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• W1993896317 abstract "Benz(othi)azepine (BTZ) derivatives constitute one of three major classes of L-type Ca2+ channel ligands. Despite intensive experimental studies, no three-dimensional model of BTZ binding is available. Here we have built KvAP- and KcsA-based models of the Cav1.2 pore domain in the open and closed states and used multiple Monte Carlo minimizations to dock representative ligands. In our open channel model, key functional groups of BTZs interact with BTZ-sensing residues, which were identified in previous mutational experiments. The bulky tricyclic moiety occupies interface between domains III and IV, while the ammonium group protrudes into the inner pore, where it is stabilized by nucleophilic C-ends of the pore helices. In the closed channel model, contacts with several ligand-sensing residues in the inner helices are lost, which weakens ligand-channel interactions. An important feature of the ligand-binding mode in both open and closed channels is an interaction between the BTZ carbonyl group and a Ca2+ ion chelated by the selectivity filter glutamates in domains III and IV. In the absence of Ca2+, the tricyclic BTZ moiety remains in the domain interface, while the ammonium group directly interacts with a glutamate residue in the selectivity filter. Our model suggests that the Ca2+ potentiation involves a direct electrostatic interaction between aCa2+ ion and the ligand rather than an allosteric mechanism. Energy profiles indicate that BTZs can reach the binding site from the domain interface, whereas access through the open activation gate is unlikely, because reorientation of the bulky molecule in the pore is hindered. Benz(othi)azepine (BTZ) derivatives constitute one of three major classes of L-type Ca2+ channel ligands. Despite intensive experimental studies, no three-dimensional model of BTZ binding is available. Here we have built KvAP- and KcsA-based models of the Cav1.2 pore domain in the open and closed states and used multiple Monte Carlo minimizations to dock representative ligands. In our open channel model, key functional groups of BTZs interact with BTZ-sensing residues, which were identified in previous mutational experiments. The bulky tricyclic moiety occupies interface between domains III and IV, while the ammonium group protrudes into the inner pore, where it is stabilized by nucleophilic C-ends of the pore helices. In the closed channel model, contacts with several ligand-sensing residues in the inner helices are lost, which weakens ligand-channel interactions. An important feature of the ligand-binding mode in both open and closed channels is an interaction between the BTZ carbonyl group and a Ca2+ ion chelated by the selectivity filter glutamates in domains III and IV. In the absence of Ca2+, the tricyclic BTZ moiety remains in the domain interface, while the ammonium group directly interacts with a glutamate residue in the selectivity filter. Our model suggests that the Ca2+ potentiation involves a direct electrostatic interaction between aCa2+ ion and the ligand rather than an allosteric mechanism. Energy profiles indicate that BTZs can reach the binding site from the domain interface, whereas access through the open activation gate is unlikely, because reorientation of the bulky molecule in the pore is hindered. Benz(othi)azepines (BTZs) 2The abbreviations used are: BTZ, benz(othi)azepine; MCM, Monte Carlo-minimization; SAR, structure-activity relationships. represent one of three main classes of ligands of L-type calcium channels (1Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar, 2Lacinova L. Gen. Physiol. Biophys. 2005; 1: 1-78Google Scholar). Tonic (resting) block of Ca2+ channels is usually measured by applying infrequent depolarization stimuli. Frequency-dependent block is measured during more rapid trains of depolarization stimuli. Increasing the stimulation frequency generally leads to more effective inhibition by BTZs (3Lee K.S. Tsien R.W. Nature. 1983; 302: 790-794Crossref PubMed Scopus (676) Google Scholar, 4Uehara A. Hume J.R. J. Gen. Physiol. 1985; 85: 621-647Crossref PubMed Scopus (156) Google Scholar, 5Herzig S. Lullmann H. Sieg H. Pharmacol. Toxicol. 