Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Dispatched uses Na+ flux to power release of lipid-modified Hedgehog

Abstract

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5,6,7,8,9,10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Structure and lipid binding sites of DISP1.
Fig. 2: Three Na+ densities in the transmembrane domain.
Fig. 3: A transmembrane Na+ permeation channel is disrupted in the structure of DISP1-A-NNN.
Fig. 4: DISP1-A binds ShhN between its distal ECDs.

Data availability

Coordinates for the DISP1-A R conformation, DISP1-A T conformation, DISP1-A-NNN, and ShhN–DISP1-A complex models reported in this paper have been deposited in the Protein Data Bank under accessions 7RPH, 7RPI, 7RPJ and 7RPK, respectively. The maps of DISP1-A R conformation, DISP1-A T conformation, DISP1-A-NNN and ShhN–DISP1-A complex have been deposited in the Electron Microscopy Data Bank under accession codes EMD-24614, EMD-24615, EMD-24616 and EMD-24617, respectively. Further information and requests for data should be directed to the corresponding authors. PDB IDs of cited structures are provided either in the related figure legends or in the Methods.

References

  1. 1.

    Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched. Cell 175, 1352–1364.e14 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Carstea, E. D. et al. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228–231 (1997).

    CAS  PubMed  Google Scholar 

  3. 3.

    Nikaido, H. & Takatsuka, Y. Mechanisms of RND multidrug efflux pumps. Biochim. Biophys. Acta 1794, 769–781 (2009).

    CAS  PubMed  Google Scholar 

  4. 4.

    Yamaguchi, A., Nakashima, R. & Sakurai, K. Structural basis of RND-type multidrug exporters. Front. Microbiol. 6, 327 (2015).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Burke, R. et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signaling cells. Cell 99, 803–815 (1999).

    CAS  PubMed  Google Scholar 

  6. 6.

    Ma, Y. et al. Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of Dispatched. Cell 111, 63–75 (2002).

    CAS  PubMed  Google Scholar 

  7. 7.

    Kawakami, T. et al. Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling. Development 129, 5753–5765 (2002).

    CAS  PubMed  Google Scholar 

  8. 8.

    Roessler, E. et al. Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans. Hum. Genet. 125, 393–400 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Caspary, T. et al. Mouse dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling. Curr. Biol. 12, 1628–1632 (2002).

    CAS  PubMed  Google Scholar 

  10. 10.

    Nakano, Y. et al. Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the zebrafish embryo. Dev. Biol. 269, 381–392 (2004).

    CAS  PubMed  Google Scholar 

  11. 11.

    Riddle, R. D., Johnson, R. L., Laufer, E. & Tabin, C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401–1416 (1993).

    CAS  PubMed  Google Scholar 

  12. 12.

    Echelard, Y. et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430 (1993).

    CAS  PubMed  Google Scholar 

  13. 13.

    Krauss, S., Concordet, J.-P. & Ingham, P. W. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in Zebrafish embryos. Cell 75, 1431–1444 (1993).

    CAS  PubMed  Google Scholar 

  14. 14.

    Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413 (1996).

    ADS  CAS  PubMed  Google Scholar 

  15. 15.

    Mann, R. K. & Beachy, P. A. Novel lipid modifications of secreted protein signals. Annu. Rev. Biochem. 73, 891–923 (2004).

    CAS  PubMed  Google Scholar 

  16. 16.

    Porter, J. A., Young, K. E. & Beachy, P. A. Cholesterol modification of Hedgehog signaling proteins in animal development. Science 274, 255–260 (1996).

    ADS  CAS  PubMed  Google Scholar 

  17. 17.

    Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human sonic Hedgehog. J. Biol. Chem. 273, 14037–14045 (1998).

    CAS  PubMed  Google Scholar 

  18. 18.

    Chamoun, Z. et al. Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal. Science 293, 2080–2085 (2001).

    ADS  CAS  PubMed  Google Scholar 

  19. 19.

