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Crystal structure of the human sterol transporter ABCG5/ABCG8


ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines1,2,3,4,5. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.

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Figure 1: Structure of the G5G8 heterodimer and comparison with other ABC transporters.
Figure 2: Interface between NBDs and TMDs of G5 and G8.
Figure 3: TMD polar relay of G5 and G8.
Figure 4: CLANS network of the ABC2 exporter superfamily and ABCG transporters.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structural factors for the reported crystal structure have been deposited in the Protein Data Bank under the accession number 5DO7.


  1. Berge, K. E. et al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 290, 1771–1775 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Lee, M. H. et al. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption. Nature Genet. 27, 79–83 (2001)

    Article  CAS  PubMed  Google Scholar 

  3. Lu, K. et al. Two genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am. J. Hum. Genet. 69, 278–290 (2001)

    Article  PubMed  PubMed Central  Google Scholar 

  4. Graf, G. A. et al. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface. J. Clin. Invest. 110, 659–669 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Graf, G. A. et al. ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion. J. Biol. Chem. 278, 48275–48282 (2003)

    Article  CAS  PubMed  Google Scholar 

  6. Theodoulou, F. L. & Kerr, I. D. ABC transporter research: going strong 40 years on. Biochem. Soc. Trans. 43, 1033–1040 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dean, M., Rzhetsky, A. & Allikmets, R. The human ATP-binding cassette (ABC) transporter superfamily. Genome Res. 11, 1156–1166 (2001)

    Article  CAS  PubMed  Google Scholar 

  8. Caffrey, M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009)

    Article  CAS  PubMed  Google Scholar 

  9. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Lin, D. Y.-W., Huang, S. & Chen, J. Crystal structures of a polypeptide processing and secretion transporter. Nature 523, 425–430 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Chen, S., Oldham, M. L., Davidson, A. L. & Chen, J. Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography. Nature 499, 364–368 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Johnson, B. J. H., Lee, J.-Y., Pickert, A. & Urbatsch, I. L. Bile acids stimulate ATP hydrolysis in the purified cholesterol transporter ABCG5/G8. Biochemistry 49, 3403–3411 (2010)

    Article  CAS  PubMed  Google Scholar 

  13. Faham, S. et al. Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 14, 836–840 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hohl, M., Briand, C., Grütter, M. G. & Seeger, M. A. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nature Struct. Mol. Biol. 19, 395–402 (2012)

    Article  CAS  Google Scholar 

  15. Ward, A. B. et al. Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain. Proc. Natl Acad. Sci. USA 110, 13386–13391 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hollenstein, K., Frei, D. C. & Locher, K. P. Structure of an ABC transporter in complex with its binding protein. Nature 446, 213–216 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  17. Hvorup, R. N. et al. Asymmetry in the structure of the ABC transporter-binding protein complex BtuCD-BtuF. Science 317, 1387–1390 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Wang, F., Li, G., Gu, H.-M. & Zhang, D.-W. Characterization of the role of a highly conserved sequence in ATP binding cassette transporter G (ABCG) family in ABCG1 stability, oligomerization, and trafficking. Biochemistry 52, 9497–9509 (2013)

    Article  CAS  PubMed  Google Scholar 

  19. Yu, L. et al. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion. Proc. Natl Acad. Sci. USA 99, 16237–16242 (2002)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hollenstein, K., Dawson, R. J. P. & Locher, K. P. Structure and mechanism of ABC transporter proteins. Curr. Opin. Struct. Biol. 17, 412–418 (2007)

    Article  CAS  PubMed  Google Scholar 

  21. Wang, J. et al. Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport. J. Biol. Chem. 286, 7308–7314 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Oldham, M. L. & Chen, J. Crystal structure of the maltose transporter in a pretranslocation intermediate state. Science 332, 1202–1205 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Ovchinnikov, S., Kamisetty, H. & Baker, D. Robust and accurate prediction of residue-residue interactions across protein interfaces using evolutionary information. eLife 3, e02030 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Wang, B., Dukarevich, M., Sun, E. I., Yen, M. R. & Saier, M. H., Jr. Membrane porters of ATP-binding cassette transport systems are polyphyletic. J. Membr. Biol. 231, 1–10 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ren, Q., Chen, K. & Paulsen, I. T. TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels. Nucleic Acids Res. 35, D274–D279 (2007)

