Solution Structure of the NaV1.2 C-terminal EF-hand Domain
2009; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês
10.1074/jbc.m807401200
ISSN1083-351X
AutoresVesselin Z. Miloushev, Joshua A. Levine, Mark A. Arbing, J.F. Hunt, Geoffrey S. Pitt, Arthur G. Palmer,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoVoltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human NaV1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four α-helices separated by two short anti-parallel β-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the NaV1.2 C-terminal domain does not bind Ca2+ through the canonical EF-hand loops, as evidenced by monitoring 1H,15N chemical shifts during aCa2+ titration. Backbone chemical shift resonance assignments and Ca2+ titration also were performed for the NaV1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca2+ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of NaV1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the NaV1.2 (1777-1882) structure. Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human NaV1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four α-helices separated by two short anti-parallel β-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the NaV1.2 C-terminal domain does not bind Ca2+ through the canonical EF-hand loops, as evidenced by monitoring 1H,15N chemical shifts during aCa2+ titration. Backbone chemical shift resonance assignments and Ca2+ titration also were performed for the NaV1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca2+ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of NaV1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the NaV1.2 (1777-1882) structure. Voltage-gated sodium channels (VGSCs) 5The abbreviations used are: VSGC, voltage-gated sodium channel; NaV1, VSGC type 1; CTD, C-terminal domain; LQT3, Long QT syndrome type 3; CaM, calmodulin; HSQC, heteronuclear single quantum spectroscopy; NOESY, nuclear Overhauser effect (NOE) spectroscopy. are molecular assemblies that span the plasma membrane of excitable cells and conduct sodium current selectively in response to depolarizing stimuli. Mutations in VGSCs underlie a variety of diseases, including the cardiac arrhythmogenic Long-QT3 and Brugada syndromes (1Brugada J. Brugada P. Brugada R. Europace. 1999; 1: 156-166Crossref PubMed Scopus (110) Google Scholar, 2Terrenoire C. Simhaee D. Kass R.S. J. Cardiovasc. 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Specific α subunit isoforms are expressed differentially in skeletal muscle (NaV1.4), cardiac muscle (NaV1.5) and the nervous system (NaV1.1, NaV1.2, NaV1.3, splice variants of NaV1.5, and NaV1.6-NaV1.9) and control the rapid upstroke of action potentials (7Goldin A.L. Barchi R.L. Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). VGSC activity is characterized by two open states and several inactivated states (8The Y.K. Fernandes J. Popa M.O. Alekov A.K. Timmer J. Lerche H. Biophys. J. 2006; 90: 3511-3522Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Kinetics of channel inactivation occur on timescales ranging from milliseconds to seconds and determine multiple aspects of action potentials (9Goldin A.L. Curr. Opin. 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Res. 1998; 83: 141-146Crossref PubMed Scopus (166) Google Scholar, 20Deschenes I. Trottler E. Chahine M. Circulation. 1999; 100: 278-279Google Scholar, 21Motoike H.K. Liu H.J. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (123) Google Scholar). Specific disease-causing mutations within the CTD affect channel function by altering kinetics of channel inactivation (22Wang D.W. Makita N. Kitabatake A. Balser J.R. George A.L. Circ. Res. 2000; 87: 37-43Crossref PubMed Google Scholar). The CTD is predicted by sequence analysis (23Babitch J.A. Anthony F.A. J. Theor. Biol. 1987; 127: 451-459Crossref PubMed Scopus (4) Google Scholar, 24Babitch J. Nature. 