    Woods, I. G. & Talbot, W. S. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in Zebrafish. PLoS Biol. 3, e66 (2005).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Creanga, A. et al. Hedgehog signal in soluble form Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form. Genes Dev. 26, 1312–1325 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Tukachinsky, H., Kuzmickas, R. P., Jao, C. Y., Liu, J. & Salic, A. Dispatched and Scube mediate the efficient secretion of the cholesterol-modified Hedgehog ligand. Cell Rep. 2, 308–320 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Stewart, D. P. et al. Cleavage activates Dispatched for Sonic Hedgehog ligand release. eLife 7, e31678 (2018).

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).

    CAS  PubMed  Google Scholar 

  24. 24.

    Cannac, F. et al. Cryo-EM structure of the Hedgehog release protein Dispatched. Sci. Adv. 6, eaay7928 (2020).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Chen, H., Liu, Y. & Li, X. Structure of human Dispatched-1 provides insights into Hedgehog ligand biogenesis. Life Sci. Alliance 3, e202000776 (2020).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Zheng, H. et al. CheckMyMetal: a macromolecular metal-binding validation tool. Acta Crystallogr. D 73, 223–233 (2017).

    CAS  Google Scholar 

  27. 27.

    Myers, B. R., Neahring, L., Zhang, Y. & Roberts, K. J. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc. Natl Acad. Sci. USA 114, E11141–E11150 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Petrov, K., Wierbowski, B. M., Liu, J. & Salic, A. Distinct cation gradients power cholesterol transport at different key points in the Hedgehog signaling pathway. Dev. Cell 55, 314–327.e7 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hall, T. M. T., Porter, J. A., Beachy, P. A. & Leahy, D. J. A potential catalytic site revealed by the 1.7-Å crystal structure of the amino–terminal signalling domain of Sonic hedgehog. Nature 378, 212–216 (1995).

    ADS  CAS  PubMed  Google Scholar 

  31. 31.

    McLellan, J. S. et al. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455, 979–983 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Zhulyn, O., Nieuwenhuis, E., Liu, C. Y., Angers, S. & Hui, C. Ptch2 shares overlapping functions with Ptch1 in Smo regulation and limb development. Dev. Biol. 397, 191–202 (2015).

    CAS  PubMed  Google Scholar 

  33. 33.

    Deshpande, I. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Qi, X. et al. Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi. Nature 571, 279–283 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Qi, C., et al Structural basis of sterol recognition by human hedgehog receptor PTCH1. Sci. Adv. 5, eaaw6490 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Whalen, D. M., Malinauskas, T., Gilbert, R. J. C. & Siebold, C. Structural insights into proteoglycan-shaped Hedgehog signaling. Proc. Natl Acad. Sci. USA 110, 16420–16425 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    PubMed  Google Scholar 

  38. 38.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Asarnow, D., Palovcak, E. & Cheng, Y. https://doi.org/10.5281/zenodo.3576630 (2019).

  42. 42.

    Dang, S. et al. Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature 552, 426–429 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D D66, 486–501 (2010).

    Google Scholar 

  44. 44.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D D66, 213–221 (2010).

    Google Scholar 

  45. 45.

    Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D D74, 519–530 (2018).

    Google Scholar 

  46. 46.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modelling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Berman, H., Henrick, K. & Nakamura, H. Announcing the worldwide Protein Data Bank. Nat. Struct. Biol. 10, 980 (2003).

    CAS  PubMed  Google Scholar 

  48. 48.

    Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  PubMed  Google Scholar 

  50. 50.

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  PubMed  Google Scholar 

  51. 51.

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Goodrich, L. V., Milenkovic, L., Higgins, K. M. & Scott, M. P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113 (1997).

    CAS  PubMed  Google Scholar 

  54. 54.

    Zhang, X. M., Ramalho-santos, M. & McMahon, A. P. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell 105, 781–792 (2001).

    CAS  PubMed  Google Scholar 

  55. 55.

    Zhang, Y. et al. Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit. Proc. Natl Acad. Sci. USA 117, 28838–28846 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    McLellan, J. S. et al. Structure of a heparin-dependent complex of Hedgehog and Ihog. Proc. Natl Acad. Sci. USA 103, 17208–17213 (2006).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Qi, X., Hassan, A., Liang, Q., Brabander, J. K. D. & Li, X. Structural basis for itraconazole-mediated NPC1 inhibition. Nat. Commun. 11, 152 (2020).

