Article pubs.acs.org/biochemistry
Characterization of the Cation Binding Sites in the NCKX2 Na+/Ca2+K+ Exchanger Hristina Zhekova,† Chunfeng Zhao,†,§ Paul P. M. Schnetkamp,‡ and Sergei Yu. Noskov*,† †
Center for Molecular Simulations, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4 Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada
‡
S Supporting Information *
ABSTRACT: NCKX1−5 are proteins involved in K+-dependent Na+/ Ca2+ exchange in various signal tissues. Here we present a homology model of NCKX2 based on the crystal structure of the NCX_Mj transporter found in Methanoccocus jannaschii. Molecular dynamics simulations were performed on the resultant wild-type NCKX2 model and two mutants (D548N and D575N) loaded with either four Na+ ions or one Ca2+ ion and one K+ ion, in line with the experimentally observed transport stoichiometry. The selectivity of the active site in wild-type NCKX2 for Na+, K+, and Li+ and the electrostatic interactions of the positive Na+ ions in the negatively charged active site of wild-type NCKX2 and the two mutants were evaluated from free energy perturbation calculations. For validation of the homology model, our computational results were compared to available experimental data obtained from numerous prior functional studies. The NCKX2 homology model is in good agreement with the discussed experimental data and provides valuable insights into the structure of the active site, which is lined with acidic and polar residues. The binding of the potassium and calcium ions is accomplished via Asp 575 and 548, respectively. Mutation of these residues to Asn alters the functionality of NCKX2 because of the elimination of the favorable carboxylate−cation interactions. The knowledge obtained from the NCKX2 model can be transferred to other isoforms of the NCKX family: newly discovered pathological mutations in NCKX4 and NCKX5 affect residues that are involved in ion binding and/or transport according to our homology model.
hampered by the absence of X-ray or nuclear magnetic resonance structures of the NCKX isoforms, which leads to insufficient structural information about the cation binding and transport processes occurring in NCKX. Functional studies of the NCKX1 (in situ) and NCKX2 (in cell lines) isoforms have shown that the extrusion of one Ca2+ ion from the cytosol is accompanied by cotransport of a K+ ion and antitransport of four Na+ ions.20,21 Transport is bidirectional and dependent on the transmembrane Na+ and K+ gradients, leading to either Ca2+ efflux (forward exchange) or Ca2+ influx (reverse exchange) mediated by NCKX with comparable Km values of approximately 1−4 μM in the absence of competing ions.22−25 Detailed analysis of the effects of various alkali cations on NCKX1-mediated Ca2+ fluxes resulted in a three-site model for NCKX1 in which of all alkali cations only Na+ competed with Ca2+ for a common site while Na+ competed for the other two sites with other alkali cations. Transport was observed only in the following two cation occupancies: one Ca2+ with one K+ or four Na+ ions.26
NCKX1−5 are proteins encoded by the SLC24 gene family and responsible for K+-dependent Ca2+/Na+ exchange in various excitable or signal tissues, including rod and cone photoreceptors (NCKX1 and NCKX2), brain synapses (NCKX2), smooth muscle (NCKX3), ameloblasts (NCKX4), melanocytes (NCKX4 and NCKX5), and retinal pigment epithelium (NCKX5).1−4 It is thought that the NCKX isoforms contribute to physiological functions such as dim light and daylight vision, light adaptation of the eye,4 synaptic plasticity, motor memory,5 salt-sensitive artery hypertension,6 olfactory response,7 melanocortin-4-receptor-dependent signaling (involved in the feeling of satiety),8 amelogenesis and enamel formation,9−12 and skin, hair, and eye pigmentation.13−16 Some deficits associated with mutations in the SLC24A1−5 genes are congenital stationary night blindness (NCKX1),4 impaired memory and motor learning in mice (NCKX2),5 amelogenesis imperfecta (NCKX4),9−12 and nonsyndromic oculocutaneous albinism (NCKX5). 17,18 In addition, mutations in the Drosophila NCKX-X genes have been linked to increased susceptibility to seizures.19 Neither the pathological mechanisms of these mutations nor the NCKX transport in healthy cells is well understood, and ongoing studies are trying to address such issues. These investigations, however, are © XXXX American Chemical Society
Received: June 10, 2016 Revised: October 31, 2016 Published: November 2, 2016 A
DOI: 10.1021/acs.biochem.6b00591 Biochemistry XXXX, XXX, XXX−XXX
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Figure 1. Topology model of WT NCKX2. The two α-repeats are colored yellow. The residues discussed in this work are also shown.