1990; 346: 321-322Crossref PubMed Scopus (68) Google Scholar) and homology modeling (25Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 26Wingo T.L. Shah V.N. Anderson M.E. Lybrand T.P. Chazin W.J. Balser J.R. Nat. Struct. Mol. Biol. 2004; 11: 219-225Crossref PubMed Scopus (116) Google Scholar, 27Glaaser I.W. Bankston J.R. Liu H.J. Tateyama M. Kass R.S. J. Biol. Chem. 2006; 281: 24015-24023Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) to contain a paired EF-hand domain and was observed to contain a distal calmodulin binding IQ motif (4Fujiwara T. Sugawara T. Mazaki-Miyazaki E. Takahashi Y. Fukushima K. Watanabe M. Hara K. Morikawa T. Yagi K. Yamakawa K. Inoue Y. Brain. 2003; 126: 531-546Crossref PubMed Scopus (269) Google Scholar, 12Tan H.L. Kupershmidt S. Zhang R. Stepanovic S. Roden D.M. Wilde A.A.M. Anderson M.E. Balser J.R. Nature. 2002; 415: 442-447Crossref PubMed Scopus (193) Google Scholar, 28Mori M. Konno T. Ozawa T. Murata M. Imoto K. Nagayama K. Biochemistry. 2000; 39: 1316-1323Crossref PubMed Scopus (105) Google Scholar, 29Young K.A. Caldwell J.H. J. Physiol. (Lond.). 2005; 565: 349-370Crossref Scopus (87) Google Scholar, 30Biswas S. Deschenes I. DiSilvestre D. Tian Y. Halperin V.L. Tomaselli G.F. J. Gen. Physiol. 2008; 131: 197-209Crossref PubMed Scopus (34) Google Scholar, 31Kim J. Ghosh S. Liu H.J. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Structural modeling also predicts that specific interactions between helix I and helix IV control channel inactivation (27Glaaser I.W. Bankston J.R. Liu H.J. Tateyama M. Kass R.S. J. Biol. Chem. 2006; 281: 24015-24023Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 32Tateyama M. Liu H. Yang A.S. Cormier J.W. Kass R.S. Biophys. J. 2004; 86: 1843-1851Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). A recent model, based on NMR chemical shift perturbations, fluorescence spectroscopy, and electrophysiology, suggests that inactivation is regulated by Ca2+ binding to the proximal EF-hand, which is strongly influenced in turn by interactions with the distal IQ motif and calmodulin (33Shah 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 (97) Google Scholar). Nevertheless, whether Ca2+ binds specifically to the putative CTD EF-hand and any resultant contribution to channel regulation is controversial (12Tan H.L. Kupershmidt S. Zhang R. Stepanovic S. Roden D.M. Wilde A.A.M. Anderson M.E. Balser J.R. Nature. 2002; 415: 442-447Crossref PubMed Scopus (193) Google Scholar, 26Wingo T.L. Shah V.N. Anderson M.E. Lybrand T.P. Chazin W.J. Balser J.R. Nat. Struct. Mol. Biol. 2004; 11: 219-225Crossref PubMed Scopus (116) Google Scholar, 31Kim J. Ghosh S. Liu H.J. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 34Pitt B. Pitt G.S. Circulation. 2007; 115: 2976-2982Crossref PubMed Scopus (24) Google Scholar). Constructs of the Nav1.2 CTD were designed by limited proteolysis and H/D exchange experiments. Briefly, the CTD of Nav1.2, residues 1777-1937 with the amino acid substitutions I1877A/Q1878A and an N-terminal His6 tag MGSSHHHHHHSSGLVPRGSHMAS (31Kim J. Ghosh S. Liu H.J. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), was subjected to proteolytic digestion with proteinase K at 4 °C for 15-60 min using a protein:protease ratio of 50:1-100:1. The termini of the protected proteolytic fragments were mapped by matrix-assisted laser desorption ionization time-of-flight time-of-flight mass spectrometry and N-terminal sequencing. H/D exchange experiments were performed by ExSAR (Monmouth Junction, NJ) and showed protection for proteolytic fragments extending from residues 1789 to 1879. The construct encompassing residues 1777-1882 of the Nav1.2 CTD defined by the above experiments, including the N-terminal His tag, was used for structure determination by solution NMR spectroscopy. [U-13C,U-15N] NaV1.2 CTD (1777-1882) was overexpressed in Escherichia coli (BL21 DE3) transformed with a pET28 vector (EMD Biosciences) using M9 minimal media prepared with 15NH4 Cl and [13C6]glucose (35Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Cultures were grown at 37 °C to A600 nm = 