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• W2021253110 abstract "ATP-binding cassette transporters use the free energy of ATP hydrolysis to transport structurally diverse molecules across prokaryotic and eukaryotic membranes. Computer simulation studies of the “real-time” dynamics of the ATP binding process in BtuCD, the vitamin B12 importer from Escherichia coli, demonstrate that the docking of ATP to the catalytic pockets progressively draws the two cytoplasmic nucleotide-binding cassettes toward each other. Movement of the cassettes into closer opposition in turn induces conformational rearrangement of α-helices in the transmembrane domain. The shape of the translocation pathway consequently changes in a manner that could aid the vectorial movement of vitamin B12. These results suggest that ATP binding may indeed represent the power stroke in the catalytic mechanism. Moreover, occlusion of ATP at one catalytic site is mechanically coupled to opening of the nucleotide-binding pocket at the second site. We propose that this asymmetry in nucleotide binding behavior at the two catalytic pockets may form the structural basis by which the transporter is able to alternate ATP hydrolysis from one site to the other. ATP-binding cassette transporters use the free energy of ATP hydrolysis to transport structurally diverse molecules across prokaryotic and eukaryotic membranes. Computer simulation studies of the “real-time” dynamics of the ATP binding process in BtuCD, the vitamin B12 importer from Escherichia coli, demonstrate that the docking of ATP to the catalytic pockets progressively draws the two cytoplasmic nucleotide-binding cassettes toward each other. Movement of the cassettes into closer opposition in turn induces conformational rearrangement of α-helices in the transmembrane domain. The shape of the translocation pathway consequently changes in a manner that could aid the vectorial movement of vitamin B12. These results suggest that ATP binding may indeed represent the power stroke in the catalytic mechanism. Moreover, occlusion of ATP at one catalytic site is mechanically coupled to opening of the nucleotide-binding pocket at the second site. We propose that this asymmetry in nucleotide binding behavior at the two catalytic pockets may form the structural basis by which the transporter is able to alternate ATP hydrolysis from one site to the other. ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain.1The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain. transporters are a large group of proteins that facilitate the permeation of solutes across cell membranes. The energy for this process is provided by the hydrolysis of ATP (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3323) Google Scholar). Members of the family so far identified include importers, exporters, receptors, and channels. Defects in a number of ABC transporters have been associated with serious hereditary disorders, of which cystic fibrosis is the best known (2Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5810) Google Scholar). In addition, increased expression levels of certain members of this ubiquitous family are a common cause of bacterial resistance to antibiotics and the resistance of human tumor cells to anticancer drugs (3Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1318) Google Scholar, 4Gottesman M.M. Fojo T. Bates S.E. Nat. Rev. Cancer. 2002; 2: 48-58Crossref PubMed Scopus (4442) Google Scholar). Recent successes in the determination of high resolution structures (5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F.-L. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 6Armstrong S.R. Tabernero L. Zhang H. Hermodson M. Stauffacher C.V. Pediatr. Pulmonol. 1998; 26: 91-92Crossref Google Scholar, 7Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar, 8Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar, 9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 10Karpowich N. Martsinkevich O. Millen L. Yuan Y.R. Dai P.L. MacVey K. Thomas P.J. Hunt J.F. Structure. 2001; 9: 571-586Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar, 12Moody J.E. Millen L. Binns D. Hunt J.F. Thomas P.J. J. Biol. Chem. 2002; 277: 21111-21114Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 14Verdon G. Albers S.V. Dijkstra B.W. Driessen A.J.M. Thunnissen A.