Conformational Dynamics and Protein–Substrate Interaction of ABC

Nov 22, 2016 - ABSTRACT: ATP-binding cassette (ABC) transporters are ubiquitous in all three kingdoms of life and are implicated in many clinically re...
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Conformational Dynamics and Protein−Substrate Interaction of ABC Transporter BtuCD at the Occluded State Revealed by Molecular Dynamics Simulations Chao Pan, Jingwei Weng,* and Wenning Wang* Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Department of Chemistry, and Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China S Supporting Information *

ABSTRACT: ATP-binding cassette (ABC) transporters are ubiquitous in all three kingdoms of life and are implicated in many clinically relevant physiological processes. They couple the energy released by ATP hydrolysis to facilitate substrate translocation across cell membranes. The crystal structures of type II ABC importers have revealed their unique transmembrane domain architecture consisting of 10 transmembrane helices and their structurally conserved nucleotidebinding domains among all ABC transporters. However, molecular details of the interactions between the importers and their substrate remain largely elusive. Taking vitamin B12 importer BtuCD as an exemplar of type II importers, we investigated the dynamics of its occluded state and the detailed protein−substrate interactions using molecular dynamics simulation. Our trajectories show that the importer accommodates the substrate through a nonspecific binding mode as the substrate undergoes evident vertical and tilt motions inside the translocation cavity. Extensive hydrogen bond and hydrophobic interactions were observed between the substrate and the importer; however, most of these interactions are weak, with 55° (Figure 2c). The relaxation of the initial model structure is also observed during the initial stage of the simulations. Two major tilt states can be identified centered at 18° and 35°, with almost equivalent populations. Different orientations imply various binding modes of the substrate. The cyano group points approximately toward the TM5aA helix in the more tilted state (Figure 2d) but reorients toward the TM3A helix in the less tilted state (Figure 2e). The general features of the vertical and tilt motions remain stable during the late stage of the simulations (Figure 2b,c), indicating that our sampling really reflects the nature of B12 binding. Consistent with the evident vertical and tilt motions of B12, the hydrogen bond interactions between BtuCD and B12 are also weak and variable, with occurrences of most hydrogen bonds of 4 Å, indicating evident vertical motion that implies that the substrate is very loosely bound in the cavity. Rotational motions were also observed, though there is no evident correlation between the rotational and vertical motions. E

DOI: 10.1021/acs.biochem.6b00386 Biochemistry XXXX, XXX, XXX−XXX

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Figure 4. Conformational changes in the BtuCD-F-ATP-B12 system. (a) Density map of all five 120 ns trajectories projected on the two-dimensional space spanned by dS143 and dL85. The projection corresponding to the snapshot of trajectory 1 at 118.15 ns is represented as a magenta cross. (b) Average radius profiles of the translocation pathway in trajectory 1. The zero point of the pore axis is placed at the center of mass of the TMD dimer. (c) Variation of the distance between the mass centers of the WAC motif and the juxtaposed signatureD motif over simulation time in the trajectories. The black, red, blue, dark green, and magenta curves stand for the values of trajectories 1−5, respectively. The values of the crystal structures are denoted by horizontal lines. (d) Variation of the distance between the mass centers of the WAD motif and the juxtaposed signatureC motif over simulation time along the trajectories.

hydrogen bond interactions, and TMDA is slightly more involved than TMDB. For both tilt states, residues on the exTM3 stretches, TM3 and TM5 helices, and the carbamino groups of the substrate were most frequently observed in the interactions (Table S3 and Figure 2a). Glu87A, Ser94A, and Ala252B are more favorable for B12 in the more tilted state, whereas preference is shown for Asn95A, Ser157A, Glu87B, Asn95B, Ser157B, and Met179B in the less tilted state (Figure 2d,e and Table S3). It is worth noting that even for the hydrogen bonds involving the same residue, the interactions can be very variable, involving different atoms from the residue and the substrate. A typical example is Asn95B. Its carbonyl oxygen on the side chain can interact with one carbamino group of B12, whereas its amino group can also donate hydrogen bonds to the cyano group or another carbamino group of B12 (Table S3). The hydrophobic interactions between BtuCD and the corrin ring of B12 are also variable, as their occurrences were also 18 Å (Figure 4a). Therefore, cytoplasmic gate I is generally more flexible and has high probability of adopting more opened conformations in the presence of B12 (Figure 4a and Table S2). On the other hand, F