    Article  CAS  PubMed  Google Scholar 

  26. Frickey, T. & Lupas, A. CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 20, 3702–3704 (2004)

    Article  CAS  PubMed  Google Scholar 

  27. Morgan, T. H. Sex limited inheritance in Drosophila . Science 32, 120–122 (1910)

    Article  ADS  CAS  PubMed  Google Scholar 

  28. Webb, B. & Sali, A. Protein structure modeling with MODELLER. Methods Mol. Biol. 1137, 1–15 (2014)

    Article  CAS  PubMed  Google Scholar 

  29. Ewart, G. D., Cannell, D., Cox, G. B. & Howells, A. J. Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster. Implications for structure-function relationships. J. Biol. Chem. 269, 10370–10377 (1994)

    Article  CAS  PubMed  Google Scholar 

  30. Mackenzie, S. M. et al. Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration. Biochim. Biophys. Acta 1419, 173–185 (1999)

    Article  CAS  PubMed  Google Scholar 

  31. Chloupková, M. et al. Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity. Biochemistry 46, 7992–8003 (2007)

    Article  PubMed  CAS  Google Scholar 

  32. Zhang, D.-W., Graf, G. A., Gerard, R. D., Cohen, J. C. & Hobbs, H. H. Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8. J. Biol. Chem. 281, 4507–4516 (2006)

    Article  CAS  PubMed  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  34. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes. Acta Crystallogr. D 62, 859–866 (2006)

    Article  PubMed  CAS  Google Scholar 

  35. Otwinowski, Z., Borek, D., Majewski, W. & Minor, W. Multiparametric scaling of diffraction intensities. Acta Crystallogr. A 59, 228–234 (2003)

    Article  PubMed  CAS  Google Scholar 

  36. Borek, D., Minor, W. & Otwinowski, Z. Measurement errors and their consequences in protein crystallography. Acta Crystallogr. D 59, 2031–2038 (2003)

    Article  PubMed  Google Scholar 

  37. Borek, D., Ginell, S. L., Cymborowski, M., Minor, W. & Otwinowski, Z. The many faces of radiation-induced changes. J. Synchrotron Radiat. 14, 24–33 (2007)

    Article  CAS  PubMed  Google Scholar 

  38. Borek, D., Cymborowski, M., Machius, M., Minor, W. & Otwinowski, Z. Diffraction data analysis in the presence of radiation damage. Acta Crystallogr. D 66, 426–436 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Borek, D., Dauter, Z. & Otwinowski, Z. Identification of patterns in diffraction intensities affected by radiation exposure. J. Synchrotron Radiat. 20, 37–48 (2013)

    Article  CAS  PubMed  Google Scholar 

  40. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Otwinowski, Z. Maximum likelihood refinement of heavy atom parameters. In Proc. CCP4 Study Weekend: Isomorphous Replacement and Anomalous Scattering (Eds Evans, P. R., Wolf, W., Leslie, A. G. W. ) 80–86 (Daresbury Laboratory: Science and Engineering Research Council, 1991)

  42. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Cowtan, K. & Main, P. Miscellaneous algorithms for density modification. Acta Crystallogr. D 54, 487–493 (1998)

    Article  CAS  PubMed  Google Scholar 

  44. Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)

    Article  CAS  PubMed  Google Scholar 

  45. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    Article  PubMed  CAS  Google Scholar 

  47. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  PubMed  Google Scholar 

  48. Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70, 2487–2499 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  50. Chivian, D. et al. Prediction of CASP6 structures using automated Robetta protocols. Proteins 61 (Suppl. 7), 157–166 (2005)

    Article  CAS  PubMed  Google Scholar 

  51. Stone, D. K., Xie, X. S. & Racker, E. Inhibition of clathrin-coated vesicle acidification by duramycin. J. Biol. Chem. 259, 2701–2703 (1984)

    Article  CAS  PubMed  Google Scholar 

  52. Wang, J. et al. Relative roles of ABCG5/ABCG8 in liver and intestine. J. Lipid Res. 56, 319–330 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kamisetty, H., Ovchinnikov, S. & Baker, D. Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era. Proc. Natl Acad. Sci. USA 110, 15674–15679 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ovchinnikov, S. et al. Large-scale determination of previously unsolved protein structures using evolutionary information. eLife 4, e09248 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  55. Vehlow, C. et al. CMView: interactive contact map visualization and analysis. Bioinformatics 27, 1573–1574 (2011)

    Article  CAS  PubMed  Google Scholar 

  56. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983)

    Article  ADS  CAS  Google Scholar 

  57. Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993)

    Article  CAS  Google Scholar 

  58. Case, D. A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668–1688 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wang, J., Wang, W., Kollman, P. A. & Case, D. A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graph. Model. 25, 247–260 (2006)