0.7, induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside, transferred to 16 °C, and harvested after 72 h. Cells were lysed using a French press, and the NaV1.2 CTD was purified with Ni+-affinity, gel-filtration (Superdex 200), and ion-exchange (Mono Q 5/50 GL) chromatography (GE Healthcare). The N-terminal tag was not removed. Sample buffer consisted of 20 mm d11-Tris (pH 7.4), 100 mm d5-glycine, 0.1 mm d16-EDTA, 1 mm d10-DTT, 0.02% NaN3, and 10% D2O. Proteins were exchanged into this buffer using centrifugal concentrators (Amicon Inc.), flash-frozen in liquid N2, and stored at -80 °C. Samples for calcium titrations were subsequently exchanged into 20 mm d11-Tris (pH 7.4), 100 mm d5-glycine, 10 μm d16-EDTA, 1 mm d10-dithiothreitol, 0.02% NaN3, and 10% D2O. Protein concentrations of 0.5 and 0.2 mm were used for structural experiments and calcium titrations, respectively. The NaV1.5 CTD construct, residues 1773-1878, was designed by sequence alignment to NaV1.2, using bl2seq (36Tatusova T.A. Madden T.L. FEMS Microbiol. Lett. 1999; 174: 247-250Crossref PubMed Google Scholar), and protein samples were prepared by the same protocol. Sample temperatures were calibrated using 99.8% MeOD to a splitting of 1.616 ppm for NaV1.2 (290.5 K) and 1.545 ppm NaV1.5 (298.0 K) (37Findeisen M. Brand T. Berger S. Magn. Reson. Chem. 2007; 45: 175-178Crossref PubMed Scopus (196) Google Scholar). Backbone assignments for the NaV1.2 and NaV1.5 CTDs were obtained with HNCO, HNCA, HNCACB, HNCOCA, HNCACO, and CBCA(CO)NH experiments; side-chain assignments for NaV1.2 CTD were obtained with HBHA(CB-CACO)NH and HCCH-two-dimensional total correlation spectroscopy (TOCSY) experiments (38Cavanagh J. Fairbrother W. Palmer A.I. Rance M. Skelton N. Protein NMR Spectroscopy: Principles and Practice.2nd Ed. Academic Press, Boston, MA2007Google Scholar). A 10% 13C sample was used for stereospecific assignment of Leu and Val methyl groups (39Neri D. Szyperski T. Otting G. Senn H. Wüthrich K. Biochemistry. 1989; 28: 7510-7516Crossref PubMed Scopus (563) Google Scholar). NOE connectivities were obtained with 15N-NOESY-HSQC (80-ms mixing time), 13Caliphatic-NOESY-HSQC (100 ms), and 13Caromatic-NOESY-HSQC (80 ms). Residual dipolar coupling constants were measured in a sample containing 15 mg/ml Pf1 phage (Asla Biotech) using two-dimensional In-phase/Antiphase 1H,15N HSQC for 1H,15N (40Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (836) Google Scholar), three-dimensional HNCO for 13C′-13Cα (41Permi P. Rosevear P.R. Annila A. J. Biomol. NMR. 2000; 17: 43-54Crossref PubMed Scopus (75) Google Scholar), quantitative three-dimensional HNCO for 15N-13C′ (42Chou J.J. Delaglio F. Bax A. J. Biomol. NMR. 2000; 18: 101-105Crossref PubMed Scopus (50) Google Scholar), and HCACO for 1Hα-13Cα residual dipolar coupling constants (43Cicero D.O. Contessa G.M. Paci M. Bazzo R. J. Magn. Reson. 2006; 180: 222-228Crossref PubMed Scopus (8) Google Scholar). Fitting of the 15N-13C′ coupling constants was performed with Mathematica v5.2 (Wolfram Research, Inc.). Chemical shifts were referenced using DSS (44Markley J.L. Bax A. Arata Y. Hilbers C.W. Kaptein R. Sykes B.D. Wright P.E. Wüthrich K. J. Biomol. NMR. 1998; 12: 1-23Crossref PubMed Scopus (298) Google Scholar). An initial structure of NaV1.2 CTD was calculated from dihedral angle and NOE distance restraints, with several iterations to resolve ambiguity using ARIA 2.2 (45Rieping W. Habeck M. Bardiaux B. Bernard A. Malliavin T.E. Nilges M. Bioinformatics. 2007; 23: 381-382Crossref PubMed Scopus (393) Google Scholar) and CNS 1.2 (46Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). The structure was refined with XPLOR-NIH 2.18 using dihedral angle, NOE distance, and residual dipolar coupling constants restraints (47Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1832) Google Scholar, 48Schwieters C.D. Kuszewski J.J. Clore G.M. Prog. Nucl. Magn. Reson. Spectrosc. 