-M.W.H. J. Mol. Biol. 2003; 330: 343-358Crossref PubMed Scopus (133) Google Scholar, 15Verdon G. Albers S.V. van Oosterwijk N. Dijkstra B.W. Driessen A.J.M. Thunnissen A. J. Mol. Biol. 2003; 334: 255-267Crossref PubMed Scopus (74) Google Scholar, 16Lewis H.A. Buchanan S.G. Burley S.K. Conners K. Dickey M. Dorwart M. Fowler R. Gao X. Guggino W.B. Hendrickson W.A. Hunt J.F. Kearins M.C. Lorimer D. Maloney P.C. Post K.W. Rajashankar K.R. Rutter M.E. Sauder J.M. Shriver S. Thibodeau P.H. Thomas P.J. Zhang M. Zhao X. Emtage S. EMBO J. 2004; 23: 282-293Crossref PubMed Scopus (329) Google Scholar) have made it possible to use computer modeling techniques to study ABC transporters (17Jones P.M. George A.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12639-12644Crossref PubMed Scopus (127) Google Scholar, 18Campbell J.D. Biggin P.C. Baaden M. Sansom M.S.P. Biochemistry. 2003; 42: 3666-3673Crossref PubMed Scopus (51) Google Scholar, 19Stenham D.R. Campbell J.D. Sansom M.S.P. Higgins C.F. Kerr I.D. Linton K.J. FASEB J. 2003; 17Crossref PubMed Scopus (120) Google Scholar, 20Seigneuret M. Garnier-Suillerot A. J. Biol. Chem. 2003; 278: 30115-30124Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Campbell J.D. Koike K. Moreau C. Sansom M.S.P. Deeley R.G. Cole S.P.C. J. Biol. Chem. 2004; 279: 463-468Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Here, we present extensive molecular dynamics (22Berendsen H.J.C. Science. 2001; 294: 2304-2305Crossref PubMed Scopus (16) Google Scholar) simulation studies of the ATP binding process in Escherichia coli BtuCD, an integral inner membrane protein that mediates the import of vitamin B12. Although ATP-driven dimerization of the ATP-binding cassettes has been predicted to represent the power stroke in the catalytic cycle (13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar), the crystal structure of an intact dimer (including the transmembrane segments) in the ATP-bound state is unfortunately yet to be reported. Two computer simulations were designed to investigate the hypothesis that the transition from a weak to a tight ATP binding state may elicit the conformational changes needed to unidirectionally force vitamin B12 through the transmembrane pathway. The biologically functional unit of ABC transporters is typically dimeric. It consists of two transmembrane domains (TMDs) that function as a pathway for the permeation of solute and two cytoplasmic nucleotide-binding domains (NBDs). The highly conserved NBDs are the nucleotide-hydrolyzing engines that drive transport through the TMDs (see Fig. 1). Conserved segments found in the NBD include the Walker A motif (or P-loop), the Walker B motif (23Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4212) Google Scholar), the Q-loop, the D-loop, the H-motif, and the LSGGQ sequence. The Walker A and Walker B motifs are found in other ATP-binding proteins as well, whereas the LSGGQ segment is the signature motif of ABC transporters (24Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (485) Google Scholar). The crystal structure of the NBD of a histidine importer, HisP (5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F.-L. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar), showed that each monomer has an overall “L” shape. The Walker A and B motifs are located in arm I, whereas the signature motif is in arm II. Other structures of NBDs that have since been solved include the structure of Rad50, an ATPase involved in DNA repair, which revealed the formation of a head-to-tail dimer in the presence of bound ATP but without the Mg2+ cofactor needed for hydrolysis (13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). The nucleotide was sandwiched between the Walker A and B motifs of one NBD and the signature motif of the opposing NBD. This had been predicted earlier through modeling studies by Jones and George (25Jones P.M. George A.M. FEMS Microbiol. Lett. 1999; 179: 187-202Crossref PubMed Google Scholar). Recent structures of dimeric MJ0796 and MalK have shown that the head-to-tail sandwich dimer can exist in true ABC transporters (9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 26Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). In the case of MJ0796, the dimer was obtained only after an E171Q mutation of the highly conserved catalytic glutamate at the end of the Walker B motif. The fact that the E171Q mutant is defective in ATP hydrolysis is believed to have stabilized the dimeric form. This was supported by studies on the glucose importer GlcV from Sulfolobus solfataricus (15Verdon G. Albers S.V. van Oosterwijk N. Dijkstra B.W. Driessen A.J.M. Thunnissen A. J. Mol. Biol. 2003; 334: 255-267Crossref PubMed Scopus (74) Google Scholar). The NBD dimer of MalK, on the other hand, was obtained from the wild-type protein. Nevertheless, ATP hydrolysis was prevented by excluding