1992; 71: 229-235Crossref PubMed Scopus (10) Google Scholar). Voltage and activation dependence of the block are consistent with the idea that different functional states of the channel have different affinities for the drug in the order inactivated > open > closed state. These observations are consistent with the “modulated receptor” hypothesis (6Hille B. J. Gen. Physiol. 1977; 69: 497-515Crossref PubMed Scopus (1376) Google Scholar), which was initially proposed to explain the action of local anesthetics on Na+ channels. The effect of BTZs also depends on the ionic environment. Raising concentrations of Ca2+ or Ba2+ antagonize diltiazem block (3Lee K.S. Tsien R.W. Nature. 1983; 302: 790-794Crossref PubMed Scopus (676) Google Scholar). However, the block is more pronounced when Ca2+ rather than Ba2+ is used as a charge carrier (3Lee K.S. Tsien R.W. Nature. 1983; 302: 790-794Crossref PubMed Scopus (676) Google Scholar, 7Dilmac N. Hillard N. Hockerman J.H. Mol. Pharmacol. 2003; 64: 491-501Crossref PubMed Scopus (30) Google Scholar), and thus the block is considered potentiated by Ca2+ (7Dilmac N. Hillard N. Hockerman J.H. Mol. Pharmacol. 2003; 64: 491-501Crossref PubMed Scopus (30) Google Scholar). The conductance with Ba2+ is higher than with Ca2+, because Ca2+ binds more tightly to the outer-pore glutamates (8Almers W. McCleskey E.W. J. Physiol. 1984; 353: 585-608Crossref PubMed Scopus (477) Google Scholar). These experiments suggest that the high affinity binding of BTZs requires a cation binding in the channel. Extracellular applications of both the tertiary and quaternary BTZ analogs effectively block Ca2+ channels. In contrast, intracellular application of the same compounds does not result in significant block, leading to the conclusion that BTZs block the channels via an extracellular pathway (9Seydl K. Kimball D. Schindler H. Romanin C. Pflugers Arch. 1993; 424: 552-554Crossref PubMed Scopus (22) Google Scholar, 10Kurokawa J. Adachi-Akahane S. Nagao T. Mol. Pharmacol. 1997; 51: 262-268Crossref PubMed Scopus (32) Google Scholar, 11Hering S. Savchenko A. Strubing C. Lakitsch M. Striessnig J. Mol. Pharmacol. 1993; 43: 820-826PubMed Google Scholar). Mutational studies have largely delimited BTZ-sensing residues to the inner helices and P-loops of domains III and IV (see Table 1). However, applying experimental data for mapping the BTZ receptor is complicated because of unequal effects of mutations on different drugs and different characteristics of the block. For example, residue I4i8 in position 8 of the inner helix of domain IV (see footnote 3We use a residue-labeling scheme, which is universal for P-loop channels. A residue label includes the domain (repeat) number (1Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar, 2Lacinova L. Gen. Physiol. Biophys. 2005; 1: 1-78Google Scholar, 3Lee K.S. Tsien R.W. Nature. 1983; 302: 790-794Crossref PubMed Scopus (676) Google Scholar, 4Uehara A. Hume J.R. J. Gen. Physiol. 1985; 85: 621-647Crossref PubMed Scopus (156) Google Scholar), segment type (p, P-loop; i, the inner helix; and o, the outer helix), and relative number of the residue in the segment (Table 1). for residue labels) was reported to affect diltiazem unblocking in the resting state (12Berjukow S. Gapp F. Aczel S. Sinnegger M.J. Mitterdorfer J. Glossmann H. Hering S. J. Biol. Chem. 1999; 274: 6154-6160Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Mutations F3i22A and V3i23A reduce diltiazem block, which was considered to slow inactivation kinetics and accelerate the recovery of the V3i23A mutant from drug blockade (13Kraus L.R. Hering S. Grabner M. Ostler D. Striessnig J. J. Biol. Chem. 1998; 273: 27205-27212Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The selectivity filter mutations do not alter diltiazem block of closed channels in Ba2+ but disrupt frequency-dependent block and potentiation by Ca2+ (7Dilmac N. Hillard N. Hockerman J.H. Mol. Pharmacol. 