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Figure 5. Conformational changes in the BtuCD-F system. (a) Variation of the distance between the mass centers of the WAC motif and the juxtaposed signatureD motif over simulation time. The black, red, blue, dark green, and magenta curves stand for the values of trajectories 1−5, respectively. The values of the crystal structures are denoted by horizontal lines. (b) Variation of the distance between the mass centers of the WAD motif and the juxtaposed signatureC motif. (c) Density map of all five trajectories projected on the two-dimensional space spanned by the distances between the conserved motifs at two nucleotide-binding sites. (d) Density map of all five trajectories projected on the two-dimensional space spanned by dS143 and dL85. The projections of the snapshot in trajectory 2 with the most closed cytoplasmic gate I are represented by magenta crosses.

the occluded state well, as indicated by the undisturbed Cα RMSD (Figure S5b). The distances between the conserved motifs at the NBD interface fluctuated around 9 Å at one site and increased slightly at the other site (Figure S7), though the detailed interactions between the nucleotide and the binding site were mostly preserved. At the TMDs, the translocation pathway was tightly occluded at both ends (Figure S8). The cytoplasmic gates were still flexible as observed in the system with BtuF bound (Table S1 and S2), and cytoplasmic gate I was slightly more opened (Table S2), similar to the difference between the crystal structures of outII-BtuCD and occ-BtuCD states (Figure 1g). Opening of the periplasmic ends of TM5 helices in the outII-BtuCD state was not observed in our trajectories (Figure S9), possibly because of the limited length of the simulation. Removal of the Nucleotides Evidently Destabilizes the NBD Interface and the Cytoplasmic Gate. To inspect the effect of ATP binding on the stability of the occluded state, we removed the nucleotides and launched five parallel trajectories. The Cα RMSD increased to >2.5 Å, exhibiting the largest variations among all the simulation systems (Figure S5c). The most evident change in the BtuCD-F system occurred at the NBD interface. In three of the trajectories, at least one of the nucleotide-binding sites visited the semiopen state with the distance between the conserved motifs increased to >13 Å (Figure 5a,b). Intriguingly, one of the sites demonstrates an accelerated tendency to open (Figure 5b) relative to that of the other site (Figure 5a), leading to an asymmetrical arrangement at the NBD dimer interface (Figure 5c). The asymmetrical behavior was also observed in other ABC transporters18,23,53 and may be attributed to the subtle differences between two NBDs in the crystal structure. In spite of the asynchronous opening motion, the sites were also

cytoplasmic gate II also shows enhanced conformational flexibility upon B12 binding. The most populated dL85 value is around 8 Å, but a small population sampled the region with a dL85 value of >12 Å. For example, in a snapshot at 118.15 ns of trajectory 1, dL85 reached one of its maxima at 13.4 Å while cytoplasmic gate I closed slightly relative to the crystal structure (the magenta cross in Figure 4a). Overall, the presence of B12 expands the conformational space of both cytoplasmic gates I and II and could result in a 1−2 Å increase in the average radius at the cytoplasmic constriction (Figure 4b), though the dilation is still not sufficient for substrate permeation. In spite of the changes at the translocation cavity and the cytoplasmic gates, other conformational features of the occluded state were almost unaffected. The periplasmic side of the translocation pathway remained fully sealed (Figure 4b and Figure S6), and the distance between TM5a helices was only slight disturbed (Figure 3g−i). The BtuF−BtuC interface was almost invariable as the buried surface area between the subunits fluctuates around 5900 Å2, without any sign of dissociation. Both nucleotide-binding sites remained intact in most trajectories (Figure 4c,d), tightly sandwiching the ATP molecules with extensive interaction networks. The only exception is trajectory 3, whose site formed by WAD and signatureC motifs became separated by 1 Å in the last 30 ns (blue curve in Figure 4d). However, the contacts between the signature motif and ATP were still retained with Ser128 on the signature motif reorienting from the γ-phosphate group of ATP to the α-phosphate group. Removal of BtuF Does Not Affect the Fully Closed Arrangement of the NBD Interface. To test the effect of BtuF on the structural stability of the occluded state, we removed the PBP from the system and conducted the simulations. The BtuCD-ATP system retains the features of G