Mg2+ using EDTA. It is also notable that MalK is different from MJ0796 and several other ABC transporters in that it has an additional regulatory domain, which may stabilize the nucleotide-bound dimer. Unlike the strongly conserved NBDs, the TMDs show significant variation. This divergence may account for the diverse range of molecules translocated by members of ABC transporter family. Known transport substrates vary from anions, lipids, and amino acids to peptides, polysaccharides, and whole proteins (24Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (485) Google Scholar). Three structures of complete transporters have been reported: the lipid A exporter MsbA from E. coli (Eco-MsbA) (8Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (581) Google Scholar) and Vibrio cholera (VC-MsbA) (7Chang G. J. Mol. Biol. 2003; 330: 419-430Crossref PubMed Scopus (247) Google Scholar) and the E. coli vitamin B12 importer BtuCD (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). Although all three had no nucleotides bound at their catalytic sites, the structures were significantly different in conformation. In the Eco-MsbA structure, the NBDs are ∼50 Å apart with the nucleotide-binding sites facing away from the dimer axis. In VC-MsbA and BtuCD, they are in close opposition, facing each other in the same way as Rad50. However, the orientation of arm I of the NBD of VC-MsbA relative to arm II differs from that of BtuCD. A conformational arrangement similar to that of VC-MsbA has not been reported in previously solved structures of isolated NBDs with or without nucleotide. These variations may be indicative of the ability of the NBD to sample a large conformational space. Alternatively, they could be indicative of the crystal packing forces in MsbA. Although the rapidly growing body of structural data has helped to clarify the nature of subunit-subunit and nucleotide-protein interactions, details regarding the mechanism by which binding, hydrolysis, and release of nucleotide are coupled to steps in the transport cycle remain unresolved or controversial. We have simulated the transition of the NBDs of BtuCD from a semi-open state, as observed in the crystal structure, to a MgATP-bound closed state. Our simulations show that concomitant conformational transitions take place in the transmembrane domain, leading to the closure of the periplasmic end of the transportation pathway and initiating the opening of the cytoplasmic gate of the pathway. Although a similar mechanistic model was suggested for BtuCD by Locher et al. (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar), it has recently been disputed by Chen et al. (9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar) based on their results on the ATP-bound dimer of the maltose transporter, MalK. They have instead proposed a tweezer-like model in which nucleotide binding leads to the reverse effect, i.e. closure of the cytoplasmic gate and opening of the periplasmic end. Our findings on conformational changes associated with MgATP binding to BtuCD are likely representative of bacterial importers in general and may be extensible to other ABC transporters. Starting coordinates for the simulations were obtained from the x-ray structure of BtuCD (Protein Data Bank entry 1L7V) (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). Cyclotetravanadate was located in the ATP-binding sites in the crystal structure but did not affect the conformation of the NBD (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). We docked a MgATP molecule at the Walker A and B motifs of each of the two NBDs for the first simulation by superimposing the monomeric ATP-bound crystal structure of the nucleotide-binding domain of HisP (5Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F.-L. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar) onto the equivalent domains of BtuCD. A preequilibrated bilayer of 288 palmitoyloleoylphosphatidylethanolamine lipids solvated in single point charge water (28Hermans J. Berendsen H.J.C. van Gunsteren W.F. Postma J.P.M. Biopolymers. 1984; 23: 1513-1518Crossref Scopus (697) Google Scholar) was used as a model of the biological membrane. Lipids were removed to generate a hole in the center of the bilayer. In a short simulation using the procedure of Faraldo-Gomez et al. (29Faraldo-Gomez J.D. Smith G.R. Sansom M.S.P. Eur. Biophys. J. 2002; 31: 217-227Crossref PubMed Scopus (148) Google Scholar), remaining lipids were removed from the hole to generate a BtuCD-shaped cavity. The protein was inserted into the cavity. The entire system was then resolvated, and 18 chloride counter ions were added. The system (98,094 atoms) was equilibrated for 250 ps with positional restraints applied on the protein atoms (Fig. 1) to allow the solvent to relax. A second independent simulation without MgATP was set up from the same protein and lipid coordinates, but water and 20 chloride ions were added. The production runs, without any restraints, were 15 ns long for each of the two simulations. Both simulations were run with GROMACS (30Berendsen H.J.C. van der Spoel D. van Drunen R. Comput. Phys. Commun. 