2003; 64: 491-501Crossref PubMed Scopus (30) Google Scholar).TABLE 1Sequence alignment and BTZ-sensing residuesChannelSegmentResidue label prefixSequence31121KcsAM1HWRAAGAATVLLVIVLLAGSYLAVLAEKvAPS5DKIRFYHLFGAVMLTVLYGAFAIYIVECav1.2IS51oAMVPLLHIALLVLFVIIIYAIIGLELFIIS52oSLRSIASLLLLLFLFIIIFSLLGMQLFIIIS53oAIRTIGNIVIVTTLLQFMFACIGVQLFIVS54oSFQALPYVALLIVMLFFIYAVIGMQVF334151KcsAPLITYPRALWWSVETATTVGYGDLKvAPPIKSVFDALWWAVVTATTVGYGDVCav1.2IP1pFDNILFAMLTVFQCITMEGWTDVIIP2pFDNFPQSLLTVFQILTGEDWNSVIIIP3pFDNVLAAMMALFTVSTFEGWPELIVP4pFQTFPQAVLLLFRCATGEAWQDI21121KcsAM2WGRLVAVVVMVAGITSFGLVTAALATWFVKvAPS6IGKVIGIAVMLTGISALTLLIGTVSNMFQCav1.2IS61iLPWVYFVSLVIFGSFFVLNLVLGVLSGEFIIS62iLVCIYFIILFISPNYILLNLFLAIAVDNLIIIS63iEISIFFIIYIIIIAFFMMNIFVGFVIVTFIVS64iFAVFYFISFYMLCAFLIINLFVAVIMDNF Open table in a new tab BTZs have a rigid bulky tricyclic core with several substituents. The structure-activity relationships (SARs) of BTZs have been rigorously analyzed in many previous studies (e.g. Refs. 14Floyd D.M. Kimball S.D. Krapcho J. Das J. Turk C.F. J. Med. Chem. 1992; 35: 756-772Crossref PubMed Scopus (70) Google Scholar, 15Das J. Floyd D.M. Kimball S.D. Duff K.J. Lago M.W. Krapcho J. White R.E. Ridgewell R.E. Obermeier M.T. Moreland S. McMullen D. Normandin D. Hedberg S.A. Schaeffer T.R. J. Med. Chem. 1992; 35: 2610-2617Crossref PubMed Scopus (20) Google Scholar, 16Das J. Floyd D.M. Kimball S.D. Duff K.J. Vu T.C. Lago M.W. Moquin R.V. Lee V.G. Gougoutas J.Z. Malley M.F. Moreland S. Brittain R.J. Hedberg S.A. Cucinotta G.G. J. Med. Chem. 1992; 35: 773-780Crossref PubMed Scopus (30) Google Scholar, 17Kimball S.D. Floyd D.M. Das J. Hunt J.T. Krapcho J. Rovnyak G. Duff K.J. Lee V.G. Moquin R.V. Turk C.F. Hedberg S.A. Moreland S. Brittain R.J. McMullen D.M. Normandin D.E. Cucinotta G.G. J. Med. Chem. 1992; 35: 780-793Crossref PubMed Scopus (45) Google Scholar, 18Kimball S.D. Hunt J.T. Barrish J.C. Das J. Floyd D.M. Lago M.W. Lee V.G. Spergel S.H. Moreland S. Hedberg S.A. Gougoutas J.Z. Malley M.F. Laud W.F. Bioorg. Med. Chem. 1993; 1: 285-307Crossref PubMed Scopus (16) Google Scholar). Blocking potency is affected by the configurations of the chiral centers and any modification of the substituents of the BTZ rings. These SAR data provide a pharmacophore model but do not provide detailed information on how specific BTZ modifications affect the electrophysiological characteristics of block. Despite numerous studies, the atomic level mechanism of action of BTZs remains unclear. A particularly intriguing question is why BTZs do not block the channel from the cytoplasm despite mutations of many pore-facing residues affect the channel blockade. Molecular determinants of the state dependence of drug action also remain unresolved. The effect of Ca2+ potentiation of the block is another puzzle: divalent cations antagonize BTZ binding (19Galizzi J.P. Fosset M. Lazdunski M. Biochem. Biophys. Res. Commun. 1985; 132: 49-55Crossref PubMed Scopus (41) Google Scholar), but the presence of Ca2+ in the selectivity filter is required for maximal drug effectiveness. Current knowledge about the BTZ action in Ca2+ channels suffers from the lack of structural models that can integrate results of mutational analysis, SAR data, and other experimental observations. There are several reasons for this situation. First, the structure of the L-type Ca2+ channel at the atomic resolution is still unavailable. Second, BTZs are complex molecules with many different functional groups. Third, the BTZ action is also complex: it involves tonic and state-dependent block and potentiation by Ca2+. Combination of these factors precluded elaborating a reliable model of BTZ receptor so far. To our knowledge, this work is the first attempt to obtain insights into the BTZ mechanism of action from the viewpoint of structural analysis. The L-type Ca2+ channel model for analysis in this study consists of transmembrane segments S5 and S6 and P-loops contributed by each of four repeats (I, II, III, and IV) of Cav1.2 (Table 1). Among available x-ray structures of the open K+ channels (MthK, KvAP, and Kv1.2), we have chosen KvAP (20Jiang Y. Lee A. Chen J. Ruta V. Cadene M. Chait B.T. MacKinnon R. Nature. 