DOI: 10.1021/acs.biochem.6b00386 Biochemistry XXXX, XXX, XXX−XXX

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Figure 6. Conformational changes in the BtuCD-F-ADP-B12 system. (a) Variation of the distance between the mass centers of the WAC motif and the juxtaposed signatureD motif over simulation time in five 120 ns trajectories. The black, red, blue, dark green, and magenta curves stand for the values of trajectories 1−5, respectively. The values of the crystal structures are denoted by horizontal lines. (b) Variation of the distance between the mass centers of the WAD motif and the juxtaposed signatureC motif over simulation time. (c) Top view of the snapshot of the NBD dimer interface at 102.725 ns of trajectory 1, which shows the most opened nucleotide-binding site. The protein subunits are represented by ribbons with NBDC colored orange and NBDD yellow. ADP and inorganic phosphate are shown as a licorice model with carbon, oxygen, nitrogen, and phosphate atoms colored cyan, red, blue, and tan, respectively. Mg2+ is represented as green spheres. Glu131 on signature motifD sharing hydrogen bond interactions with ADP (denoted by black dashed lines) is shown as a licorice model and is colored similarly. (d) Density plot of all five 120 ns trajectories projected on the two-dimensional space spanned by dS143 and dL85. (e) Average radius profile of the translocation pathway in trajectory 1.

phosphates in the simulation system to monitor the conformational changes. The Cα RMSDs of this BtuCD-F-ADP-B12 system increased by >2.5 Å at the end of the simulations (Figure S5d). One of the nucleotide-binding sites is hardly affected (Figure 1a), but the other site exhibits opening motion with a significant increase in the distance between WA and signature motifs (Figure 6b). All interdomain hydrogen bond interactions between the nucleotide and the signature motif at the opening site were disrupted, whereas the interactions at the other site were partially retained (Figure 6c). Therefore, these simulations demonstrate that ATP hydrolysis is effective in triggering the opening motion of the nucleotide-binding site, and the asymmetric opening of the NBD dimer is favorable, similar to the case in which the nucleotides were removed. Besides the changes in NBDs, the cytoplasmic gates of the translocation pathway also respond to ATP hydrolysis. Compared with that of the BtuCD-F system, the closing of cytoplasmic gate I is less evident, and there is some population with dS143 shorter than that of the crystal structure (Figure 6d and Tables S1 and S2). Despite the conformational variation at cytoplasmic gates, the translocation pathway was tightly occluded throughout the simulation (Figure 6e and Figure S11). Taken together, the posthydrolysis system shows evident asymmetrical opening motion of nucleotide-binding sites and complete detachment of ADP from the signature motif at one