1995; 91: 43-56Crossref Scopus (6848) Google Scholar, 31Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar) using the ffG43a2 force field with a 2-fs time step. SETTLE (for water) and LINCS (32Hess B. Bekker H. Berendsen H.J.C. Fraaije J. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (10840) Google Scholar, 33Miyamoto S. Kollman P.A. J. Comput. Chem. 1992; 13: 952-962Crossref Scopus (4951) Google Scholar) were used to constrain covalent bond lengths. Long range electrostatic interactions were computed with the Particle-Mesh Ewald method (34Darden T. York D. Pedersen L. J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (19718) Google Scholar). The temperature was kept at 300 K by separately coupling the protein, lipids, nucleotide, and solvent to an external temperature bath (τ = 0.1 ps) (35Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (22543) Google Scholar). The pressure was kept constant at 1 bar by weak coupling (τ = 1.0 ps) to a pressure bath (35Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (22543) Google Scholar) in the z-dimension with constant area. The protein proved to be stable during both simulations. Molecular graphics were made using VMD (36Humphrey W. Dalke A. Schulten K. J. Mol. Graphics. 1996; 14: 33-38Crossref PubMed Scopus (35726) Google Scholar). Overall Conformational Changes—Snapshots of the ATP-bound and ATP-free protein at 15 ns are shown in Fig. 2 and in two supplementary movies (see supplemental materials). Fig. 2, B and C, are different orientations of the MgATP-bound snapshot showing the position of each docked ATP molecule at 15 ns. In Fig. 2, the yellow space-filling profile represents the pathway through which vitamin B12 is believed to pass during transport. The space between the two opposing NBDs is similarly presented in gray. It is evident from a visual comparison of Fig. 2A (nucleotide-free) with Fig. 2, B or C (MgATP-bound) that the docking of MgATP changes the overall shape of the protein. Conformational transitions occur in both the nucleotide-binding domains and the membrane-spanning domains. Differences in the size of the gray space-filling profile at the dimer interface of the NBDs after 15 ns of simulation without nucleotide (Fig. 2A) and with MgATP bound at the active site (Fig. 2, B and C) indicate that the docking of MgATP closes the gap between the two ATP-binding cassettes. The results of our simulation reveal that whereas the centers of mass of the two NBDs show a trend of movement toward each other in the MgATP-bound case, the two cassettes drift apart during the course of the MgATP-free simulation (details not shown). The small magnitudes of the overall center of mass motion recorded (1–2 Å) suggest that the TMD-NBD interface acts as a pivot about which the two NBDs rotate in a rigid body fashion toward each other. In effect, the dimension of the long axis of the intact transporter is shortened. Side chain displacements localized at the interface of the two NBDs contribute further to tighter dimerization of the NBDs. A striking feature was observed at the nucleotide-binding pockets. MgATP binding across the dimer interface occurred at only one of the two binding sites (Fig. 2C). At the other catalytic site, the binding pocket opened up, preventing the formation of hydrogen bonds between ATP and the signature motif of the opposing cassette (Fig. 2B). This occurred despite the fact that both nucleotides were docked in identical positions relative to the Walker A and B motifs of each NBD at the beginning of the simulation. Indeed, the separation distance between the centers of mass of the P-loop and the signature motif in the opposing NBD decreases by 0.4 Å at the catalytic site shown in Fig. 2C. The equivalent distance increases by 1.6 Å at the alternate site, shown in Fig. 2B. In contrast, the corresponding distances in the nucleotide-free simulation both increase by 2.2 and 2.6 Å, respectively (Fig. 2A). Events at the cytoplasmic end of the transmembrane pathway were analyzed by measuring the distance between the centers of mass of