2006; 48: 47-62Abstract Full Text Full Text PDF Scopus (605) Google Scholar). Dihedral angle restraints were derived from chemical shifts using TALOS (49Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). Distance restraints were obtained from NOE intensities corrected for multiplicity of the 1H spins. NOE connectivities were categorized into three classes (50Guntert P. Wüthrich K. J. Biomol. NMR. 1991; 1: 447-456Crossref PubMed Scopus (335) Google Scholar). Class I contains all intra-residue HN-Hα and all intra-residue, sequential, and medium range Hβ-HX NOEs, where X is not a methyl proton. Class III contains all NOEs involving a methyl group, and class II includes all other NOEs. A calibration factor, kI, was obtained by equating the average class I intensity to 3.4 Å. The class II calibration factor kII = kI/2.42. The class III calibration factor kIII = kII/2. Class I was averaged with a 1/6 order exponent, whereas classes II and III were averaged using a 1/4 exponent (50Guntert P. Wüthrich K. J. Biomol. NMR. 1991; 1: 447-456Crossref PubMed Scopus (335) Google Scholar, 51Mumenthaler C. Guntert P. Braun W. Wüthrich K. J. Biomol. NMR. 1997; 10: 351-362Crossref PubMed Scopus (134) Google Scholar). A standard 10% error term was applied to the upper bound of each restraint. All distances were constrained to the range (1.8, 5.5 Å). Pseudo atom corrections were applied to upper distance restraints for geometric considerations (52Fletcher C.M. Jones D.N.M. Diamond R. Neuhaus D. J. Biomol. NMR. 1996; 8: 292-310Crossref PubMed Scopus (125) Google Scholar). The 1H,15N, 15N-13C′, and 1Hα-13Cα residual dipolar coupling constants were included in the structure calculations. The residual dipolar coupling magnitude and rhombicity were set to -12.5 Hz and 0.55, respectively, during the initial minimization and were refined in the final all-atom minimization. The final average residual dipolar coupling magnitude and rhombicity are -12.8 ± 0.23 Hz and Rh = 0.56 ± 0.01, respectively, for the 200 conformers. Structural quality statistics refer to residues Leu-1790-Glu-1868 of the 15 lowest-energy structures of 200 total structures calculated. NOE completeness was determined with aqua3.2 (53Doreleijers J.F. Raves M.L. Rullmann T. Kaptein R. J. Biomol. NMR. 1999; 14: 123-132Crossref PubMed Scopus (70) Google Scholar). The Pearson correlation coefficient (R) and the quality factor (Q) were computed with PALES (54Zweckstetter M. Bax A. J. Am. Chem. Soc. 2000; 122: 3791-3792Crossref Scopus (589) Google Scholar) from 64 C′-Cα dipolar couplings that were not included in the structure calculation. MolProbity scores were calculated for the lowest energy structure (55Davis I.W. Leaver-Fay A. Chen V.B. Block J.N. Kapral G.J. Wang X. Murray L.W. Arendall W.B. Snoeyink J. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2007; 35: 375-383Crossref PubMed Scopus (2953) Google Scholar). Average root mean square deviation values were calculated to the average coordinates with VMD (56Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (35757) Google Scholar). Interhelical distances and angles (rounded to the nearest degree) were computed using interhlx. 6K. Yap, University of Toronto. Structural alignments were performed with CE (57Shindyalov I.N. Bourne P.E. Protein Eng. 1998; 11: 739-747Crossref PubMed Scopus (1680) Google Scholar), and structure figures were prepared with VMD (56Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (35757) Google Scholar) and MOLMOL (58Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6454) Google Scholar). The isolated NaV1.2 CTD (1777-1882) and NaV1.5 CTD (1773-1878) constructs each contain the region just after their respective predicted IVS6 transmembrane helix and extend to a region highly conserved among all VGSCs just before the IQ motif. Assignments of 1H,15N resonances for the NaV1.2 CTD and the NaV1.5 CTD are, respectively, 99 and 97% complete. Notably, Asn-1835 could not be assigned in the 1H,15N HSQC of NaV1.2. The resonances for Asn-1831 (the homologue of Asn-1835) and Gln-1832 were not assigned, and the resonance for Ile-1833 appears broadened