2003; 423: 33-41Crossref PubMed Scopus (1643) Google Scholar) as a template. Like Ca2+ channels, KvAP is a voltage-gated channel whose inner helices lack the PVP motif, which affects the open pore geometry. The closed channel was modeled using the KcsA structure (21Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5770) Google Scholar) as a template. When viewed from the extracellular side, domains I-IV are arranged clockwise around the pore axis (22Dudley Jr., S.C. Chang N. Hall J. Lipkind G. Fozzard H.A. French R.J. J. Gen. Physiol. 2000; 116: 679-690Crossref PubMed Scopus (90) Google Scholar). Extracellular linkers are far from the BTZ binding site and, thus, were not included in our model. The sequences of P-loops and inner helices were aligned as in Refs. 23Zhorov B.S. Tikhonov D.B. J. Neurochem. 2004; 88: 782-799Crossref PubMed Scopus (104) Google Scholar and 24Zhorov B.S. Folkman E.V. Ananthanarayanan V.S. Arch. Biochem. Biophys. 2001; 393: 22-41Crossref PubMed Scopus (61) Google Scholar. The sequences of the outer helices were aligned as proposed by Huber et al. (25Huber I. Wappl E. Herzog A. Mitterdorfer J. Glossmann H. Langer T. Striessnig J. Biochem. J. 2000; 347: 829-836Crossref PubMed Scopus (68) Google Scholar). The conformational energy expression included van der Waals, electrostatic, H-bonding, hydration, and torsion components, as well as the energy of deformation of the ligands' bond angles. Hydration energy was calculated by the implicit-solvent method (26Lazaridis T. Karplus M. Proteins. 1999; 35: 133-152Crossref PubMed Scopus (1120) Google Scholar). Nonbonded interactions were calculated using the AMBER force field version (27Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Chio C. Alagona G. Profeta S. Weiner P.K. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4895) Google Scholar, 28Weiner S.J. Kollman P.A. Nguen D.T. Case D.A. J. Comput. Chem. 1986; 7: 230-252Crossref PubMed Scopus (3604) Google Scholar), which is consistent with the implicit-solvent approach. More recent versions of the AMBER force field were parameterized for explicit-waters environment. A large number of degrees of freedom associated with explicit waters would reduce efficiency of the Monte Carlo minimization (MCM) method, which we use in this study (see below). To take into account that atomic charges may be screened by water molecules, electrostatic interactions were calculated with a distance- and solvation-dependent dielectric function, ϵ = d·(4-3s), where d is the distance between interacting atoms and s is a screening factor calculated using a modified algorithm of Lazaridis and Karplus (26Lazaridis T. Karplus M. Proteins. 1999; 35: 133-152Crossref PubMed Scopus (1120) Google Scholar). The screening factor value varies from 0 for a pair of water-exposed atoms to 1 for a pair of protein-buried atoms. All ionizable residues were modeled in their neutral forms except for the selectivity filter glutamates in position p50 of the P-loops. Nonbonded interactions were truncated at distances >8 Å. This cutoff distance speeds up calculations without noticeable decrease in the precision of energy calculations (29Bruhova I. Zhorov B.S. BMC Struct. Biol. 2007; 7: 1-13Crossref PubMed Scopus (26) Google Scholar). The cutoff was not applied to electrostatic interactions involving Ca2+ ions and ionized groups; these interactions were computed at all distances. The energy was minimized in the space of generalized coordinates, which include all torsion angles, bond angles of the ligand, positions of ions, and positions and orientations of root atoms of free molecules (30Zhorov B.S. J. Struct. Chem. 1981; 22: 4-8Crossref Scopus (44) Google Scholar). Energy minimizations were terminated when the energy gradient became <1 kcal mol-1 rad-1. The channel models were optimized by the MCM method (31Li Z. Scheraga H.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6611-6615Crossref PubMed Scopus (1284) Google Scholar). Complexes of the channel with ions and ligands were searched using a multi-MCM method described in our recent report (32Tikhonov D.B. Zhorov B.S. Biophys. J. 2007; 93: 1557-1570Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Calculations were performed using the ZMM program. Model Building—The starting backbone geometry of the open and closed channel models was taken from the KvAP and KcsA K+ channels, respectively. The all-trans conformations were used as starting approximations for side chains of residues that mismatched between the K+ channel templates and Cav1.2. Previous mutations in the selectivity filter DEKA locus rendered Na+ channel Ca2+ selectivity (33Heinemann S.H. Terlau H. Stuhmer W. Imoto K. Numa S. Nature. 