observed to be simultaneously semiopened at the last 20 ns in trajectory 3 (blue curves in Figure 5a,b), leading to a symmetrical NBD interface similar to the outI-BtuCD and asym-BtuCD states (Figure 5c). Taken together, the presence of a semiopen site strongly indicates that the nucleotides are crucial for a fully closed NBD interface. Conformational flexibility at the cytoplasmic gates was also observed in the BtuCD-F system (Tables S1 and S2), though the translocation pathway remained tightly occluded at both ends throughout the simulations (Figure S10). Trajectory 2 of the system shows the most closed cytoplasmic gate I in all trajectories. The minimum of dS143 decreased to 10.0 Å, 2.2 Å smaller than that of the BtuCD-F-ATP system (Table S2 and the magenta cross in Figure 5d). Closing of gate I was accompanied by partial opening of cytoplasmic gate II (dL85 = 11.5 Å), with the cytoplasmic gates more similar to the asymBtuCD state rather than the in-MolBC state (Figure 5d). The closing motion of gate I demonstrates the notable effect of nucleotide binding on the conformation of cytoplasmic gates. ATP Hydrolysis Induces Evident Opening of One Nucleotide-Binding Site. It has been supposed that ATP hydrolysis would trigger the conformational changes of the occBtuCD state and facilitate the translocation of B12.13 To examine the effect of ATP hydrolysis (distinct from nucleotide binding), we also replaced ATPs with ADPs and inorganic H

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Cytoplasmic gate II could vary by >5 Å (Figure 1g and Table S1) as does cytoplasmic gate I (Table S2), indicating that the conformational transitions in these regions would occur more easily than the crystal structures demonstrate. We did not observe significant conformational changes upon removal of BtuF (Figure S7); however, removal of nucleotides (the BtuCD-F system) evidently destabilized the conformational state. In three of five trajectories, at least one of the nucleotide-binding sites turned to semiopen as observed in the outI-BtuCD and asym-BtuCD states (Figure 5a−c). In one of five 120 ns trajectories, we also observed evident closing motion of cytoplasmic gate I (Figure 5d and Table S2), accompanied by partial opening of cytoplasmic gate II. The motion leads the cytoplasmic gates to being more similar to the asym-BtuCD state rather than the in-MolBC state (Figure 5d), though the asymmetrical arrangement of TM5 helices was not observed (data not shown). The cytoplasmic motions are in agreement with the results of the electron paramagnetic resonance (EPR) experiments,14,15 which show that apo BtuCD-F complex in liposomes has more closed cytoplasmic gate I and more opened cytoplasmic gate II than the ATPbinding state does. Similarly, in the ADP-bound system, evident opening (>7 Å) was observed in one nucleotide-binding site (Figure 6b), with the nucleotide completely detached from the signature motif, whereas the other site remained closed (Figure 6a,c). The conformational changes of the cytoplasmic gates upon hydrolysis are similar to those of the nucleotide-free state, but less evident. These conformational changes observed in the nucleotidefree and ADP-bound systems are physiologically important as they are likely to occur in a futile transport cycle (with no substrate binding) after the release of hydrolysis products. The cycle may start with the outII-BtuCD state13 because of the proliferation of ATP in cells. As BtuCD still shows a higher apparent affinity for substrate-free BtuF revealed by microscale thermophoresis experiments,13 the occluded state of the BtuCD-F complex is formed.8 ATP hydrolysis and the subsequent release of hydrolysis products lead to the dissociation of the NBD interface and induce further changes at the TMDs. Because of the absence of the substrate, simultaneous opening of cytoplasmic gates I and II may not be activated as mentioned above (Figure 1d vs Figure 4a), and the inward-facing state that is analogous to the MolBC crystal structure16 may be skipped. The importer would evolve toward the asym-BtuCD state7 instead by opening cytoplasmic gate II and closing cytoplasmic gate I, as indicated by our trajectory 2 for the BtuCD-F system (Figure 5d). Then the transport cycle is restarted by BtuF dissociation and nucleotide binding, during which the importer may briefly remain in the outI-BtuCD state.6 In spite of the accumulating evidence, many questions about the detailed translocation mechanism remain to be answered. One important question is how B12 dissociates from BtuF and enters the translocation cavity. On the basis of the current crystal structures, the TM5a helices have to separate to open the periplasmic gate and allow access, but how the separation occurs is largely unknown. Other questions include how nucleotide hydrolysis triggers the simultaneous opening of both cytoplasmic gates and how the substrate passes through the gates and diffuses into the cytoplasm. Further experimental and computational efforts are required to elucidate these questions.

site, though these motions are not enough to open the cytoplasmic gates and facilitate substrate translocation.