in 1H,15N HSQC of NaV1.5. Moreover, homologous resonances Leu-1855 in NaV1.2 and Met-1851 in NaV1.5 have liminal intensities in 1H,15N HSQC spectra. These observations suggest conserved dynamics between isoforms. For the Nav1.2 CTD (1777-1882), 13Cα and 13Cβ assignments are 100% complete, 13C′ assignments are 97.1% complete, 1H aromatic assignments are 89.1% complete, and non-aromatic 1H assignments are 97.7% complete. The NaV1.2 CTD construct contains six proline residues, of which Pro-1789, Pro-1807, Pro-1827, and Pro-1845 are in a trans conformation, whereas Pro-1828 and Pro-1834 are in a cis conformation. The cis conformation is evidenced by stronger X-Pro Hα-Hα than X-Pro Hα-Hδ NOE contacts and differences of Cβ-Cγ chemical shifts of 9.4 and 8.5 ppm, respectively (59Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley, New York1986Crossref Google Scholar, 60Schubert M. Labudde D. Oschkinat H. Schmieder P. J. Biomol. NMR. 2002; 24: 149-154Crossref PubMed Scopus (271) Google Scholar). Medium range 1H-1H NOEs, steady-state {1H}-15N NOE, and 13Cα secondary chemical shifts for NaV1.2 indicate that the CTD forms a well folded domain between residues Leu-1790 and Glu-1868, with a less well ordered region between residues Ser-1869 and Arg-1882 and a disordered N-terminal region between residues Gly-1777 and Pro-1789 (Fig. 1 and supplemental Fig. S1). Secondary chemical shifts indicate that the NaV1.5 CTD has a similar secondary structural architecture as NaV1.2 CTD (Fig. 1C). The structure of NaV1.2 CTD is presented in Fig. 2 and supplemental Fig. S1 with statistical details of the calculation presented in Table 1. The structure contains four α-helices and two short anti-parallel β-strands, consistent with homology models based on structures of paired EF-hand domains (25Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 26Wingo T.L. Shah V.N. Anderson M.E. Lybrand T.P. Chazin W.J. Balser J.R. Nat. Struct. Mol. Biol. 2004; 11: 219-225Crossref PubMed Scopus (116) Google Scholar). Comparison of interhelical angles of helices I and II of NaV1.2 CTD and the N-terminal lobe of the prototypical EF-hand protein calmodulin suggests that the isolated NaV1.2 CTD most closely resembles the canonical apoEF-hand conformation (Fig. 3D and Table 2). The hydrophobic interface between helices I and IV predicted through mutational analysis (27Glaaser I.W. Bankston J.R. Liu H.J. Tateyama M. Kass R.S. J. Biol. Chem. 2006; 281: 24015-24023Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) is observed with direct NOE contacts between residues Phe-1795, Phe-1798, and Tyr-1799 in helix I and Leu-1855, Ile-1857, and Leu-1858 in helix IV.TABLE 1Structural statistics for Nav 1.2 CTDQuantityValueUnique NOE distance restraints1772Intra-residual699Sequential442Medium range (2 ≤ i ≤ 5)321Long range (i > 5)310Residual violations >0.3 Å per structure (n = 15)1.2 ± 1.1Maximum violation (Å)0.27 ± 0.2NOE completeness per shell2.0-2.5 Å (%)872.5-3.0 Å (%)693.0-3.5 Å (%)573.5-4.0 Å (%)44TALOS dihedral restraints (φ, ψ)65, 65Residual dipolar coupling restraintsH-N41N-C′64Hα-Cα44R-value; Q-factor (64 C′-Cα couplings)0.93 ± 0.02; 0.38 ± 0.05PROCHECKMost favored (%)82.2 ± 1.6Allowed (%)15.8 ± 1.7Generously allowed (%)1.41 ± 0.8Disallowed (%)0.5 ± 0.9MolProbity score; all-atom clash score (percentiles)3.09 (20th); 20.33 (31th)Average backbone r.m.s.d. (Å)0.80Average all atom r.m.s.d. (Å)1.29 Open table in a new tab FIGURE 3Ca2+ titration of NaV1. 2 (1777-1882) (panel A) and NaV1.5 (1773-1878) (panel B). The plots show joint 1H,15N chemical shift deviations from resonance assignments in 0 mm Ca2+. The titration was performed by serial addition of Ca2+ obtaining the following concentrations: 0 (red), 0.1 (orange), 0.5 (maroon), 1.5 (magenta), 2.5 (cyan), 3.5 (blue), and 4.5 mm (green) for NaV1.2 (panel A) and (0 (red), 0.1 (orange), 0.5 (maroon), 2.5 (magenta), 3.5 (cyan), 4.5 (blue), and 5.5 mm (green) for NaV1.5. Insets show resonances Phe-1812-Ile-1813 and Phe-1808-Ile-1809 for NaV1.2 and NaV1.5, respectively. Titration curves are shown in supplemental Fig. S2. In panel C the joint 1H,15N chemical shift changes for NaV1.2 (1777-1882) at 4.5 mm Ca2+ are mapped onto the lowest energy structure, interpolated between 0 ppm (blue) and 0.1 ppm (red).