. Biochim. Biophys. Acta. 1993; 1161: 272-278Crossref PubMed Scopus (20) Google Scholar, 45Orlov V.N. Rostkova E.V. Nikolaeva O.P. Drachev V.A. Gusev N.B. Levitsky D.I. FEBS Lett. 1998; 433: 241-244Crossref PubMed Scopus (9) Google Scholar) of proteins in the calorimeter cell or significant changes in protein structure, which occur with release of energy such as chain exchange (45Orlov V.N. Rostkova E.V. Nikolaeva O.P. Drachev V.A. Gusev N.B. Levitsky D.I. FEBS Lett. 1998; 433: 241-244Crossref PubMed Scopus (9) Google Scholar). In our experiments, the appearance of this exothermic peak cannot be explained by aggregation because after the peak, in the temperature region between 60 and 80 °C, the experimental curve follows the baseline and does not have any noise. Furthermore, the sample extracted from the cell after heating did not show any discernible traces of aggregation. This extremely unusual behavior was reproduced in a mixture of C0 with RLC-MyBS (data not shown), suggesting that it is indeed the interaction of C0 with the RLC that causes such an unusual thermodynamic signature. Furthermore, control experiments of miniHMM or RLC-MyBS with C1 did not show any sign of interaction let alone such an unusual exothermic event (data not shown). The same mixtures investigated by DSC were also studied by ITC, and the resulting titration curves are shown in Fig. 4C for C0 + miniHMM and in Fig. 4D for C0 + S2Δ. A very clear binding curve with saturation at an excess of C0 over miniHMM of around 1.5–2.0 confirms an interaction. Fitting the binding curve in Fig. 4C yields a dissociation constant of 3.2 ± 1.7 μm and a stoichiometry of C0:miniHMM of 1:0.81 ± 0.16. In contrast, as shown in Fig. 4D, no interaction can be detected between domain C0 and S2Δ. Initial interaction studies were performed with purified RLC alone by re" @default.
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• W2016296503 title "Structure and Interactions of Myosin-binding Protein C Domain C0" @default.
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