    Article  ADS  PubMed  CAS  Google Scholar 

  60. Wickstrom, L., Okur, A. & Simmerling, C. Evaluating the performance of the ff99SB force field based on NMR scalar coupling data. Biophys. J. 97, 853–856 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Dickson, C. J. et al. Lipid14: the amber lipid force field. J. Chem. Theory Comput. 10, 865–879 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Sagui, C., Pedersen, L. G. & Darden, T. A. Towards an accurate representation of electrostatics in classical force fields: efficient implementation of multipolar interactions in biomolecular simulations. J. Chem. Phys. 120, 73–87 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  63. Ryckaert, J. P., Ciccotti, G. & Berendsen, H. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 23, 321–341 (1977)

    Article  ADS  Google Scholar 

  64. Uberuaga, B. P., Anghel, M. & Voter, A. F. Synchronization of trajectories in canonical molecular-dynamics simulations: observation, explanation, and exploitation. J. Chem. Phys. 120, 6363–6374 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  65. Bahar, I., Lezon, T. R., Bakan, A. & Shrivastava, I. H. Normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins. Chem. Rev. 110, 1463–1497 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods Mol. Biol. 1079, 263–271 (2014)

    Article  PubMed  PubMed Central  Google Scholar 

  67. Katoh, K. & Standley, D. M. MAFFT: iterative refinement and additional methods. Methods Mol. Biol. 1079, 131–146 (2014)

    Article  PubMed  Google Scholar 

  68. Pei, J. & Grishin, N. V. AL2CO: calculation of positional conservation in a protein sequence alignment. Bioinformatics 17, 700–712 (2001)

    Article  CAS  PubMed  Google Scholar 

  69. Lazarevic, V. & Karamata, D. The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids. Mol. Microbiol. 16, 345–355 (1995)

    Article  CAS  PubMed  Google Scholar 

  70. Evans, I. J. & Downie, J. A. The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes. Gene 43, 95–101 (1986)

    Article  CAS  PubMed  Google Scholar 

  71. Goldman, B. S., Beck, D. L., Monika, E. M. & Kranz, R. G. Transmembrane heme delivery systems. Proc. Natl Acad. Sci. USA 95, 5003–5008 (1998)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  72. Ajdic´, D. et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl Acad. Sci. USA 99, 14434–14439 (2002)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  73. Han, C. S. et al. Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis . J. Bacteriol. 188, 3382–3390 (2006)

    Article  PubMed  PubMed Central  Google Scholar 

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We thank the University of Texas Southwestern Structural Biology Laboratory and the staff of the Advanced Photon Source (beamlines 19ID and 23ID) for support during data collection. We thank C. Zelasko, L. Donnelly, F. Xu, L. Nie, Z. Wang, Y. Ma, and C. Zhao for technical assistance. We also thank S. Wilkens for comments, and L. Rice, Y. Jiang, and R. Hibbs for providing reagents and equipment. This project was supported by grants from the American Heart Association South Central Affiliate- (0825285F; J.-Y.L.), the American Heart Association Texas Affiliate Beginning Grant-in-Aid (0463130Y; I.L.U.), the Welch Foundation (I-1770; D.M.R.), the Packard Foundation (D.M.R.), the Howard Hughes Medical Institute (H.H.H., N.V.G.), and the National Institutes of Health (HL72304 and P01-HL20948 (H.H.H., J.C.C., X.-S.X.), GM094575 (N.V.G.), GM053163 and GM117080 (Z.O.), and GM113050 (D.M.R.)). The Advanced Photon Source is a US Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory (DE-AC02-06CH11357).

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Authors and Affiliations



J.-Y.L. and Ji.W. performed the experiments described in this paper with guidance from H.H.H., D.M.R., J.C.C., I.L.U., and X.-S.X. X-ray data were collected by J.-Y.L., and structure determination/refinement was performed by Z.O. and D.M.B. with help from J.-Y.L. L.N.K. and N.V.G. performed bioinformatics and coevolution analysis. Ju.W. performed molecular dynamics simulations and modelling calculations. H.H.H. and D.M.R. supervised the overall project. All authors contributed to the writing of the paper.