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DISCUSSION Molecular details of the interactions between ABC transporters and their substrates are crucial for the understanding of the translocation mechanism. For type I ABC importers such as maltose transporter MalFGK2, the interaction details are provided well by crystal structures. A binding site was clearly identified between TM helices establishing specific hydrogen bonds and van der Waals interactions with the sugar rings54,55 and shows a moderate affinity of 1 mM.56 Moreover, the crystal structures also reveal a continuous pathway extending from the binding pocket on PBP to the TM helices, which probably facilitates the diffusion of the substrate to the TM site. For type II importer BtuCD, however, the translocation process between PBP and the importer is different. First, no continuous pathway between the binding pocket in BtuF and the binding site in BtuCD is observed in any available crystal structure,6−8,57 or that of the homologous importers.16,58 The TM5a helices always disrupt the pathway and shield the translocation cavity from the periplasmic space, which would be largely responsible for the unmeasurable binding affinity of B12 for BtuCD or BtuCD-F.8,11 Second, as we demonstrate in this work, the occluded state of BtuCD-F can readily accommodate B12, with all parts constituting the cavity participating in substrate binding. However, most hydrogen bond and hydrophobic interactions between the cavity and the substrate are weak and variable, with an occurrence of ≤38% (Tables S3 and S4). Therefore, our simulations suggest that B12 cannot form specific bonds to the cavity. Moreover, the binding of B12 in BtuCD is entirely different from those of other proteins cocrystallized with B12, including the cofactor-binding pockets of B12-dependent enzymes,59−62 the B12-transporting protein intrinsic factor63 and transcobalamin,64,65 cobalamin outer membrane transporter BtuB, 66,67 and the B 12 PBP BtuF.30,35,51,68 We found that the cobalt−corrin complex undergoes evident global motions inside the cavity of BtuCD, including a 4 Å vertical motion (Figure 2b) and a 55° tilt motion (Figure 2c), which are consistent with the weak interactions. Two major tilt states that show quite different binding patterns were identified (Figure 2d,e and Tables S3 and S4). The evident motions indicate that even when the crystal structure of the occluded state is obtained with B12 binding, it is still probable that the atoms of the cobalt−corrin complex remain invisible as their positions are averaged out in the electron density map. The global motions and the weak and nonspecific interactions also explain the absence of a continuous translocation pathway in BtuCD. If the TM5a helices did not disrupt the pathway, B12 would probably dissociate from the cavity and diffuse back to the binding pocket in BtuF. Therefore, tightly closing the periplasmic gate (as illustrated by Figures 1d and 4b and Figures S3, S6, S8, and S10 with the radius at the region invariably kept under 1 Å) might be functionally important to facilitate unidirectional translocation. Besides protein−substrate interactions, our simulations also revealed conformational dynamics of the transporter. The main features of the occluded state, including the occluded cavity and the fully closed NBD interface, are well retained with Mg·ATPbound form even when the chemical modifications are removed (Figure 1d−f and Figure S3). Conformational flexibility at the cytoplasmic ends of the translocation pathway is worth noting. I