    ADS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Wang, X. et al. TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes. Cell 151, 372–383 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Altmann, S. W. et al. Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol absorption. Science 303, 1201–1204 (2004).

    ADS  CAS  PubMed  Google Scholar 

  60. 60.

    Chaudhry, A. et al. Phenotypic spectrum associated with PTCHD1 deletions and truncating mutations includes intellectual disability and autism spectrum disorder. Clin. Genet. 88, 224–233 (2015).

    CAS  PubMed  Google Scholar 

  61. 61.

    Fan, J. et al. Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3. Biochem. Biophys. Res. Commun. 363, 757–761 (2007).

    CAS  PubMed  Google Scholar 

  62. 62.

    Chung, J. H., Larsen, A. R., Chen, E. & Bunz, F. A PTCH1 homolog transcriptionally activated by p53 suppresses Hedgehog signaling. J. Biol. Chem. 289, 33020–33031 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Seeger, M. A. et al. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298 (2006).

    ADS  CAS  PubMed  Google Scholar 

  64. 64.

    Sennhauser, G., Bukowska, M. A., Briand, C. & Grütter, M. G. Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J. Mol. Biol. 389, 134–145 (2009).

    CAS  PubMed  Google Scholar 

  65. 65.

    Su, C. et al. Structures and transport dynamics of a Campylobacter jejuni multidrug efflux pump. Nat. Commun. 8, 171 (2017).

    ADS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Su, C. et al. Cryo-electron microscopy structure of an Acinetobacter baumannii multidrug efflux pump. mBio 10, e01295-19 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Su, C. et al. Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature 470, 558–562 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Su, C. et al. MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine. Proc. Natl Acad. Sci. USA 116, 11241–11246 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Kumar, N., Su, C., Chou, T., Radhakrishnan, A. & Delmar, J. A. Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN. Proc. Natl Acad. Sci. USA 114, 6557–6562 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Tsukazaki, T. et al. Structure and function of a membrane component SecDF that enhances protein export. Nature 474, 235–238 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Ishii, E. et al. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria. Proc. Natl Acad. Sci. USA 112, E5513-E5522 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank K. Roberts and A. Kershner for technical assistance and advice, and V. Korkhov and X. Li for sharing data and information prior to publication. Y.C. is an investigator of the Howard Hughes Medical Institute. Supported by NIH grants R01GM102498, Stanford Department of Urology, and Ludwig Cancer Institute (to P.A.B.) and R35GM140847, S10OD020054 and S10OD021741 (to Y.C.).

Author information

Affiliations

Authors

Contributions

Q.W. purified and characterized DISP1-A, DISP1-A-NNN and ShhN–DISP1-A complex and SCUBE2 protein, and developed and performed ShhNp binding and release assays. D.E.A. prepared cryo-EM grids, collected and processed cryo-EM data, and performed the 3DVA. R.M. devised and aided in the SCUBE2 purification. J.H. performed ShhNp release assays. K.D., D.E.A., Y.Z. and Q.W. built the models. Y.M. provided mouse genetic epistasis analysis while in the laboratory of P.A.B. at Johns Hopkins University School of Medicine. All authors participated in discussion and analysis of the data. P.A.B., Q.W., D.E.A., K.D., Y.C. and Y.Z. prepared the manuscript.

Corresponding authors

Correspondence to Yifan Cheng or Philip A. Beachy.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Philip Ingham, Poul Nissen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Structurally related transporters Dispatched and Patched in Hedgehog signalling.