Figure 2. Sequence alignment of the two α-repeats in NCKX2, NCKX4, NCX1, and NCX_Mj. Colored red are the residues preserved in all four systems. Boxed are the residues that form the ion binding sites in NCKX2 and NCX_Mj. Colored blue are additional residues preserved between NCX1 and NCX_Mj.
the NCX_Mj protein found in Methanoccocus jannaschii, whose crystal structure was resolved in 2012.36 Figure 2 shows the sequence alignment of the two α-repeats relevant for the binding and transport of Ca2+ and Na+ (in NCX_Mj and NCX1) or Ca2+, K+, and Na+ (in NCKX2 and NCKX4). The full sequence alignment between NCKX2 and NCX_Mj can be found in Figure S1. Although the overall degree of sequence homology between NCKX2 and NCX_Mj is low (∼16%), the two functionally important α-repeats are significantly more preserved (∼34%). The residues that form the binding site in NCX_Mj (based on the available crystal structure)36 are almost identical to those in NCKX2 (Figure 2, boxed residues). The most pronounced difference is found in the α1 repeat, where residue T50 in NCX_Mj is substituted with a Gly (G184) in NCKX2. In addition, the 10 short TMS in NCX_Mj and NCKX2 form a tight compact structure. Thus, the crystal structure of NCX_Mj can be considered an adequate template for molecular modeling of NCKX2 and can provide valuable information about the three-dimensional (3D) organization and functionality of NCKX1−5, which is inaccessible with the available experimental methods. In the work presented here, we strive to elucidate the experimentally observed functionality in the NCKX isoforms by constructing and analyzing a homology model of NCKX2 using the crystal structure of NCX_Mj as a template. We have chosen the NCKX2 isoform because of the abundance of experimental data available from prior functional and structural−topological studies for this isoform (reviewed in ref 23). The high degree of conservation of residues in the transmembrane domains of the five NCKX isoforms and recent comparative studies of the
Hydropathy analysis of the NCKX1−5 proteins indicates the presence of two large hydrophilic loops and 11 hydrophobic sections (Figure 1), which are presumed to be α-helical transmembrane segments (TMS). The first TMS located at the N-terminus is thought to be a cleavable signal peptide or an uncleaved signal anchor important for targeting of protein to the cell membrane.27 Topological studies of NCKX2 expressed in cell lines have shown that the 10 remaining TMS contain highly conserved amino acid residues among the NCKX isoforms and are organized in two sets of five TMS each, separated by a large hydrophilic loop positioned in the cytosol.28,29 The most conserved and central sequences in the two sets of TMS are known as the α1 and α2 repeats (yellow, Figure 1) and are believed to have arisen from an ancient gene duplication event.30 The repeats feature several residues of potential significance for the binding of Na+, K+, and Ca2+ ions (Asp, Glu, Ser, Thr, and Asn) or for conformational changes consistent with ion transport (Gly, Ala, and Pro). Mutations of 25 residues found in the two repeats have been shown to reduce transport activity by more than 80% compared to that of WT NCKX2.31 Subsequent more detailed kinetic analysis of single-residue NCKX2 substitutions identified several residues that appeared to be important for Ca 2+, K+ , and Na + coordination in NCKX2.32−34 NCKX1−5 share functional but limited sequence similarity with members of the SLC8 gene family of K+-independent Na+/Ca2+ exchangers (NCX) that operate with a 3:1 Na+:Ca2+ stoichiometry.35 Recent studies determined that NCX and NCKX have a topological arrangement of the TMS identical to that of a distantly related archaebacterial Na+/Ca2+ exchanger, B
DOI: 10.1021/acs.biochem.6b00591 Biochemistry XXXX, XXX, XXX−XXX
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piston mass of 2000 kcal mol−1 ps2) and in the NPAT ensemble (P = 1 atm, maintained with Langevin pressure control53 with a piston collision frequency of 20 ps−1 and a piston mass of 9578 amu). The simulations were performed for 120 ns. The SHAKE algorithm54 was used to maintain bonds involving hydrogens, and the integration time step was set to 2 fs. The nonbonded interactions were evaluated under constant dielectric conditions with an ε of 1 and with the following cutoffs: 10 Å for smoothing potentials, 12 Å for force-based switching functions, and 16 Å for list pair generation. An Ewald mesh55,56 with dimensions of 90 Å × 90 Å × 120 Å, a κ of 0.34, and B-spline interpolation of the sixth order was used for the calculation of the electrostatic interactions. The drift of the protein was controlled with a harmonic potential of cylindrical shape (force constant of 1 kcal mol−1 Å−2) applied on the center of mass of all non-hydrogen and non-protein atoms in the system. The equilibrated WT NCKX2 model with four Na+ ions was used for analysis, evaluation of the ion selectivity (see below), and preparation of a WT NCKX2 model loaded with one K+ and one Ca2+ by removal of the Na+ ions at positions 1 and 3 (Figure 3) and substitution of the Na+ at positions 4 and 2 with