1992; 356: 441-443Crossref PubMed Scopus (635) Google Scholar). These results indicate that there is likely a similar folding of the selectivity filter region of Cav1.2, channel, which has a ring of four glutamates (EEEE) in place of DEKA in Na+ channels. Given the similarity, we modeled the backbones of the ascending limbs in P-loops of Cav1.2 using our previously reported tetrodotoxin receptor model of Na+ channel (34Tikhonov D.B. Zhorov B.S. Biophys. J. 2005; 88: 184-197Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This choice of the starting point was not critical for the present study, because BTZs do not bind in the outer pore. The coordination pattern of Ca2+ ions in the EEEE ring is unknown. Asymmetric split (24Zhorov B.S. Folkman E.V. Ananthanarayanan V.S. Arch. Biochem. Biophys. 2001; 393: 22-41Crossref PubMed Scopus (61) Google Scholar) and symmetric single-file (35Lipkind G.M. Fozzard H.A. Biochemistry. 2001; 40: 6786-6794Crossref PubMed Scopus (59) Google Scholar) models were considered. Recent calculations predict that both models are energetically possible (36Cheng R.C. Zhorov B.S. Biophys. J. 2008; 94 (3147-Pos)PubMed Google Scholar). However, because substitutions of individual selectivity filter glutamates have unequal effects on BTZ binding (7Dilmac N. Hillard N. Hockerman J.H. Mol. Pharmacol. 2003; 64: 491-501Crossref PubMed Scopus (30) Google Scholar), we used the split model in this study with the following coordination pattern: E1p50:Ca2+:E2p50 and E3p50:Ca2+:E4p50. The channel models in the open and closed states were optimized by MCM trajectories, which were terminated when 10,000 consecutive energy minimizations did not improve the energy of the apparent global minimum. Random Docking—For the ligand-docking computational experiments we have chosen one of the most potent BTZ derivatives, SQ32910 (14Floyd D.M. Kimball S.D. Krapcho J. Das J. Turk C.F. J. Med. Chem. 1992; 35: 756-772Crossref PubMed Scopus (70) Google Scholar, 18Kimball S.D. Hunt J.T. Barrish J.C. Das J. Floyd D.M. Lago M.W. Lee V.G. Spergel S.H. Moreland S. Hedberg S.A. Gougoutas J.Z. Malley M.F. Laud W.F. Bioorg. Med. Chem. 1993; 1: 285-307Crossref PubMed Scopus (16) Google Scholar). Random docking of the ligand in the open channel was performed using the multi-MCM method as described previously (29Bruhova I. Zhorov B.S. BMC Struct. Biol. 2007; 7: 1-13Crossref PubMed Scopus (26) Google Scholar, 32Tikhonov D.B. Zhorov B.S. Biophys. J. 2007; 93: 1557-1570Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The resulting low energy structures fall into two categories in which the BTZ rings occur either in the inner pore (Fig. 1, A and B) or in the III/IV domain interface (Fig. 1, C and D). The ligand-channel interaction energy is most favorable when the BTZ rings are in the III/IV domain interface. However, the energy difference between the binding modes is not significant enough to favor the domain-interface model based on the energetics alone. More importantly, ligand binding in the III/IV domain interface correlates better with available data on BTZ-sensing residues and with the proposed access pathway of the drug to the binding site as discussed below. The models with the BTZ rings in the inner pore cannot easily explain why the BTZ binding site is accessible form the outside but not the inside the cell. It should be noted that, in these models, the ligand interacts with all four S6 segments, but some BTZ-sensing residues in the domain interface do not contact the drug molecule (Fig. 1, A and B) (Structure 1).STRUCTURE 1SQ32910View Large Image Figure ViewerDownload Hi-res image Download (PPT) Energy Profile—The experimental data on the sidedness of BTZ action and on BTZ-sensing residues evidence that BTZs access their binding site through the III/IV domain interface. The inner pore of K+ channels has four radial niches between neighboring S6 segments and P-helices. Mutations in these niches affect binding of some K+ channel blockers (37Dreker T. Grissmer S. Mol. Pharmacol. 