a, b, Opposing functions of Dispatched and Patched in Hedgehog signalling. a, The HH protein signal, covalently modified by cholesterol and palmitate, requires the action of DISP1 and SCUBE for release from the membrane of producing cells. HH then uses its palmitoyl adduct to clog the sterol transport conduit and block the function of its receptor PTCH1 in responding cells. The loss of PTCH1 sterol transport activity permits accumulation of cholesterol within the inner leaflet to levels that activate SMO by binding within its seven transmembrane helix bundle, resulting in activation of the GLI transcriptional effector of Hedgehog signalling. b, As Ptch1 is a target for GLI activation, the X-Gal staining of a Ptch1LacZ knock-in allele53 provides an indication of Hedgehog pathway activity (leftmost embryo). Homozygous mutation of Disp16,7,8,9,10 causes a loss of nearly all embryonic Hedgehog pathway activity (2nd embryo from left)54, whereas homozygous disruption of Ptch1 leads to unregulated ectopic pathway activity, regardless of the functional status of Disp1 (rightmost two embryos)53. cf, Truncated DISP1 protein. As murine full-length DISP1 protein was poorly expressed in HEK293 cells, we tested a variety of constructs, and ultimately settled on an N-terminal truncation. Although its export activity was partially reduced, truncated DISP1-A protein nevertheless mediated efficient release of ShhNp (autoprocessed, lipid-modified Shh protein) into cell culture medium containing SCUBE2 (mouse SCUBE2, lacking amino acids 30-281) upon transfection into Disp-/- mouse embryonic fibroblasts6,20 (MEFs), indicating preservation of its function. c, DISP1-mEGFP and DISP1-A-mEGFP in HEK293T cells with magnified insets showing expression on the cell membrane. d, Western blot showing high level of DISP1-A expression relative to DISP1 (both proteins were SBP and HA-tagged at the C-terminus). e, Functional assay (Methods, ShhNp release endpoint assay) in Disp-/- MEFs. Culture media and cell lysates from transiently transfected Disp-/- MEFs were probed by immunoblotting for expression of DISP-mEGFP (SBP, HA and mEGFP tagged at the C-terminus, see panel c), ShhNp, and SCUBE2. DISP1 and DISP1-A both released ShhNp in the presence of SCUBE2. β-ACTIN, loading control. f, Size exclusion chromatography of purified DISP1-A, together with the SDS-PAGE of the indicated fraction, corresponding to monomeric DISP1-A. Panels cf, show representative results (n = 4 biologically independent replicates). g, Structural comparison between DISP1-A and mouse PTCH155 (PDB ID: 7K65). The ECDs, closely apposed in PTCH1 to form a conduit for sterol transport, in DISP1-A are splayed apart. Conserved ferredoxin-like α + β open-faced sandwich folds are highlighted (lime for ECD1 and pink for ECD2). Magnified views in spectral sequence from N to C termini of the α + β open-faced sandwich folds in DISP1-A ECD1 (bottom left) and ECD2 (bottom right). Distal structures inserted into the peripheral loops of the ferredoxin-like folds are structurally unrelated to each other or to distal PTCH1 ECD structures. Red symbols indicate the five N-linked glycosylation sites, three in ECD1 (N362, N390 and N475) and two in ECD2 (N834 and N915), which can be inferred from additional densities that extend from N-X-S/T sequences in the extracellular loops. Panels d, e, f, see Supplementary Fig. 1 for gel source data.

Extended Data Fig. 2 Dispatched protein sequence alignment and structural features.

Sequence alignment of Dispatched proteins from Mus musculus (mouse), Homo sapiens (human), Danio rerio (zebrafish) and Drosophila melanogaster (fruit fly). Secondary structure elements and other features are indicated above the sequence, with subdomains coloured according to Fig. 1a. Protein sequences were aligned using Clustal Omega51 and the ESPript server52. TM, transmembrane helix.

Extended Data Fig. 3 Cryo-EM data and image processing flow for DISP1-A and DISP1-A-NNN.

a, A representative cryo-EM micrograph (n = 4487 for DISP1-A, n = 4467 for DISP1-A-NNN) and several highly-populated, reference-free 2D class averages are shown for DISP1-A (left) and DISP1-A-NNN (right). The micrograph for DISP1-A-NNN has been contrast-stretched for display in order to account for the presence of a gold edge in the upper left corner of the image (the DISP1-A-NNN particle distribution on this grid necessitated targeting of the gold edge). b, Schematic flow-chart representing the image processing approach for DISP1-A. Thumbnail images of each 3D class or refinement are shown along with global GS-FSC resolution in black, particle counts in red, and dashed black boxes to indicate selected 3D classes. After separation of the R and T conformations in the first round of 3D classification, the identical processing flows for the two conformations are shown in parallel. Cryo-EM map (Red box) and atomic model of R conformation are used in main figures to present DISP1-A features. Subclasses of R and T conformations, R1, R2, T1 and T3, are labeled.