cation dependencies of transport in NCKX1−422 suggest that NCKX2 can serve as a structural model of the whole SLC24 family. Homology models with either four Na+ ions or one Ca2+ and one K+ have been created for the sake of consistency with the experimentally observed 4Na+/1Ca2+-1K+ transport stoichiometry. The structures were relaxed with molecular dynamics37 (MD), and the selectivity of the active site for a number of ions (Na+, K+, Li+, and Ca2+) was studied with the free energy perturbation (FEP) method.38 Two critical mutants, D548N and D575N, were also included in our investigation. The replacement of the charged Asp residue with the neutral Asn residue of similar size and geometry has been shown either to abolish the K+ dependence of Ca2+ transport (D575N) or to eliminate Ca2+ transport entirely (D548N).33,34 Equilibrated mutant structures were obtained from MD simulations and used for structural comparison to their wildtype analogues. The electrostatic component of the free energy of binding of Na+ ions to the active site of wild-type (WT) NCKX2 and the two mutants was calculated with the FEP− GSBP method39 and used for discussion of the coexistence of four positive charges and the effects of mutation on the binding in NCKX2.
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MATERIALS AND METHODS Homology Modeling of NCKX2. The homology model of NCKX2 reported here is based on the crystal structure of M. jannaschii [Protein Data Bank (PDB) entry 3V5U]. Modeler 9.1040 was used for both alignment and folding of the NCKX2 model. The following restraints from experimental studies were adopted for the construction of the 3D structure: an α-helical conformation for the protein backbone containing residues 598−607 and 641−648, a distance of 7 Å between the αcarbons of residues 205 and 651, and a disulfide bond between Cys 605 and Cys 657. A total of 500 structures were generated, and their quality was assessed with the DOPE41 and GA34142 methods. The residues forming the active site in the best NCKX2 model (based on the DOPE and GA341 scores) were then set at the same coordinates as their counterparts in the NCX_Mj crystal structure. This NCKX2 model was used as a starting point for CHARMM-GUI43 (see below). MD Simulations of Wild-Type NCKX2 and Its Mutants. The CHARMM-GUI43 interface was used to generate the simulation system for the MD studies of the NCKX2 protein. The initial model contained four Na+ ions in the active site mapped on the crystal structure of NCX_Mj (PDB entry 3V5U). The protein was then embedded in a lipid membrane consisting of 177 DPPC molecules (89 in the upper leaflet and 88 in the lower leaflet).44−46 The system was immersed in a 0.15 M NaCl aqueous solution containing 48 Na+ ions, 48 Cl− ions, and 17158 water molecules. All simulations were run with rectangular periodic boundary conditions with a primary cell size of 83 Å × 83 Å × 116 Å. The system was subjected to the standard six short (25−50 ps) consecutive cycles of restrained minimization and equilibration set up by the CHARMM-GUI43 interface with the CHARMM3847 program and the CHARMM22/CMAP48,49 and CHARMM3650 force fields for proteins and lipids, respectively (these force fields were used in all MD calculations described in this work). The constrained simulations were followed by a longer unrestrained production run of 400 ps (see refs 44 and 51) prior to the production runs. Molecular dynamics simulations were performed at a constant temperature (298.15 K, controlled with a Hoover thermostat52 with a
Figure 3. Position of the active site in the WT NCKX2 protein with a load of four Na+ ions (left). Enlarged representation of the active site with all residues found within 3 Å of the Na+ ions (right). The residues and the protein backbone are color-coded: red for acidic, orange for polar, and gray for nonpolar. The Na+ ions are shown as yellow spheres and numbered.
