Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein

Abstract

ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD)1,2,3,4. Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron–electron resonance5,6 and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy7. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.

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Figure 1: Pgp alternating access.
Figure 2: Conformational dynamics of the NBSs.
Figure 3: Intrinsic local asymmetry at the NBSs reflects catalytic asymmetry.
Figure 4: Model of energy input and alternating access in Pgp.

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Acknowledgements

We thank R. Stein for advice on the global analysis of DEER data, I. L. Urbatsch for donation of Pichia pastoris expression construct, and S. Wilkens for providing three expressed mutants (615–1276, 627–1260 and 613–1258) in P. pastoris. This work was supported by National Institutes of Health grants U54-GM087519 (to H.S.M., R.K.N. and E.T.) and P41-GM104601 (to E.T.). We also acknowledge computing resources provided by Blue Waters at National Center for Supercomputing Applications, Department of Energy Innovative and Novel Computational Impact on Theory and Experiment, and Extreme Science and Engineering Discovery Environment (grant TG-MCA06N060 to E.T.).

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Authors

Contributions

H.S.M., B.V. and R.K.N. designed the experimental studies. B.V. and R.D. purified the mutants and performed the experiments. R.D. and B.V. analysed the data. S.T. and E.T. designed and performed the molecular dynamics simulations. H.S.M., R.D., B.V., S.T. and E.T. wrote the paper. Y.P. cloned and expressed the mutants. H.A.K. and B.V. initially contributed in the optimization, cloning and expression of Pgp.

Corresponding author

Correspondence to Hassane S. Mchaourab.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 ATPase activity.

a, Representative ATPase assays on Pgp mutants. Basal (black) and Ver-stimulated (blue) ATP hydrolysis were measured by Pi-release. The red dots represent the release rate under Vi-trapping condition (5 mM ATP, 1 mM Vi). b, c, The maximum activity (Vmax) values for mutants with intact NBSs (b) and with catalytic glutamate substitutions (c) are determined by fitting the data with simple Michaelis–Menten equation. Error bars are fitting errors or standard deviation (Cys-less Pgp).

Extended Data Figure 2 NBDs form a closed dimer in the Vi-trapped transition state.

From left to right, ribbon representation of NBDs viewed from the cytosolic side (PDB accession 4M1M) with residues mutated to Cys represented as purple sticks, and DEER distance distributions. Inward-facing and outward-facing (OF) distance distributions predicted from the X-ray structure of the inward-facing, and from the outward-facing model, are shown as dotted black and red traces, respectively. Residues 627 and 1276 were not resolved in the X-ray structure. The datasets on spin-labelled pairs 607–1252 and 613–1258 are repeated from Fig. 1b.

Extended Data Figure 3 Opening of the extracellular side of the TMD in the transition state.

a, b, Spin-labelled pairs designed to monitor distances between structurally nonequivalent helices (a) as well as equivalent helices (b) in the two leaflets are represented as purple spheres. Changes in the distance distributions are predominantly indicative of increased distances as predicted by the MsbA outward-facing (OF) structure and consistent with the Pgp outward-facing model. However, even for residues where distance changes are observed, the distributions are broad and components similar to those of the apo state are present, suggesting a heterogeneous transitions state. Inward-facing and outward-facing distance distributions predicted from the X-ray structure of the inward-facing and from the outward-facing model are shown as dotted black and red traces, respectively.

Extended Data Figure 4 Local asymmetry of the NBSs.

a, Positions of the mutated residues in each NBS are represented as purple sticks. be, Distance distributions monitoring each NBS in the wild-type (WT; b), E552Q (c), E1197Q (d) and E552Q/E1197Q (e) backgrounds. Spin-labelled pairs 400–1121 and 478–1043 have greatly reduced ATPase activity (see Extended Data Fig. 1). However, comparing the distributions of these spin-labelled pairs to those of 400–1156 and 511–1043, the pattern of changes in distance distributions is consistent, confirming the asymmetry is localized to the region of the A-loop. The data sets on spin-labelled pairs 400–1156 and 511–1043 are repeated from Fig. 3.

Extended Data Figure 5 Assembly of the NBSs upon Vi-trapping of the transition state.

a, Ribbon representation of NBSs viewed from the side with the mutated residues represented as purple sticks. be, Distance distributions monitoring each NBS in the wild-type (WT; b, repeated from Fig. 2b), E552Q (c), E1197Q (d) and E552Q/E1197Q (e) backgrounds. The pattern and amplitude of the distance changes at these locations are similar and consistent with the proximity of the Walker A and signature motifs in the outward-facing (OF) conformation as previously observed for MsbA and Sav1866. Introduction of the catalytic glutamate substitutions does not affect the distance changes. Moreover, these substitutions stabilize the transitions state so that it is populated under turnover conditions—that is, in the presence of ATP/Mg2+ and Ver (ATP-Ver, blue). These results demonstrate that the asymmetry identified in Extended Data Fig. 4 and its dependence on the catalytic activity of each NBS is localized to the A-loop region.