Extended Data Fig. 4 Cryo-EM density and atomic model quality.

Fourier shell correlation curves (a), particle orientation distributions (b), and local resolution maps (c, d) are shown for R and T conformations of DISP1-A, DISP1-A-NNN, and for ShhN–DISP1-A complex. The ‘gold-standard’ independent half-map FSC curves and orientation distributions were determined during refinement in cryoSPARC, map-to-model FSC curves were calculated in PHENIX using protein chains only, and directional FSC curves were estimated as in ref. 42. The orientation distributions are plotted such that an elevation angle of 0˚ corresponds to a ‘side-view’ perpendicular to the transmembrane helices; in each case the predominant views are ‘side-views’ at a wide range of azimuthal angles. Local resolution estimates were computed using the BLOCRES algorithm as implemented in cryoSPARC.

Extended Data Fig. 5 Coincident modes of DISP1 conformational flexibility revealed by 3D classification and 3D variability analysis (3DVA).

a, Overlay comparing front view of R and T conformations (khaki and salmon, respectively) from 3D classification. Major conformational changes are localized to the extracellular domains. b, Cut-away view showing the formation of a ‘kink’ in the back-side linker of the T conformation, with an accompanying shift of about one helix turn that breaks a hydrogen bond between linker residue H777 and the backbone carbonyl of K767. c, Extracellular view of the R and T conformations, highlighting the movement of secondary structure elements in ECD1 (> 5 Å) and ECD2 (~2 Å). The shift of ECD1 and the formation of the inter-ECD linker ‘kink’ appear intimately related. Numbers indicate distances (Å) between the Cα of F772 in R and T (marked by ▲ in b) and R382 and S898 (marked by and , respectively, in c). d, Comparison of the most extreme R and T conformations from 3D classification (right) and the two extreme ends of PC2, PC2+ (R-like) and PC2- (T-like), from 3DVA left) shows that a nearly identical mode of motion is captured by both techniques. e, Distributions of DISP1-A particles stratified by their 3D class along the PC2 axis of 3DVA, demonstrating colinearity between the 3DVA trajectory and the 3D subclasses ordered by apparent conformation. T1 and T3, and R1 and R2, respectively, are subclasses of T and R conformations resolved by 3D classification (Extended Data Fig. 3).

Extended Data Fig. 6 Representative cryo-EM densities from selected structural features.

ad, Representative cryo-EM densities from 3D reconstruction of DISP1-A, conformation R. a, Densities of all transmembrane helices. b, Representative densities of beta-sheets from ECD1 and ECD2. c, Cryo-EM densities of three representative disulfide bonds. d, Cryo-EM densities of five representative CHS molecules. e, Representative cryo-EM densities in ShhN from 3D reconstruction of ShhN–DISP1-A complex. f, g, Representation of surface hydrophobicity, viewed from top (f, left) or a top-front position of DISP1-A (g, left). Close-up views of hydrophobic cavities in both ECD1 (f, right) and ECD2 (g, right). A hydrophobic track beginning near the front-side lifted sterol (cyan) extends outward from the membrane (dotted shape). The enclosed hydrophobic conduit employed by the PTCH1 protein for sterol movement away from the membrane is formed by the juxtaposition of ECD1 and ECD2. In DISP1, although ECD1 and ECD2 are split apart in a manner that would bisect this conduit, the ECD2 portion retains a series of hydrophobic residues that line its inner surface, which could perhaps form a partial hydrophobic conduit to the hydrophobic cavities near the distal tips of ECD1 and ECD2, analogous to the sterol conduit within the center of the conjoined ECDs of PTCH1.