K+ and Ca2+, respectively. Another Cl− ion was removed from the surrounding solution to preserve the overall neutrality of the model. The K+- and Ca2+-loaded model was then equilibrated for 105 ns under the conditions described above. The last 100 ns of equilibration for both NCKX2 models was performed with NAMD 2.957 because of its higher speed. The NAMD calculations were performed at 298.15 K and 1 atm pressure (with a flexible periodic cell), controlled by Langevin dynamics37 (thermostat damping coefficient of 5 ps−1, barrostat piston decay of 50 fs). All bonds involving H atoms were restrained with the SHAKE algorithm.54 The SETTLE algorithm58 was used to make the water molecules rigid. The evaluation of the electrostatic interactions was performed on an Ewald mesh as described above at an ε of 1. The cutoff for the van der Waals and electrostatic interactions was set to 12 Å, and smoothing functions were applied at 10 Å. The pair list cutoff was 13.5 Å. D575N and D548N mutants were created from the equilibrated four-Na+ and one-K+/one-Ca2+ WT models with the help of CHARMM-GUI.43 A single sodium ion was removed from the water solution for preservation of the overall neutrality upon substitution of the Asp with the Asn residue. C
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Biochemistry No other changes were made in the protein, lipid, and water environments. The mutants were then equilibrated for 20−40 ns with NAMD 2.957 as described above. Binding of Four Na+ Ions to the Active Site: Free Energy Perturbation Simulations with the GSBP Model. The GSBP method39 developed for reduced system simulations with realistic boundary potentials was used to perform free energy perturbation simulations on the NCKX2 transporter with one or four bound Na+ ions. The starting structures were extracted after equilibration from all-atom MD simulations trajectories, obtained as described above. The inner region of the GSBP system included part of the macromolecule, bound cations, and ligating water molecules confined within an 18 Å region and simulated explicitly with MD. The lipid molecules were removed, and the effect of the lipid bilayer was treated implicitly as described previously.59 The contributions from the outer electric fields are calculated only once for macromolecules of arbitrary geometry using the finite-difference Poisson−Boltzmann equation.60 This approach provides an accurate and computationally efficient hybrid MD/continuum method for simulation of a small region of a large biological macromolecular system. Dielectric constants of 80 and 4 were used for the solvent and the protein in the outer region, respectively. The static field arising from the protein charges in the outer region and the generalized reaction field matrix, including seven electric multipoles, were calculated using the PBEQ module of CHARMM38.47 Each GSBP-reduced NCKX2/Nax system (where x was set to either 1 or 4 bound ions) was re-equilibrated for 50 ns at 310 K using Langevin dynamics. A friction coefficient of 25 ps−1 was assigned to all heavy atoms. The center of mass constraint was used to restrain ion dynamics in the protein binding site. The charging and uncharging FEP simulations were performed for five different systems containing four Na+ ions or one Na+ in sites Na1, Na2, Na3, or Na4 (Figure 3). We also performed charging− uncharging simulations for two of the NCXK2 mutants, D575N and D548N. A linear coupling parameter λ was introduced to control the electrostatic interactions between the bound ion and the rest of the system. We used 21 perturbation (λ) windows running FEP for 5 ns per window. Both forward and backward perturbations were considered. The thermodynamic Integration (TI) method was used for computation of the electrostatic component of the ion binding free energy, Gsite. A similar protocol was used to obtain the free energy of solvation, Gsolv, for Na+ in a spherical system (SSBP) containing 512 TIP3P water molecules. The