Thr-142 and Ser-143 located at the turn between membrane-spanning helices TM4 and TM5 in each half of the transporter. These residues were identified in the crystal structure as defining the gate region leading from the transmembrane pathway into the cytoplasmic water-filled channel at the interface of the NBDs and the TMDs (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). As the NBDs are drawn closer to each other due to ATP binding, the size of the gate region opening increases. This suggests that tighter ATP-induced association of the NBDs and the change in gate aperture are coupled. Analysis of the evolution of hydrogen-bonding patterns between ATP and the binding pocket and between the two NBDs suggests that closer association of the NBDs is aided by the formation of hydrogen bond contacts between residues of the two ATP-binding cassettes. ATP Binding—Over a time span of only 4 ns, significant conformational changes occur to optimize ATP binding at one of the two active sites in the ABC dimer. Surrounding water molecules were progressively displaced from hydrogen-bonding sites within the nucleotide-binding pocket located at the interface of the two opposing ATP-binding cassettes (Fig. 3, A and B). The signature motif in the opposite subunit reached out toward the docked ATP molecule. The nucleotide also shifted toward the signature motif and reoriented itself to form stable hydrogen bonds between the two side chain oxygen atoms of the glutamate residue in the LSGGE signature motif and the two hydroxyl hydrogen atoms in the ribose moiety of ATP. This is consistent with equivalent hydrogen bonds formed between the ribose hydroxyls and the corresponding Gln of the LSGGQ motifs in the ATP-bound dimers of MalK and E171Q-MJ0796 (26Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). The Glu residue of the LSGGE signature motif of BtuCD also forms a water-mediated hydrogen bond with one of the γ-phosphate oxygen atoms of ATP. Coordination of the magnesium ion cofactor occurred via the α-, β-, and γ-phosphate oxygen atoms of ATP, Gln-80 of the Q-loop, as well as Ser-40 of the Walker A motif and a solvent water molecule. Direct contact between metal ion cofactors and Gln-80 has been similarly observed in the dimeric crystal structures of Rad50-AMPPNPMG2+ and E171Q-MJ0796-Na+ (13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar, 26Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Allosteric Control of Translocation Pathway—The transmembrane translocation pathway becomes constricted as the crystal structure relaxes to new conformations in solution without bound MgATP (Fig. 4, A and B). It is, however, evident from a comparison of the MgATP-bound and MgATP-free structures at 15 ns (Fig. 4, B and C) that further constriction of the translocation pathway occurs as an effect of nucleotide occupancy. Fig. 4, A–C, show that the reorientation of transmembrane helix 5 (TM5) in each monomeric unit plays an important role in mediating changes in pathway profile. To quantitatively map out the changes taking place in the membrane-spanning pathway, separation distances between equivalent C-α atoms of TM5 from each subunit were measured at approximately every turn of the helix from the cytoplasmic to the periplasmic end. The results are presented in Fig. 5. In the crystal structure, the periplasmic end of the translocation pathway was open wide enough to accommodate a vitamin B12 molecule (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). Our results show that the periplasmic opening collapses by ∼7 Å in the ligand-free simulations. This implies that the periplasmic entrance may be more constricted in solution than it is in the crystal structure. The observed mobility may also explain why residues at the entrance to the periplasm were only partly resolved in the crystal structure (11Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (920) Google Scholar). Fig. 5 shows that the docking of MgATP at the catalytic sites allosterically induces further constriction of the translocation pathway by up to 5 Å. Profile differences of interest are also observable at the gate region (Thr-142 and Ser-143) that leads into the water-filled channel at the center of the transporter. Vitamin B12 is believed to escape into the cytoplasm of the cell through this channel. The dimension of the opening at the gate region increases in response to the docking of MgATP, whereas in the absence of MgATP, it becomes slightly narrower, as evident in the segment containing residues 142–150 in Fig. 5. High resolution structures have played an important role in aiding our understanding of the mechanism of ABC transporters. Protein function, however, involves movement, and molecular dynamics simulations are one way of probing potential functional motions starting from rigid crystal structures. Several recent computer simulation studies have demonstrated the usefulness of accessible conformational states in unraveling the functional mechanics of protein systems comparable with BtuCD (22Berendsen H.J.C. Science. 