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Comparison of helix orientations in EF-hand proteinsMoleculeHelix IIHelix IIIHelix IVHelix ICa-CaM85 (20.4)−134 (25.5)91 (14.6)IQ-Ca-CaM92 (18.3)−161 (21.9)117 (10.1)ApoCaM136 (12.9)−93 (21.2)126 (11.9)Nav1.2 CTD152 ± 2 (10.9 ± 0.1)−103 ± 6 (20.9 ± 0.3)143 ± 1 (15.7 ± 0.2)Helix IICa-CaM83 (10.1)−20 (16.7)IQ-Ca-CaM107 (11.2)−41 (18.6)ApoCaM125 (11.9)−49 (12.9)Nav1.2 CTD100 ± 5 (10.8 ± 0.4)−46 ± 2 (13.4 ± 0.1)Helix IIICa-CaM65 (15.3)IQ-Ca-CaM80 (16.5)ApoCaM129 (14.1)Nav1.2 CTD98 ± 6 (14.4 ± 0.4) Open table in a new tab Helices I and IV contribute to the hydrophobic core of the protein, with a majority of aromatic side chains contributed from helix I. The segments between Gln-1811-Glu-1814 and Arg-1851-His-1853 participate in an anti-parallel β-sheet. An additional anti-parallel β-sheet contribution from residues Met-1846-Val-1847 is not present in all conformers of the structural ensemble. The helix II-III interhelical segment, delimited by two cis proline residues, Pro-1828 and Pro-1834, is well ordered in the structural ensemble. The conformation of residues Asp-1826-Leu-1829 is consistent with a type VI tight-turn (61Chou K.C. Anal. Biochem. 2000; 286: 1-16Crossref PubMed Scopus (258) Google Scholar), also called a βαR turn (62Wilmot C.M. Thorton J.M. Protein Eng. 1990; 3: 479-493Crossref PubMed Scopus (396) Google Scholar). The unique di-proline-leucine motif, Pro-1827-Leu-1829 extends the helix II-III interhelical segment by forming a small handle at the base of helix II (Figs. 3, D and E). The absence of long-range NOE contacts for residues Ser-1848 and Gly-1849 is represented by disorder of this region in the ensemble. The segment from residues Ser-1869 to Arg-1882 is predicted to have residual helical content based on secondary 13C chemical shifts and characteristic dαN(i, i+3) and dαβ(i, i+3) NOEs (Fig. 1). A short helix V is observed in the final ensemble extending from Gly-1870 to Arg-1876 with a backbone root mean square deviation of 0.59 Å when superposed on itself (supplemental Fig. S1). However, the reduced magnitudes of the secondary 13C chemical shifts and the {1H}-15N NOEs for helix V compared with helices I to IV, suggest that the helical conformation is not fully populated in solution. Furthermore, helix V does not exhibit residual dipolar couplings or long-range NOE contacts and, hence, is not well defined relative to the core EF-hand domain structure (supplemental Fig. S1). Additional interactions present in longer constructs of the CTD or in complexes with other components of the VGSC may stabilize helix V. Binding of Ca2+ by the NaV1.2 (1777-1882) and NaV1.5 (1773-1878) CTDs was assessed by monitoring 1H,15N chemical shifts as a function of Ca2+ concentration (0-4.5 mm). Chemical shift perturbations exhibit titration behavior suggesting that the interaction occurs on a fast-exchange timescale with equilibrium constants of 1.65 ± 0.03 mm for NaV1.2 CTD and 3.28 ± 0.13 mm for NaV1.5 CTD (Fig. 3 and supplemental Fig. S2), consistent with a previous report for the NaV1.5 CTD (33Shah 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 (97) Google Scholar). However, resonance assignments were not obtained previously, and the structure of NaV1.2 CTD now reveals that chemical shift perturbations >0.05 ppm are localized to residues in the N terminus of helix I, the linker between helices II and III, the C terminus of helix IV and the partially structured helix V. Thus, this weak Ca2+ binding site is distal to the canonical EF-hand loop motifs. In contrast, the average chemical shift change between the end points of the titration is <0.01 ppm in the N-terminal EF-hand loop (residues 1806-
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