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with their substrate binding proteins. Proc. Natl. Acad. Sci. U. S. A. 110, 5440−5445. (13) Korkhov, V. M., Mireku, S. A., Veprintsev, D. B., and Locher, K. P. (2014) Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F. Nat. Struct. Mol. Biol. 21, 1097−1099. (14) Joseph, B., Jeschke, G., Goetz, B. A., Locher, K. P., and Bordignon, E. (2011) Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle. J. Biol. Chem. 286, 41008−41017. (15) Joseph, B., Korkhov, V. M., Yulikov, M., Jeschke, G., and Bordignon, E. (2014) Conformational cycle of the vitamin B12 ABC importer in liposomes detected by double electron-electron resonance (DEER). J. Biol. Chem. 289, 3176−3185. (16) Pinkett, H. W., Lee, A. T., Lum, P., Locher, K. P., and Rees, D. C. (2007) An inward-facing conformation of a putative metal-chelatetype ABC transporter. Science 315, 373−377. (17) Stansfeld, P. J., and Sansom, M. S. (2011) Structure 19, 1562− 1572. (18) Aittoniemi, J., de Wet, H., Ashcroft, F. M., and Sansom, M. S. (2010) Asymmetric switching in a homodimeric ABC transporter: a simulation study. PLoS Comput. Biol. 6, e1000762. (19) Becker, J. P., Van Bambeke, F., Tulkens, P. M., and Prevost, M. (2010) Dynamics and structural changes induced by ATP binding in SAV1866, a bacterial ABC exporter. J. Phys. Chem. B 114, 15948− 15957. (20) Ferreira, R. J., Ferreira, M. J. U., and dos Santos, D. (2012) Insights on P-Glycoprotein’s Efflux Mechanism Obtained by Molecular Dynamics Simulations. J. Chem. Theory Comput. 8, 1853− 1864. (21) Furukawa-Hagiya, T., Furuta, T., Chiba, S., Sohma, Y., and Sakurai, M. (2013) The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations. J. Phys. Chem. B 117, 83−93. (22) Jones, P. M., and George, A. M. (2011) Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and mechanistic basis for the asymmetric occluded state. Biophys. J. 100, 3025−3034. (23) Oliveira, A. S., Baptista, A. M., and Soares, C. M. (2011) Conformational changes induced by ATP-hydrolysis in an ABC transporter: a molecular dynamics study of the Sav1866 exporter. Proteins: Struct., Funct., Genet. 79, 1977−1990. (24) St-Pierre, J. F., Bunker, A., Rog, T., Karttunen, M., and Mousseau, N. (2012) Molecular dynamics simulations of the bacterial ABC transporter SAV1866 in the closed form. J. Phys. Chem. B 116, 2934−2942. (25) Wen, P. C., Verhalen, B., Wilkens, S., McHaourab, H. S., and Tajkhorshid, E. (2013) On the origin of large flexibility of Pglycoprotein in the inward-facing state. J. Biol. Chem. 288, 19211− 19220. (26) Oliveira, A. S., Baptista, A. M., and Soares, C. M. (2011) Interdomain communication mechanisms in an ABC importer: a molecular dynamics study of the MalFGK2E complex. PLoS Comput. Biol. 7, e1002128. (27) Wen, P. C., and Tajkhorshid, E. (2011) Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters. Biophys. J. 101, 680−690. (28) Oloo, E. O., and Tieleman, D. P. (2004) Conformational transitions induced by the binding of MgATP to the vitamin B-12 ATP-binding cassette (ABC) transporter BtuCD. J. Biol. Chem. 279, 45013−45019. (29) Ivetac, A., Campbell, J. D., and Sansom, M. S. P. (2007) Dynamics and function in a bacterial ABC transporter: Simulation studies of the BtuCDF system and its components. Biochemistry 46, 2767−2778. (30) Kandt, C., and Tieleman, D. P. (2010) Holo-BtuF stabilizes the open conformation of the vitamin B12 ABC transporter BtuCD. Proteins: Struct., Funct., Genet. 78, 738−753.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00386. Two tables of conformational changes and two tables of B12−protein interactions, one figure of five conformational states of the type II importer, one figure of B12 structure and force field parameters, and nine figures of detailed conformational changes of the importer (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

This work was supported by the National Major Basic Research Program of China (2016YFA0501702), the National Science Foundation of China (21473034 and 21403036), the Specialized Research Fund for the Doctoral Program of Higher Education (20130071140004), and the Science & Technology Commission of Shanghai Municipality (08DZ2270500). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the supercomputer center of Fudan University for their allocation of computer time. REFERENCES

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DOI: 10.1021/acs.biochem.6b00386 Biochemistry XXXX, XXX, XXX−XXX