2005; 68: 966-973Crossref PubMed Scopus (15) Google Scholar, 38Madeja M. Leicher T. Friederich P. Punke M.A. Haverkamp W. Musshoff U. Breithardt G. Speckmann E.J. Mol. Pharmacol. 2003; 63: 547-556Crossref PubMed Scopus (14) Google Scholar). In homology models of Na+ and Ca2+ channels several residues, which control access of drugs to the inner pore, line a hypothetical access pathway (34Tikhonov D.B. Zhorov B.S. Biophys. J. 2005; 88: 184-197Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 39Yamaguchi S. Zhorov B.S. Yoshioka K. Nagao T. Ichijo H. Adachi-Akahane S. Mol. Pharmacol. 2003; 64: 235-248Crossref PubMed Scopus (50) Google Scholar) that overlaps with the niche between domains III and IV. The pathway is wide enough to allow drug access to the inner pore (40Tikhonov D.B. Bruhova I. Zhorov B.S. FEBS Lett. 2006; 580: 6027-6032Crossref PubMed Scopus (42) Google Scholar). We calculated the energy profile for SQ32910 pulled with steps of 1 Å along the proposed pathway parallel to the IIIP helical axis from a starting position outside the channel toward the inner pore (Fig. 2). The energy was Monte Carlo-minimized at each position of the profile. During energy minimizations the ligand retained all internal degrees of freedom and five of the six rigid-body degrees of freedom. The pulled atom Nsp2 was constrained to a plane normal to the IIIP helical axis. To prevent flip-flop of the ligand, the flat-bottomed energy constraints were imposed at each position of the profile to keep atom Nsp3 closer to the pore axis than atom Nsp2 and the angle between line Nsp2-Nsp3, and the IIIP helical axis was retained within 0 ± 90°. In the final point of the profile (at 30 Å), atom Nsp2 is positioned at the pore axis. van der Waals interaction energy is negative all along the pathway (Fig. 2A) indicating the absence of steric hindrances. The lowest van der Waals energy was observed in the narrowest part of the pathway whose cross-sectional dimensions are similar to the drug width. The electrostatic component of the interaction energy is slightly negative at the beginning of the pathway, and a small electrostatic barrier is present between positions 12 and 20. This barrier is due to repulsion between the ligand ammonium group and the Ca2+ ion chelated by E3p50 and E4p50. To reach the inner pore, the ammonium group passes as close as 7 Å from the Ca2+ ion. Just after the barrier, the electrostatic interactions have a minimum of -13.2 kcal/mol. Two types of interactions are responsible for the minimum. First, the ligand carbonyl oxygen approaches the Ca2+ ion and binds to it. Second, the ammonium group is located at the focus of P-helices with nucleophilic C-ends. This region in P-loop channels accommodates inorganic (21Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5770) Google Scholar) and organic (40Tikhonov D.B. Bruhova I. Zhorov B.S. FEBS Lett. 2006; 580: 6027-6032Crossref PubMed Scopus (42) Google Scholar, 41Rossokhin A. Teodorescu G. Grissmer S. Zhorov B.S. Mol. Pharmacol. 2006; 69: 1356-1365Crossref PubMed Scopus (19) Google Scholar, 42Zhou M. Morais-Cabral J.H. Mann S. MacKinnon R. Nature. 2001; 411: 657-661Crossref PubMed Scopus (494) Google Scholar) cations. The hydration component of interaction energy increases as the drug enters a narrow part of the pathway. A sharp maximum at positions 22 and 23 corresponds to the location where the drug replaces water molecules of the Ca2+ inner hydration shell. The dehydration energy cost is partially compensated by the electrostatic attraction between the drug and the Ca2+ ion. Beyond position 23, all interactions weaken because the drug enters the wider inner pore and looses tight contacts with the III/IV domain interface. Notably, the energetically optimal position 22, which is marked on the plot by the vertical line, corresponds to the interface-binding mode obtained by the random docking protocol. Characteristics of BTZ Binding in the Domain Interface—Intensive multi-MCM docking of the ligand in the III/IV domain interface provided an ensemble of 42 low energy complexes (Fig. 