free energy of binding, ΔGbind, was then evaluated as ΔGbind = Gsite − Gsolv. Ion Selectivity in NCKX2 from Free Energy Perturbation Simulations. The ion selectivity of certain positions in the active site of the equilibrated native NCKX2 with either four Na+ ions or one K+ and one Ca2+ was evaluated from free energy perturbation (FEP) calculations,38 performed with CHARMM3847 and using the same setup for nonbonding interactions that was used for the MD equilibration (see above). The conversion of one ion at a time [Na+ at positions 1−4 (Figure 3), Ca2+, and K+ (Figure 4)] to another (Li+, K+, or Na+) was executed in 21 consecutive windows with an increment λ of 0.05. The postprocessing of the FEP results was done with WHAM61 as implemented in CHARMM38. The selectivity of a specific position in the protein with respect to the different ions tested (Na+, Li+, K+, and Ca2+) was determined from the sign of ΔΔGsel as reported previously:62
Figure 4. Position of the active site in the WT NCKX2 protein with a load of one Ca2+ and one K+ (left). Enlarged representation of the active site with all residues found within 3 Å of the Ca2+ and K+ ions (right). The residues and the protein backbone are color-coded: red for acidic, orange for polar, and gray for nonpolar. The purple and lime green spheres represent the K+ and Ca2+ ions, respectively.
ΔΔGsel = ΔG FEP − ΔGsolv
(1)
where ΔGFEP is the free energy obtained from the FEP calculations described above and ΔGsolv is the relative free energy of solvation obtained from FEP simulations for pairs of ions. The following protocol was used for all solvation computations. A box of 500 water molecules (TIP3P63) containing a single ion restrained to the center of the box was equilibrated for 10 ns to follow up with FEP simulations using the protocol described previously. We have used 21 perturbation windows run forward and backward with simulation for 2−5 ns per λ window.62
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RESULTS NCKX2 Model. The NCKX2 homology model was built with Modeler 9.1040 as described in Materials and Methods using the crystal structure of NCX_Mj36 as a template. The signal anchor and most of the large hydrophilic loop were omitted on the basis of their poor conservation between the isoforms of the NCKX family and between the different families (NCKX/NCX).23 Mutagenesis studies have established that they are not involved in ion binding or transport in NCKX and NCX, although the hydrophilic loop might play a role in ion regulation.23,63 The resultant 3D structure with either four Na+ ions or one Ca2+ and one K+ was equilibrated with molecular dynamics (rmsd plots illustrating the successful equilibration of the fourNa+-loaded NCKX2 model and its similarity to the NCX_Mj template are displayed in Figure S2). The four Na+ ions or, alternatively, the one Ca2+ ion and one K+ ion that were positioned within the active site as suggested by the NCX_Mj structure and previous experimental data33,34,64 remained in the vicinity of the acidic and polar residues. The N-terminus [after cleavage of signal peptide TMS0 (Figure 1)] and the Cterminus are pointing in the same direction (toward the extracellular space). The large hydrophilic loop (which is shortened in our models) is located at the intracellular side of the membrane well within the water layer that represents the cytoplasm. Previous mass-tagging studies29 with the MALPEG reagent have established that residues Q594 and S660 are positioned on the extracellular side of the membrane, on the small loop between TMS8 and TMS9 (the numbering of the TMS in Figure 1 is based on refs 23 and 63) and at the Cterminus, respectively. Residues M628 and C394 belong to the D
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Figure 5. (A) Hypothetic ion (Mn+) pathway with some relevant acidic (in red) and polar (in orange) residues. (B) Water density maps for the WT NCKX2 protein with four Na+ ions and the residues from panel A.