Extended Data Figure 6 The mutations of the catalytic glutamates do not alter the TMD conformational changes but stabilize the transition state.

a, Cytoplasmic (left three panels) and extracellular (right panel) TMD regions with spin-labelled residues represented as purple sticks. Distance distributions monitoring cytoplasmic and extracellular closing and opening in the wild-type (WT; b, repeated from Fig. 1c, d), E552Q (c), E1197Q (d) and E552Q/E1197Q (e) backgrounds. In the Vi-trapped transition state (ADP-Vi-Ver, red), the distance distributions are consistent with population of the outward-facing (OF) conformation. In the E-to-Q backgrounds, ATP/Mg2+ is sufficient to stabilize Pgp to outward-facing.

Extended Data Figure 7 Structural modelling of the unknown outward-facing state.

a, Structural alignment of all the TM helices between mouse Pgp and MsbA, which is used as a structural template in model construction. Regions highlighted with black dashed boxes indicate the missing structural segments from the template. TM helices of MsbA are coloured silver whereas TM helices 1–6 and 7–12 of Pgp are coloured yellow and cyan, respectively. The residues of TM helices and intracellular helices (IH) are labelled in colours corresponding to their structures. b, Incomplete model of Pgp outward-facing state resulted from the alignment without fixing the missing segments. Incomplete structural regions are highlighted in black dashed boxes. c, A complete model of Pgp outward-facing state after rebuilding the incomplete regions with structural information obtained from the inward-facing crystal structure of mouse Pgp (PDB accession 4M1M).

Extended Data Figure 8 Collective variables and structural features used to obtain the outward-facing state.

ag, Description of the collective variables (CVs) used to obtain a reliable outward-facing state of Pgp (ad) and tracking important structural features to verify the stability of the outward-facing state (eg). a, Orientation quaternion (β) describing the angle between the two bundles of TM helices that separate to form the outward-facing state. Distance between K185 and D993, a charged residue pair located within the translocation chamber. b, CVs used to form accurate NBD-based interactions, which include NBD–ATP interactions and X-loop interactions. Walker A (WA1 and WA2), Walker B (WB1 and WB2) and LSGGQ (L1 and L2) motifs are shown in purple, yellow and green new cartoon representations, respectively. Y397/1040 from the A-loop (white) and ATPs (cyan) are shown in stick representations, whereas Mg2+ ions and Cα carbons of X-loop residues are displayed as grey and white beads, respectively. c, Metrics used in evaluating the basic structural elements that are key to any outward-facing ABC exporter, namely, dimerized NBDs (dNBD), closed cytoplasmic (α), and opened extracellular/periplasmic (β) sides. d, Sim1 (light colours) failed to maintain these basic structural requirements within 10 ns, whereas Sim2 (dark colours) results in a stable outward-facing state for up to 300 ns. Solid and dotted horizontal lines represent the corresponding values in inward-facing and outward-facing conformations, respectively, based on crystal structures of Pgp (PDB accession 4M1M) and MsbA (PDB accession 3B60). e, Description and time series of centre of mass distance between extended TM helical regions of TM3 (V164–V175) and TM10 (E887–E898) shown in orange, and TM4 (A244–A255) and TM9 (T806–D817) shown in pink, describing the tight closing of cytoplasmic side. f, Description and time series of centre of mass distance between the residues forming the top half of TMDs that open at the extracellular side (shown with pink and orange beads). g, Salt bridge interaction between K185 (TM3) and D993 (TM12). These calculations are compared between five different simulations.

Extended Data Table 1 List of collective variables used in non-equilibrium simulations
Extended Data Table 2 Details of the five simulations performed in the present study with different combinations of collective variables

Supplementary information

Supplementary Information

This file contains Supplementary Text and additional references. Experimental design and interpretation is expanded to include rationale, detailed analysis of DEER data, and the IF/OF alternating access model. The overarching mechanistic implications of the ABC transporter diversity is discussed in relation to literature and this work. (PDF 668 kb)

Dynamics of outward-facing (OF) state of Pgp in membrane.

Dynamics of OF state of Pgp within membrane is shown from both the front and side views. Also, the restrained and free (unbiased) parts of the simulations are labeled and shown in different color modes, with the first 30 ns (restrained) part in a dimmer and the following 270 ns of free simulation shown in a brighter representations. (MP4 14257 kb)

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Verhalen, B., Dastvan, R., Thangapandian, S. et al. Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein. Nature 543, 738–741 (2017). https://doi.org/10.1038/nature21414

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