3). The scheme of ligand-channel interactions (Fig. 4A) shows the most conservative features of the ensemble: (i) interaction of the carbonyl oxygen with the Ca2+ ion chelated by E3p50 and E4p50, (ii) location of the ammonium group in the inner pore, at the focus of P-helices, and (iii) location of the BTZ rings in the III/IV domain interface that contains BTZ-sensing residues. Importantly, in our model the Ca2+ ion is the only electrophile that can interact with the carbonyl oxygen of BTZ. Furthermore, in the presence of Ca2+ in the selectivity filter, the only attractive site for the ligand ammonium group is the focus of P-helices. In the majority of structures, the ammonium group is not exactly in the focus but lies close in the vicinity.FIGURE 4Specific ligand-channel interactions of the interface binding mode. A, structure of SQ32910 and scheme of specific interactions; B, intracellular view showing that F3p49 forms cation-pi contact with the ligand ammonium group, whereas F3i22 and ring B of the ligand stack face-to-face. C, side view with domain II and transmembrane helices of domain III removed for clarity. The carbonyl oxygen of the ligand binds to the Ca2+ ion chelated by E3p50 and E4p50, whereas methoxy oxygen forms an H-bond with the side chain of Y4i11.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Despite individual low energy structures belong to the same ensemble, they differ by details of particular interactions. The methoxyphenyl group interacts with Y4i11 (Fig. 4B). Two types of interaction have been found: (i) face-to-face attraction of the aromatic rings and (ii) an H-bond between the methoxy oxygen and the side-chain hydroxyl of Y4i11 with the aromatic rings approaching each other in the edge-to-face manner. The methyl group in position 3 of the seven-membered ring faces residues A4i15 and I4i18 in the majority of structures. Thus, all three residues of the YAI motif, forming what is known as a fingerprint of the BTZ recognition site (1Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar), significantly interact with the ligand. Ring B of the ligand interacts mainly with F3i22 in the face-to-face (Fig. 4A) or edge-to-face (not shown) manner. The CF3 group, which has lipophilic properties (43Yale H.L. J. Med. Pharm. Chem. 1959; 1: 121-133Crossref PubMed Scopus (130) Google Scholar), faces the III/IV domain interface. Interaction of F3p49 at the P-loop turn with the drug varies significantly between low energy structures, because in our model the alpha carbon of F3p49 was not constrained to the template. The aromatic side chain of F3p49 can participate in pi-cation interactions with a hydrated Ca2+ ion or with the ammonium group of the ligand (Fig. 4B). In some structures, F3p49 interacts with ring B of the drug in a face-to-face manner. Residues providing strong contributions to the ligand-receptor energy are underlined in Table 1. In the proposed binding mode, the drug does not interact directly with residues I3i8, I3i11, I3i14, and I4i8, which were experimentally demonstrated to affect the action of BTZ. However, residues I3i14 and I4i8 contribute significantly to the drug-channel energy along the access pathway (Fig. 2, B and C). Thus, among experimentally defined BTZ-sensing residues, only I3i8 and I3i11 do not contribute directly to the BTZ binding site or the access pathway. These particular residues could affect BTZ binding allosterically. In our model, SQ32910 significantly interacts with residues F3p44 and T3p48 in the domain interface. Mutational studies suggest that F3p44 interacts with dihydropyridines (39Yamaguchi S. Zhorov B.S. Yoshioka K. Nagao T. Ichijo H. Adachi-Akahane S. Mol. Pharmacol. 2003; 64: 235-248Crossref PubMed Scopus (50) Google Scholar), and T3p48 interacts with verapamil (44Dilmac N. Hilliard N. Hockerman G.H. Mol. Pharmacol. 2004; 66: 1236-1247Crossref PubMed S" @default.
• W1993896317 created "2016-06-24" @default.
• W1993896317 creator A5030029005 @default.
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• W1993896317 title "Molecular Modeling of Benzothiazepine Binding in the L-type Calcium Channel" @default.
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