Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5
2008; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês
10.1074/jbc.m807747200
ISSN1083-351X
AutoresBenjamin Chagot, F Potet, Jeffrey R. Balser, Walter Chazin,
Tópico(s)Electrochemical Analysis and Applications
ResumoThe voltage-gated sodium channel NaV1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of NaV1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of NaV1.5. The voltage-gated sodium channel NaV1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of NaV1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of NaV1.5. The cardiac voltage-gated sodium channel NaV1.5 mediates the voltage-dependent sodium ion permeability of excitable membranes. NaV1.5 is responsible for the initial upstroke of the action potential in the electrocardiogram. Assuming opened or closed conformations in response to the voltage difference across the membrane, the protein allows Na+ ions to pass in accordance with their electrochemical gradient. Upon repeated stimulation, channels convert to the third state known as the inactive state. Channels must pass from the inactive to the closed state before they can be opened again. The structure of the channel is dominated by four membrane spanning domains (DI, DII, DIII, DIV), each containing six transmembrane helices linked by intracellular loops. The loop between DIII and DIV is of particular interest here because it is involved in the fast-inactivation of the channel (1Yu F.H. Catterall W.A. Genome Biology.http://genomebiology.com/2003/4/3/207Date: 2003Google Scholar). There are also substantial N- and the C-terminal domains, both located on the cytoplasmic side of the membrane. Several diseases are linked to the dysfunction of NaV1.5, such as long QT syndrome, Brugada syndrome, and idiopathic ventricular fibrillation (2Abriel H. Cabo C. Wehrens X.H. Rivolta I. Motoike H.K. Memmi M. Napolitano C. Priori S.G. Kass R.S. Circ. Res. 2001; 88: 740-745Crossref PubMed Scopus (108) Google Scholar, 3Wehrens X.H. Abriel H. Cabo C. Benhorin J. Kass R.S. Circulation. 2000; 102: 584-590Crossref PubMed Scopus (77) Google Scholar, 4Priori S.G. Napolitano C. Gasparini M. Pappone C. Della Bella P. Giordano U. Bloise R. Giustetto C. De Nardis R. Grillo M. Ronchetti E. Faggiano G. Nastoli J. Circulation. 2002; 105: 1342-1347Crossref PubMed Scopus (892) Google Scholar, 5Vatta M. Dumaine R. Antzelevitch C. Brugada R. Li H. Bowles N.E. Nademanee K. Brugada J. Brugada P. Towbin J.A. Mol. Genet. Metab. 2002; 75: 317-324Crossref PubMed Scopus (51) Google Scholar, 6Chen Q. Kirsch G.E. Zhang D. Brugada R. Brugada J. Brugada P. Potenza D. Moya A. Borggrefe M. Breithardt G. Ortiz-Lopez R. Wang Z. Antzelevitch C. O'Brien R.E. Schulze-Bahr E. Keating M.T. Towbin J.A. Wang Q. Nature. 1998; 392: 293-296Crossref PubMed Scopus (1553) Google Scholar), and some of these dysfunctions are due to mutations located in the C-terminal domain (7An R.H. Wang X.L. Kerem B. Benhorin J. Medina A. Goldmit M. Kass R.S. Circ. Res. 1998; 83: 141-146Crossref PubMed Scopus (170) Google Scholar, 8Smits J.P. Eckardt L. Probst V. Bezzina C.R. Schott J.J. Remme C.A. Haverkamp W. Breithardt G. Escande D. Schulze-Bahr E. LeMarec H. Wilde A.A. J. Am. Coll. Cardiol. 2002; 40: 350-356Crossref PubMed Scopus (329) Google Scholar, 9Petitprez S. Jespersen T. Pruvot E. Keller D.I. Corbaz C. Schlapfer J. Abriel H. Kucera J.P. Cardiovasc. Res. 2008; 78: 494-504Crossref PubMed Scopus (34) Google Scholar). Substantial evidence has accumulated showing that inactivation gating of NaV1.5 is modulated in response to changes in the level of calcium (Ca2+) in the adjacent cytosol. Ca2+-dependent effects on NaV1.5 inactivation gating have been shown to play an important role in the progression of certain arrhythmia conditions. Therefore, elucidating the mechanism for the Ca2+-dependent modulation of channel function is an important objective for understanding the basis of the Brugada and Long QT syndromes and for the long range goal of finding suitable chemotherapeutic strategies to neutralize the damaging events caused by these cardiac defects. Studies from our laboratory and others have shown that three regions of NaV1.5 as well as the ubiquitous intracellular Ca2+ sensor calmodulin are involved in generating the Ca2+-dependent effects on channel function (10Kim J. Ghosh S. Liu H. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). In addition to the DIII-DIV loop noted above, the C-terminal domain contains two regions involved in Ca2+-dependent modulation of channel gating function. The first is an EF-hand domain, a structure closely associated with intracellular calcium sensing in a wide range of proteins including calmodulin. The second region of interest is an IQ motif. IQ motifs are calmodulin (CaM) 2The abbreviations used are: CaM, calmodulin; CTD, C-terminal domain; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BME, β-mercaptoethanol; MES, 4-morpholineethanesulfonic acid; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect (NOE) spectroscopy. binding domains most commonly associated with recruitment of CaM to specific sites so that it is able to respond rapidly to calcium signals. The fact that the NaV1.5 IQ motif has important functions for both the intrinsic sensing of Ca2+ by the EF-hand domain and the recruitment of CaM led us to propose a model in which the IQ motif serves as molecular switch that couples the two Ca2+-sensing modules (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). In the present study we report the solution structure of the C-terminal domain EF-hand domain (CTD-EF) in NaV1.5 determined by NMR spectroscopy. Analysis of this structure reveals important features relating to the function of this domain and provides essential groundwork for determining why functional Ca2+ binding requires an additional 60 residues downstream from the EF-hand domain (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). The implications of these results are described with respect to the mechanism of action of the complex calcium-sensing apparatus in NaV1.5. Expression and Purification-Recombinant human CTD-EF (Glu-1773-Ser-1865) was subcloned between BamHI and EcoRI restriction sites of an in-house pBG100 plasmid (Dr. L. Mizoue, Center for Structural Biology), which is derived from pET27. To facilitate purification, this vector codes for a His6 tag and a flexible linker containing a 3C protease cleavage site. The CTD-EF construct is expressed as a 97-residue protein that contains Gly-Pro-Gly-Ser fused to its N terminus after cleavage of the His6 tag. Proteins were expressed in Escherichia coli BL21 (DE3) cells (Novagen) cells and grown at 37 °C up to 0.6 A600, then isopropyl 1-thio-β-d-galactopyranoside (1 mm) was added, and the culture was grown at 18 °C overnight. Production of unlabeled and 13C,15N-enriched proteins were carried out by growth on lysogeny broth or minimal medium, respectively. The minimal medium was supplemented with 15NH4Cl and glucose or [13C6]glucose as the sole nitrogen and carbon sources. Cell pellets were resuspended in 50 mm BisTris at pH 6.5, 200 mm NaCl, 10 mm MgCl2, 5 mm β-mercaptoethanol (BME), and 10 mm imidazole and lysed using sonication. The solution was centrifuged at 20,000 × g for 20 min. Supernatant was filtered and loaded onto a nickel affinity chromatography column (Amersham Biosciences). Nonspecifically bound proteins were removed by washing the column with 50 mm BisTris at pH 6.5, 200 mm NaCl, 10 mm MgCl2, 5 mm BME, and 10 mm imidazole, and the bound protein was eluted using a gradient to 50 mm BisTris at pH 6.5, 200 mm NaCl, 10 mm MgCl2, 5 mm BME, and 500 mm imidazole buffer. Fractions containing CTD-EF were pooled, and rhinovirus His6-tagged 3C protease was added. This solution was dialyzed at 4 °C overnight against 50 mm Bis-Tris at pH 6.5, 200 mm NaCl, 5 mm BME. The solution was then loaded onto a nickel affinity chromatography column to separate CTD-EF and the His6 tag. The flow-through was concentrated on a 3K-Centricon device to ∼0.5 ml and run over an S75 gel filtration column (Amersham Biosciences). Peptide Synthesis-A 29-residue peptide (1897RRKHE EVSAM VIQRA FRRHL LQRSL KHAS1925) containing the IQ motif of NaV1.5 was synthesized (Sigma Genosys). Peptides were purified by reversed-phase high performance liquid chromatography and analyzed by mass spectrometry to confirm purity and molecular weight. Isothermal Titration Calorimetry-Isothermal titration calorimetry (ITC) measurements were carried out with a VP-ITC MicroCalorimeter (MicroCal, Inc., Northampton, MA). Titration experiments were performed in 50 mm MES buffer at pH 6.5, 200 mm NaCl, and either 1 mm CaCl2 or 1 mm EDTA. Experiments were performed at 25 °C. Both protein and peptide were dialyzed against the same buffer. Peptide (25-28 μm) in the calorimetric cell was titrated by a series of 10-μl volume injections of CTD-EF (500-560 μm) with an interval of 240 s. The binding isotherms, ΔH versus molar ratio, were analyzed with a single-site binding model using MicroCal Origin software. The association constant, Ka (Kd = 1/Ka) was obtained directly from the fit. NMR Sample Preparation-CTD-EF was dialyzed into 100 mm phosphate at pH 6.5, 200 mm NaCl, 5 mm BME, and 0.01% NaN3. The protein concentration was 1.2 mm for structure determination and 0.2 mm for titration studies. To minimize ionic strength effects on sensitivity, 300 μl of protein solution was loaded into a 4-mm NMR tube, and this tube was inserted into a 5-mm NMR tube containing 150 μl of D2O (12Voehler M.W. Collier G. Young J.K. Stone M.P. Germann M.W. J. Magn. Reson. 2006; 183: 102-109Crossref PubMed Scopus (74) Google Scholar). NMR Spectroscopy-All NMR data were recorded at 25 °C on Bruker DRX600 and DRX800 spectrometers. Data were processed with Topspin 2.0b (Bruker) and analyzed with Sparky (13Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco2006Google Scholar). Backbone and sequential resonance assignments were obtained by the combined use of two-dimensional 15N,1H HSQC and three-dimensional HNCA, HNCACB, CBCA-(CO)NH, and HNCO experiments (for review, see Ref. 14Cavanagh J. Fairbrother W.J. Palmer A.G.I. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press Inc., New York1996Google Scholar). Aliphatic side chain resonance assignments were obtained from three-dimensional (H)CC(CO)NH, H(CCCO)NH, HCCH-TOCSY, HCCH-COSY, and HBHANH experiments. 1H chemical shift assignments of aromatic side chains were primarily based on two-dimensional homonuclear COSY, TOCSY, and NOESY experiments. To assign NOE-based distance restraints, a two-dimensional homonuclear NOESY experiment was recorded on an unlabeled sample, and three-dimensional 15N NOESY-HSQC and 13C NOESY-HSQC were recorded on a uniformly 13C,15N-enriched sample. The mixing time used in all NOESY experiments was 120 ms. Heteronuclear 15N relaxation parameters (T1, T2, NOE) were measured at 500 MHz as described previously (15Farrow N.A. Zhang O. Forman-Kay J.D. Kay L.E. J. Biomol. NMR. 1994; 4: 727-734Crossref PubMed Scopus (389) Google Scholar). The relaxation delays were 10, 25, 50, 100, 150, 250, 400, 600, 900, and 1200 ms for the T1 measurements and 11, 23, 34, 68, 79, 114, 136, and 170 ms for the T2 measurements. Steady-state {1H}-15N NOE data were obtained with and without 3 s of 1H saturation using a total recycle delay of 7 s. NOE values were extracted as the ratio of peak intensities with and without proton saturation for 88 well resolved resonances. The rate analysis functions in Sparky were used to analyze the T1 and T2 decay rates, which were computed using the two-parameter model in CurveFit (16Palmer A.G. CurveFit. Columbia University, New York1998Google Scholar) for 84 and 80 well resolved resonances, respectively. The isotropic rotational correlation time (τm) was approximated using the trimmed T1/T2 ratio computed for only well resolved resonances in the well structured regions of the protein (17Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1809) Google Scholar). The binding site of the IQ motif peptide to CTD-EF was determined by recording 15N,1H HSQC NMR spectra of CTD-EF as the peptide was titrated into the solution. Spectra were acquired for protein:peptide ratios of 1:0, 1:0.25, 1:0.5, 1:1, and 1:2. The overall chemical shift change of the 1H,15N peak was expressed as Δδ = [(ΔδHN)2 + (ΔδN/6.5)2)]1/2 (18Mulder F.A. Schipper D. Bott R. Boelens R. J. Mol. Biol. 1999; 292: 111-123Crossref PubMed Scopus (225) Google Scholar). Structure Calculations-The first stage of calculations used CYANA (19Guntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1176) Google Scholar). The standard CYANA protocol of 7 iterative cycles of calculations was performed starting from a set of manually assigned NOEs. In each iteration 100 structures started from random torsion angle values were calculated with 10,000 steps of torsion angle dynamics-driven simulated annealing. 