2001; 294: 2304-2305Crossref PubMed Scopus (16) Google Scholar, 37de Groot B.L. Grubmüller H. Science. 2001; 294: 2353-2357Crossref PubMed Scopus (801) Google Scholar, 38Böckmann R.A. Grubmüller H. Nat. Struct. Mol. Biol. 2002; 9: 198-202Google Scholar). The main limitation of modern simulations is the fact that conformations separated by high energy barriers are unlikely to be visited during the simulation on a time scale that can be simulated directly. The fast changes observed in the nucleotide-binding domains and in the transmembrane domains indicate significant driving forces for these motions. Nucleotide Binding—Nucleotide binding is a two-step process. First, MgATP diffuses and docks into the catalytic pocket, producing a weak binding conformation of the complex. The nucleotide is subsequently sequestered, and binding interactions are optimized to generate a tight binding conformation. For F1F0-ATP synthase, Antes et al. (39Antes I. Chandler D. Wang H.Y. Oster G. Biophys. J. 2003; 85: 695-706Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) have suggested that energy transduction occurs during the transition from the weak to the tight binding state. In our simulation, we circumvent the slow diffusion process and start with the MgATP weakly docked at the binding pocket. Within only 4 ns, there is evidence that the transition of the complex from the weak to the tight binding state has largely occurred. The resulting dimer likely represents the catalytically competent state of the ATP-binding cassette (13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). The short time scale in which this transition takes place is an indication of the strong attractive force involved and suggests that the energy transduced as a result may be sufficient to drive the power stroke of the transporter. However, we observe that just one ATP molecule appears capable of holding the transporter in the closed nucleotide-bound state. Tight ATP binding at one catalytic site occurs concomitantly with opening of the binding pocket at the alternate site in a manner that may help create an exit path for the release of hydrolyzed nucleotide. This finding may at first appear contrary to evidence from recent crystal structures of dimeric NBDs of Rad50, MJ0796, and MalK in which ATP is bound at each of the two catalytic sites (9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 12Moody J.E. Millen L. Binns D. Hunt J.F. Thomas P.J. J. Biol. Chem. 2002; 277: 21111-21114Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 13Hopfner K.P. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). However, it is notable that in all three cases either the protein used was a catalytically incapacitated mutant (MJ0796 (E171Q)) or the magnesium cofactor was excluded to prevent hydrolysis (Rad50 and Malk). The crystal structure of catalytically competent wild-type NBDs with MgATP stably bound at both nucleotide-binding sites has so far proven elusive. We suggest that this may be attributed to the asymmetry in ATP binding observed in our simulations. The singly occupied dimer is unstable without additional contacts provided by the transmembrane domain. Under non-hydrolyzing conditions, the two binding sites are more likely to be simultaneously occupied with ATP. Together, both ATP molecules may thus provide sufficient contacts to stabilize the dimer in the absence of transmembrane domains as observed in the crystal structures of Rad50, MJ0796, and MalK. Other lines of experimental evidence in support of this kind of asymmetry include vanadate trapping studies in which the protein is incubated with vanadate and [α-32P]MgATP (40Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubM" @default.
• W2021253110 created "2016-06-24" @default.
• W2021253110 creator A5065556977 @default.
• W2021253110 creator A5080678224 @default.
• W2021253110 date "2004-10-01" @default.
• W2021253110 modified "2023-09-27" @default.
• W2021253110 title "Conformational Transitions Induced by the Binding of MgATP to the Vitamin B12 ATP-binding Cassette (ABC) Transporter BtuCD" @default.
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