K+ (∼4 Å for Asp 548 and 80%.31 Among the studied mutants, D548N and D575N show some welldefined and peculiar characteristics: complete deactivation of NCKX2 (D548N) and loss of the K+ dependence of Ca2+ transport (D575N).33,34 From Figure 4 and the discussion of the structure of the active site (see above), it becomes clear that D548 coordinates Ca2+, D575 coordinates K+, and their chargeomitting mutations to Asp would disturb the binding of these ions. We constructed these two mutants (starting from the equilibrated WT NCKX2 structures with four Na+ ions or one Ca2+ ion and one K+ ion) and subjected them to MD equilibration to probe the effect of the mutation on the
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DISCUSSION The homology model of NCKX2 presented here is in good agreement with most of the available experimental data. The active site is lined with acidic and polar residues that can
Figure 7. Changes in the ion binding motifs upon Asp to Asn mutation at positions D548 and D575 in the NCKX2 model loaded with four Na+ ions (above) or one K+ ion and one Ca2+ ion (below). The atom coloring is as follows: cyan for C, dark blue for N, red for O, white for H, and yellow for Na. H
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ACKNOWLEDGMENTS Computations were performed on the West-Grid/Compute Canada facilities and the University of Calgary TNK cluster supported by the Canadian Foundation for Innovation.
accommodate the available cations. In particular, Asp 575 and 548 bind the K+ and Ca2+, and their replacement with the uncharged Asn residue leads to the experimentally observed altered functionality due to the weakened electrostatic attractions between the cations and the carbonyl O of the mutated residues, as revealed by our MD simulations and binding free energy calculations. Nevertheless, four Na+ ions can coexist in the limited space of the active site cavity in WT NCKX2, consistent with the known stoichiometry of transport. The position of the polar and acidic residues involved in ion binding and transport is in agreement with previous disulfide mapping results. A distinct ion pathway through the protein cannot be derived from the presented water density maps, likely because the crystal structure of NCX_Mj used as a template for the homology model of NCKX2 is in a stable occluded state and the MD simulations were not long enough to sample open conformations. The free energy perturbation results indicate that Li+ can outcompete Na+ at positions 1 and 4 but were not conclusive for the competition of Na+ and K+ ions. The results presented here can be extended to the other isoforms of the SLC24 gene family because of the highly preserved amino acid residues between them, especially in the area of the functionally significant α-repeats. In particular, eight residues in NCKX2 analogous to residues in NCKX4, NCKX5, and Drosophila NCKX-X that are linked to congenital defects were analyzed, and seven of them were found to be in significant proximity to either the binding site or the proposed ion pathway.
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ABBREVIATIONS DPPC, dipalmitoylphosphatidylcholine; FEP, free energy perturbation; MD, molecular dynamics; NCKX, Na+/Ca2+-K+ exchanger; NCX, Na+/Ca2+ exchanger; NCX_Mj, Na+/Ca2+ exchanger from M. jannaschii; NPAT, pressure control with constant membrane area; SLC, solute carrier family; TMS, transmembrane segments; VdW, van der Waals; WHAM, Weighted Histogram Analysis Method; WT, wild type.