2131 NOE-based distance and 51 backbone φ angle restraints were used for the final calculations. The angle restraints were obtained from 1Hα, 13Cα, 13Cβ, 13C′, and 15N chemical shifts using PREDITOR (20Berjanskii M.V. Neal S. Wishart D.S. Nucleic Acids Res. 2006; 34: 63-69Crossref PubMed Scopus (165) Google Scholar) with a minimum range of ±40°. The 50 lowest CYANA target function conformers were selected for refinement by restrained molecular dynamics simulations in AMBER (21Case D.A. Cheatham III, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6819) Google Scholar) using the generalized Born solvent model. The starting structures were energy minimized for 100 steps, then 20 ps of restrained molecular dynamics was performed with the following protocol: 1 ps heating the system from 0 to 600 K followed by 4 ps at 600 K with a coupling parameter TAUTP for heating and equilibration of 0.4; 15 ps of cooling to 0 K with 8 ps of slow cooling (loose coupling, TAUTP = 4.0-2.0) followed by 2 ps of faster cooling (TAUTP = 1.0) and a final 2 ps of very fast cooling (TAUTP = 0.5-0.05). The restraints were slowly ramped from 10 to 100% that of their final values over the first 3 ps. The representative ensemble corresponds to the 20 conformers with the lowest restraint and AMBER energy terms. Docking of IQ Motif to CTD-EF-A model for the CTD-EF/IQ motif complex was generated using HADDOCK2 (22Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2249) Google Scholar). The unstructured N-terminal 10 residues of the CTD-EF construct were removed from the coordinate files so as to not interfere with docking of the peptide. The central region (Glu-1901-Leu-1921) of the IQ motif was modeled as a helix with the N- and C termini in a random coil conformation in accord with secondary structure prediction (23Rost B. Yachdav G. Liu J. Nucleic Acids Res. 2004; 32: 321-326Crossref PubMed Scopus (1185) Google Scholar) and available structures of IQ motif complexes (24Fallon J.L. Halling D.B. Hamilton S.L. Quiocho F.A. Structure. 2005; 13: 1881-1886Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar,25Van Petegem F. Chatelain F.C. Minor Jr., D.L. Nat. Struct. Mol. Biol. 2005; 12: 1108-1115Crossref PubMed Scopus (205) Google Scholar). The average solvent accessibilities per residue in the ensemble of CTD-EF conformers were calculated using NACCESS (26Hubbard S. Thornton J. NACCESS. University of Manchester, U.K.1992-96Google Scholar). Residues with a solvent accessible surface higher than 40% and chemical shift perturbations more than 1 S.D. above the mean were designated as active for the HADDOCK calculations. The adjacent residues with solvent accessibility greater than 50% were designated as passive residues. In the first iteration of the calculation, an initial ensemble of 5000 rigid body docking models was generated. The 200 lowest energy models were selected for a second iteration with semi-flexible simulated annealing and were further refined in explicit water. The 10 lowest-energy models were selected from the most populated cluster with the lowest HAD-DOCK score and used for structural analysis. Computational Analysis-Multiple sequence alignments were performed with T-Coffee (27Notredame C. Higgins D.G. Heringa J. J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5545) Google Scholar) and MUSCLE (28Edgar R.C. Nucleic Acids Res. 2004; 32: 1792-1797Crossref PubMed Scopus (31483) Google Scholar) through the phylogeny web server (29Dereeper A. Guignon V. Blanc G. Audic S. Buffet S. Chevenet F. Dufayard J.F. Guindon S. Lefort V. Lescot M. Claverie J.M. Gascuel O. Nucleic Acids Res. 2008; 36: 465-469Crossref PubMed Scopus (3579) Google Scholar). Graphical analyses of the structures and figure preparation were carried out with the programs MOLMOL (30Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (and 29-32): 51-55Crossref PubMed Scopus (6503) Google Scholar) and PyMOL (31DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar). The geometric quality and stereochemistry of the CTD-EF structural ensemble was assessed using PROCHECK-NMR (32Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4507) Google Scholar). Electrostatic surface potentials were calculated with the Adaptive Poisson-Boltzmann Solver (APBS) software (33Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10037-10041Crossref PubMed Scopus (5943) Google Scholar). Electrophysiology Experiments-Site-directed mutagenesis was performed on the NaV1.5 α subunit cDNA and subcloned into the expression vector pCGI for bicistronic expression of the channel protein and green fluorescent protein as described (34Tan H.L. Bink-Boelkens M.T. Bezzina C.R. Viswanathan P.C. Beaufort-Krol G.C. van Tintelen P.J. van den Berg M.P. Wilde A.A. Balser J.R. Nature. 