Corresponding authors

Correspondence to Helen H. Hobbs or Daniel M. Rosenbaum.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purification and ATPase activity of G5G8 heterodimers.

a, G5G8 heterodimers were co-purified by sequential affinity chromatography (Ni-NTA and CBP) followed by gel-filtration chromatography using Superdex 200 (SD 200) as described in the Methods. Protein purity was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining. b, Dimerization of detergent-soluble G5G8 was analysed by size fractionation using blue native PAGE (BN-PAGE) followed by Coomassie staining of the gel (top). Proteins were transferred to nitrocellulose and immunoblotting was performed using an anti-RGSH4 monoclonal antibody (Qiagen) and anti-G8 rabbit polyclonal antibodies (bottom). Arrows point to the G5G8 dimer. c, Bile acid stimulated ATPase activity of purified recombinant WT G5 and G8 (4 μg) and WT G5 and catalytic-deficient G8 (G8-G216D) (Mut, 8 μg). Each point represents the mean ± s.d. of three separate measurements.

Extended Data Figure 2 Optimization of crystal growth and X-ray diffraction.

Crystals of G5G8 were grown using bicelle crystallization (see Methods). Optimal diffraction-quality crystals were obtained by using cholesterol as additive to the bicelle matrices. Shown here are crystals observed under a light microscope without the polarizer. Scale bar, 100 μm. a, b, In the presence of 0–1% (mol) cholesterol, crystals grew up to 500 μm, but only diffracted to 7–10 Å. c, In the presence of 2–5% (mol) cholesterol, crystals grew to 50–150 μm (longest edge) and diffracted to 3.5–4 Å. d, A diffraction frame shows structural information of G5G8 crystals out beyond 3.9 Å resolution.

Extended Data Figure 3 Experimental electron density maps and crystal packing in G5G8 bicelle crystals.

a, Side view of Cα backbone for two G5G8 dimers in an asymmetric unit (containing two transporters) with the anomalous Fourier map calculated from W-clusters plotted at 3σ. b, Orthorhombic G5G8 bicelle crystals with lateral crystal packing of TMDs indicative of G5G8 reconstitution into phospholipid bilayers (G5: orange; G8: blue). c, Electron density maps (2FoFc) show the complete G5G8 heterodimer (centre, contoured at 1.5σ), selected residues at the G5 ECD (upper left, 1.5σ), conserved three arginines of G5 (R198/R199/R200) in proximity to the Signature motif (bottom left, 1.0σ), R543 of G8 where point mutation R543S causes sitosterolaemia (upper right, 1.5σ), and Y432 on TMH5 (G5) and F561 on TMH5 (G8) at the TMD interface (bottom right, 1.5σ).

Extended Data Figure 4 G5G8 heterodimer interface.

a, Sitosterolaemia mutation positions R419 (G5) and G574 (G8) at the G5-TMH2 and G8-TMH5 interface near the extracellular surface. b, Separation between the +4 glycine of G5 Walker A (G5, GSSGSGKT) and G8 Signature (LSGGE) motifs. c, Superposition of the nucleotide-free NBDs of TM287/288 (PDB accession number 4Q4H), BtuD (PDB accession number 2QI9), and Atm1 (PDB accession number 4MYC) with varying distances between NBDs. d, Left: surface-filled view of the NBDs of G5G8 showing contacts between the two half-transporters at the cytoplasmic apex. Middle: interaction between conserved NPFDF (G5) and NPADF (G8) sequences (grey dots). Right: closest distances between the two half-transporters.

Extended Data Figure 5 Vestibules in the membrane spanning region.

a, b, Left: cartoon of G5G8 TMDs in transparent surface (orange and blue). Vestibules on opposing faces of the TMDs are highlighted in boxes. Middle: Fo − Fc difference electron density map contoured at 3.0σ showing extended features at the vestibules. TMHs are numbered. Right: highly conserved residues in G5 and G8 (sequence conservation ≥ 7, see Methods) are shown as orange (G5) and blue (G8) cartoon/sticks. c, Left: in vivo functional reconstitution assay using G5G8 KO mice (G5G8−/− mice)19. Biliary cholesterol levels from mice infected with RR (empty adenovirus), or with adenoviruses expressing human (h)G5:WT and hG8:WT, or with adenoviruses expressing hG5:A540F and hG8:WT are shown. Pictures of the bile are below the graph. Each experiment represents the mean ± s.d. from three infected mice (n = 3) in each group. Right: expression of hG5 and hG8 detected by immunoblotting using anti-hG5 monoclonal antibodies (see Methods). Connexin was used as the gel-loading control. As a positive control for the mouse and a negative control for the human anti-G5 antibody, we also immunoblotted liver membranes from G5G8+/− mice.