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REFERENCES
(1) Schnetkamp, P. P. M. (2013) The SLC24 gene family of Na +/Ca2+-K+ exchangers: From sight and smell to memory consolidation and skin pigmentation. Mol. Aspects Med. 34, 455−464. (2) Lytton, J. (2007) Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem. J. 406, 365−382. (3) Jalloul, A. H., Rogasevskaia, T. P., Szerencsei, R. T., and Schnetkamp, P. P. (2016) A Functional Study of Mutations in K +-dependent Na+-Ca2+ Exchangers Associated with Amelogenesis Imperfecta and Non-syndromic Oculocutaneous Albinism. J. Biol. Chem. 291, 13113−13123. (4) Schnetkamp, P. P. (2004) The SLC24 NA(+)/Ca(2+)-K(+) exchanger family: vision and beyond. Pfluegers Arch. 447, 683−688. (5) Li, X. F., Kiedrowski, L., Tremblay, F., Fernandez, F. R., Perizzolo, M., Winkfein, R. J., Turner, R. W., Bains, J. S., Rancourt, D. E., and Lytton, J. (2006) Importance of K+-dependent Na+/Ca2+exchanger 2, NCKX2, in motor learning and memory. J. Biol. Chem. 281, 6273−6282. (6) Citterio, L., Simonini, M., Zagato, L., Salvi, E., Delli Carpini, S., Lanzani, C., Messaggio, E., Casamassima, N., Frau, F., D’Avila, F., Cusi, D., Barlassina, C., and Manunta, P. (2011) Genes involved in vasoconstriction and vasodilation system affect salt-sensitive hypertension. PLoS One 6, e19620. (7) Stephan, A. B., Tobochnik, S., Dibattista, M., Wall, C. M., reisert, J., and Zhao, H. (2011) The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response. Nat. Neurosci. 15, 131−137. (8) Li, X. F., and Lytton, J. (2014) An Essential Role for the K +-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4receptor-dependent Satiety. J. Biol. Chem. 289, 25445−25459. (9) Wang, S., Choi, M., Richardson, A. S., Reid, B. M., Seymen, F., Yildirim, M., Tuna, E., Gencay, K., Simmer, J. P., and Hu, J. C. (2014) STIM1 and SLC24A4 Are Critical for Enamel Maturation. J. Dent. Res. 93, 94S−100S. (10) Herzog, C. R., Reid, B. M., Seymen, F., Koruyucu, M., Tuna, E. B., Simmer, J. P., and Hu, J. C. (2015) Hypomaturation amelogenesis imperfecta caused by a novel SLC24A4 mutation. Oral Surg Oral Med. Oral Pathol Oral Radiol 119, e77−81. (11) Parry, D. A., Poulter, J. A., Logan, C. V., Brookes, S. J., Jafri, H., Ferguson, C. H., Anwari, B. M., Rashid, Y., Zhao, H. Q., Johnson, C. A., Inglehearn, C. F., and Mighell, A. J. (2013) Identification of Mutations in SLC24A4, Encoding a Potassium-Dependent Sodium/ Calcium Exchanger, as a Cause of Amelogenesis Imperfecta. Am. J. Hum. Genet. 92, 307−312. (12) Seymen, F., Lee, K. E., Tran Le, C. G., Yildirim, M., Gencay, K., Lee, Z. H., and Kim, J. W. (2014) Exonal deletion of SLC24A4 causes hypomaturation amelogenesis imperfecta. J. Dent. Res. 93, 366−370. (13) Sulem, P., Gudbjartsson, D. F., Stacey, S. N., Helgason, A., Rafnar, T., Magnusson, K. P., Manolescu, A., Karason, A., Palsson, A., Thorleifsson, G., Jakobsdottir, M., Steinberg, S., Palsson, S., Jonasson, F., Sigurgeirsson, B., Thorisdottir, K., Ragnarsson, R., Benediktsdottir, K. R., Aben, K. K., Kiemeney, L. A., Olafsson, J. H., Gulcher, J., Kong, A., Thorsteinsdottir, U., and Stefansson, K. (2007) Genetic
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00591. Full sequence alignment between NCKX2 and NCX_Mj and rmsd plots, demonstrating the equilibration of the homology NCKX2 model (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Center for Molecular Simulations, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4. E-mail:
[email protected]. Phone: 1 (403) 210-7971. Present Address §
C.Z.: CGG Services Ltd., Calgary, 715 5th Ave SW, Calgary, AB T2P 5A2, Canada. Funding
This work was supported by the National Sciences and Engineering Research Council (Discovery Grant RGPIN315019 to S.Yu.N.) and the Canadian Institutes of Health Research (Grant MOP-81327 to P.P.M.S.). S.Yu.N. was also supported by the Alberta Innovates Technical Futures Strategic Chair in BioMolecular Simulations. H.Z. was supported by the Eyes High Post-Doctoral Fellowship offered by the University of Calgary. Notes
The authors declare no competing financial interest. I
DOI: 10.1021/acs.biochem.6b00591 Biochemistry XXXX, XXX, XXX−XXX
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