2001; 409: 1043-1047Crossref PubMed Scopus (344) Google Scholar). Cultured cells (tsA201) were transiently transfected with either wild-type or mutant (2X) cDNA (2.5 μg). Green cells were selected for electrophysiological analysis 24 h later. The cells were maintained on culture plates with Dulbecco's modified Eagle's medium with fetal bovine serum (10%) and 1% penicillin-streptomycin. INa was recorded at 22 °C and analyzed as described (34Tan H.L. Bink-Boelkens M.T. Bezzina C.R. Viswanathan P.C. Beaufort-Krol G.C. van Tintelen P.J. van den Berg M.P. Wilde A.A. Balser J.R. Nature. 2001; 409: 1043-1047Crossref PubMed Scopus (344) Google Scholar). The voltage-clamp protocols used are shown in the legend to Fig. 5. A Boltzmann function (I = 1/(1 + exp(Vt - V½)/μ))) was fitted to the availability curves to determine the membrane potential eliciting half-maximal inactivation (V½), where μ is the slope factor. The pipette solution used to approximate zero [Ca2+]i contained 10 mm NaF, 100 mm CsF, 20 mm CsCl, 20 mm BAPTA, 10 mm HEPES, adjusted to pH 7.35 with CsOH. For high [Ca2+]i (1 μm free Ca2+), 1 mm BAPTA was used with 0.9 mm Ca2+. The extracellular (bath) recording solution contained 145 mm NaCl, 4 mm KCl, 1 mm MgCl2, 10 mm HEPES, and 1.8 mm CaCl2, adjusted to pH 7.35 with CsOH. To avoid the time-dependent shift of the INa availability curve commonly observed during patch clamp experiments, voltage-dependent inactivation was assessed within 2 min after rupture of the membrane. Patch clamp measurements are presented as the mean ± S.E. Comparisons were made using Student's t test, with p < 0.05 considered significant. Data Deposition-1H, 13C, and 15N chemical shift assignments are deposited in the BioMagResBank under accession number 16045. The coordinates of the final ensemble of 20 structures and the full list of NMR restraints are deposited in the Protein Data Bank under accession code 2KBI. Structure of the EF-hand Domain in the NaV1.5 C Terminus-The three-dimensional solution structure of apoCTD-EF was determined by multidimensional heteronuclear NMR spectroscopy. To achieve this goal, the purification protocol and buffer conditions were extensively optimized relative to our previous report (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). 1H, 13C, and 15N resonance assignments (>98%) were obtained for all but the Cγ, Cδ, C∈, and, CδH resonances of Lys-1829 using the standard series of double- and triple-resonance backbone-based scalar correlated experiments described under "Experimental Procedures." Of note, cis conformations for two of the five proline residues (Pro-1824, Pro-1830) were identified on the basis of characteristic Cγ and Cδ chemical shifts and Hα-Hα NOEs (35Schubert M. Labudde D. Oschkinat H. Schmieder P. J. Biomol. NMR. 2002; 24: 149-154Crossref PubMed Scopus (281) Google Scholar, 36Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Qualitative analysis of the pattern of medium range NOEs revealed the presence of four α helices, which were fully consistent with analysis by the chemical shift index (37Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1919) Google Scholar) (Fig. 1A). Heteronuclear relaxation parameters were measured to characterize the backbone dynamics of CTD-EF, including steady-state {1H}-15N NOE, longitudinal 15N(T1), and transverse 15N(T2) relaxation times (supplemental Fig. S1). The average values of 0.74, 440 ms, and 77 ms, respectively, with small S.D. are consistent with the existence of a globular domain between residues 1785 and 1863. Clear trends to lower values for the NOE and T2 are clearly evident for the preceding 12 residues of CTD-EF, indicating that the N terminus of this construct is