Extended Data Figure 6 TMD polar relay in other ABC transporters and molecular dynamics simulation of G5G8 structure.

a, b, Analysis of bacterial PCAT1 (a) and maltose transporter (MalK2EGF, b) structures reveals conserved polar residues in the TMD subunits. TMD superposition of a nucleotide-free structure with a nucleotide-bound structure shows breakage of hydrogen bonding in TMD polar relay (top right) and inward movement of coupling helices (bottom right). (Nucleotide-free PCAT1 (PDB accession number 4RY2), ATPγS-bound PCAT1 (PDB accession number 4s0f), nucleotide-free MalK2EGF (PDB accession number 3PV0), and ATP-bound MalK2EGF (PDB accession number 2R6G).) c, d, Molecular dynamics simulation (100 ns) and vibrational mode analysis performed on the nucleotide-free G5G8 structure (see Methods). For each vibrational mode, the contribution of each residue was calculated by adding up the vectors of the main chain atoms. Among the lowest 20 vibrational modes, modes 9 and 10 are shown. Mode 10 (c) describes collective movements (green-to-red vectors on the Cα atoms) between CpH/CnH/E-helix bundle and the interface at ATP-binding cassette, indicating inward movement of the CpH/CnH/E-helix bundles from both G5 and G8. This is consistent with stereotypical inward motion of coupling helices in bacterial ABC transporters. Mode 9 (d) describes a collective movement (arrows) of NBD (bottom) and TMH1, 3, and 4 in G8 (top), indicating coupling between NBD and TMD. R543 of G8, a conserved polar residue in the TMD polar relay where a disease-causing mutation occurs, is highlighted together with its interacting partner, E503.

Extended Data Figure 7 Coevolution analysis of ABCG5 and ABCG8.

a, Co-evolving residue pairs from the ABCG5 and ABCG8 TMD that were closer than an all-atom cutoff of 8 Å (≤8 Å) are depicted as lines connecting Cα atoms of the residues in contact in the respective TMD structures. b, Co-evolving pairs that are more spatially distant than the defined cutoff (>8 Å). Expanded views show potential interface contacts predicted by spatially distant G5 and G8 coevolving residue pairs (red lines connecting Cα atoms) (top). Cross contacts in G8 (red) were mapped from the distant G5 GREMLIN pairs near the interface (pink) using the GREMLIN MSA, and vice versa (bottom). For example, we mapped S538 from the predicted G5 pair L436/S538 to the corresponding residue in G8-Y567, and considered G5-L436 and G8-Y567 to be a potential interface contact. Predicted interface contacts that are distant in the nucleotide-free structure could form contacts at another stage of the ABCG ATPase cycle. c, Left: in vivo functional reconstitution assay using liver-specific G5G8−/− mice52. Biliary cholesterol levels of mice infected with RR (empty adenovirus), or with adenoviruses expressing hG5:WT and hG8:WT, or with adenoviruses expressing hG5:Y432A and hG8:WT are shown. G5-Y432 is part of the TMD polar relay and predicted to interact with G8-N568 during the sterol transport cycle (b). Each experiment represents the mean ± s.d. from three infected mice (n = 3) in each group. Right: immunoblot analysis of hG5 levels in membranes isolated from the mice using an anti-hG5 monoclonal antibody (see Methods). Connexin was used as the gel-loading control.

Extended Data Figure 8 Homology model of Drosophila white/brown based on the G5G8 structure.

A model of the Drosophila melanogaster white/brown heterodimeric guanine pigment transporter was made using the program Modeller28. On the basis of the sequence alignment and comparison of scoring metrics for the two possible heterodimer models (that is, white modelled on G5 versus G8, brown on the other), we selected G8 (blue cartoon) as the white template and G5 (orange cartoon) as the brown template. At right, top/extracellular views of the G5G8 structure and white/brown model are shown, with residues important for dye interaction highlighted. At bottom is an MSA for the C-terminal region of TMH5, showing the conservation of the functionally important amino acids.

Extended Data Table 1 Data processing and refinement statistics

Supplementary information

Supplementary Figures

This file contains the uncropped gels and blots. (PDF 2453 kb)

Supplementary Data

This file contains Multiple sequence alignment (MSA) of ABCG5 (page 1) and ABCG8 (page 2). MSA of ABCG5 and ABCG8 subfamily members is colored by Jalview in bluescale according to identity. The sequences are labeled to the left according to the NCBI GI and accession number and numbers to the left and right correspond to the first and last residue number in the corresponding sequence line. The AL2CO conservation index for each position is mapped below the alignment. The first sequences are human ABCG5 (gi|11967969) and ABCG8 (gi|11967971). Top GREMLIN L/3 co-evolving residue pairs in the TMD of ABCG5 (pages 3-6) andABCG8 (pages 7-10). (PDF 8518 kb)

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Lee, JY., Kinch, L., Borek, D. et al. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 533, 561–564 (2016).

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