flexible. The trimmed T1/T2 ratio provides an estimate of 5.7 ns for the rotational correlation time, consistent with the CTD-EF tumbling as a monomeric species in solution. The three-dimensional structure of CTD-EF was calculated using a two-step protocol that involves generation of initial structures using CYANA (19Guntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1176) Google Scholar) and refinement by restrained molecular dynamics using AMBER (21Case D.A. Cheatham III, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6819) Google Scholar) as described previously (38Maler L. Sastry M. Chazin W.J. J. Mol. Biol. 2002; 317: 279-290Crossref PubMed Scopus (46) Google Scholar). In all, 2131 NOE-based distance restraints were generated (Table 1), and these were well distributed along the sequence except for the first 12 residues, which lack medium and long range restraints (Fig. 1B). In addition to NOEs, 51 backbone φ angle restraints were derived from chemical shifts, and the CYANA program provided stereo-specific assignments for 9 pairs of Leu and Val methyl groups and 23 pairs of Cβ protons. The final ensemble of 20 conformers was selected based on lowest restraint violation, and total molecular energy and is shown in Fig. 2A. Consistent with the large number of experimental restraints (>20/residue), this ensemble is well defined and exhibits low constraint violation energies, no distance violations >0.2 Å, no torsion angle violations >5°, and low molecular energies (Table 1). The structure has been refined to high precision with a root mean square deviation of 0.47 ± 0.07 Å for the backbone and 1.09 ± 0.10 Å for all heavy atoms (Table 1). The high quality of the structure is further reflected by PROCHECK analysis. For example, 99.4% of all backbone torsion angles occupy the favored regions of the Ramachandran plot.TABLE 1Structural statistics for CTD-EF in Nav1.5Restraints for calculationTotal NOE restraints2131Intraresidue466Sequential554Medium range526Long range585Dihedral angle restraint51Constraint violations, mean ± S.D.Distance violations0.1 Å < d < 0.2 Å1.6 ± 1.0d > 0.2 Å0Average maximum distance violations (Å)0.13 ± 0.03Torsion angle violations >5.0°0Average maximum torsion angle violations0(degree)AMBER energiesaSee Ref. 21., mean ± S.D. (kcal mol−1)Restraint1.8 ± 0.4van der Waals−741 ± 8Total molecular−4021 ± 10Precision, root mean square deviation from the mean (Å), ordered regionsbResidues 20-94.Backbone0.47 ± 0.07All heavy atoms1.09 ± 0.10Ramachandran statisticscPROCHECK nomenclature (32). (%)Most favored86.8Additionally allowed12.6Generously allowed0.4Disallowed (only for residues 1-16 and 55)0.2a See Ref. 21Case D.A. Cheatham III, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6819) Google Scholar.b Residues 20-94.c PROCHECK nomenclature (32Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4507) Google Scholar). Open table in a new tab The overall fold of CTD-EF is similar to other EF-hand domains, consisting of two helix-loop-helix EF-hand motifs and a short linker between them. The four helices include: I, Glu-1788-Trp-1798; II, Tyr-1811-Ala-1820; III, Gln-1832-Asn-1837; IV, Cys-1850-Leu-1862. The typical cross-strand β-like interaction between the two EF-hand loops is found involving residues Phe-1808-Glu-1810 and Arg-1947-His-1849 (Fig. 2B). The loop between helices III and IV is somewhat unusual; although it is the characteristic 12 residues in length, it possesses one proline (Pro-1841) and adopts a more extended conformation than is typical of other EF-hand domain loops. As noted above, the first 12 residues of the CTD-EF construct are unstructured. In the remainder of the discussion, CTD-EF will refer only to the globular EF-hand domain. Although CTD-EF does not have substantial sequence similarity with well characterized EF-hand calcium-binding proteins such as calmodulin, troponin C, recoverin, or S100 proteins, it does have a typical EF-hand protein structure. The Vector Alignment Search Tool (VAST) (39Gibrat J.F. Madej T. Bryant S.H. Curr. Opin. Struct. Biol. 1996; 6: 377-385Crossref PubMed Scopus (892) Google Scholar) search finds three EF-hand proteins as the most similar: Ca2+-loaded troponin-C (PDB 1TOP), Ca2+-loaded domain VI of
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