Extended Data Fig. 7 Na+-coordinating amino acid residues in DISP1, and conformational rearrangements within the transmembrane Na+ pathway in DISP1-A-NNN.

a, b, Side (a) and extracellular (b) views of three Na+ ion binding sites within the transmembrane domain, each labeled with its associated charge-neutralizing intra-membrane Asp. c, Residues in DISP1 with carbonyl oxygens or side-chain residues that coordinate Na+ and carboxylate residues that neutralize Na+ charge are conserved in PTCH1 (see also Extended Data Fig. 11). d, Solvent excluded surfaces (1.4 Å probe radius) reveal transmembrane cavities within DISP1-A (left) that are altered in DISP1-A-NNN (right). Two extracellular branches (upper insets) that provide access to the central channel can be individually opened or closed depending on alternate conformations of M557 (‘A’ and ‘B’ in DISP1-A) and alternate conformations of L1035 (‘A’ and ‘B’ in DISP1-A-NNN). Significant rearrangements also take place around a mid-membrane water-filled cavity containing the three ion coordination sites (lower insets). In DISP1-A, short kinks in TM4 and TM10 position I568 above this cavity. In DISP1-A-NNN, however, TM4 and TM10 straighten, rotating I568 and D571N and D1049N into this cavity, dramatically reducing its volume and isolating the channel from its intracellular exit (see Supplementary Discussion).

Extended Data Fig. 8 Quantitative luciferase-based assay of ShhNp release.

a, Inserting Nanoluciferase coding sequence between E131 and D132 of Shh does not affect Shh autoprocessing and associated lipid modification. Immunoblotting of ShhNp detects both precursor and processed forms of the protein. Most of the expressed nanoluciferase-inserted Shh is in the processed form. b, Coomassie blue staining of purified SCUBE2 (Δ30-281) protein. c, Composition of buffers used in this study(see Fig. 2b, c). d, Time course of NanoLuc-ShhNp release at different Na+ concentrations (see panel c) with or without SCUBE2 (Δ30-281, 1μM). One representative set of normalized data (see Methods) with linear regression (dashed lines) is shown for each condition (n = 4 biologically independent experiments). The release rate determined as the slope of the linear regression line is presented in Fig. 2b. e, Western blot-based ShhNp release assay (Methods, ShhNp release endpoint assay). Culture media and cell lysates from transiently transfected Disp-/- MEFs were probed by immunoblotting for DISP-HA (SBP and HA-tagged at the C-terminus), ShhNp, and SCUBE2. β-ACTIN, loading control. f, DISP-ShhNp binding assay using HEK293 with stably integrated constructs for doxycycline-inducible expression of full-length Shh20. DISP1-A variants tagged with SBP and HA at the C-terminus (DISP-HA) were immunoprecipitated with Streptavidin resin, and ShhNp detected by Western blot. Alterations in ion site residues as follows: DISP1-A-NNN, D571N/D572N/D1049N; DISP1-A-VVVVVA, T613V/T614V/T1087V/T1088V/T610V/S611A; Site I (NVV), D1049N/T613V/T614V; Site II (NVV), D571N/T1087V/T1088V; Site III (NVA), D572N/T610V/S611A; Site I (LVV), D1049L/T613V/T614V; Site II (LVV), D571L/T1087V/T1088V; Site III (LVA), D572L/T610V/S611A. Panels a, b, e, f, show representative results (n = 3 biologically independent replicates, see Supplementary Fig. 1 for gel source data).

Extended Data Fig. 9 Cryo-EM data and image processing flow for ShhN–DISP1-A complex, and comparison to Drosophila HhNDisp complex.

a, A representative cryo-EM micrograph (n = 1687) and highly populated reference-free 2D class averages for ShhN–DISP1-A. b, Schematic flow-chart illustrating the image processing used for ShhN–DISP1-A data. Thumbnail images are shown for reference-based 3D classes and high resolution refinements. Dashed black boxes indicate 3D classes selected for the next processing step, with class particle counts in red and refinement GS-FSC resolutions in black. The label, ‘3D classification with references’ indicates that explicit ‘apo’ and ‘complex’references were used to seed the classification. c, Left, the cryo-EM density from our ShhN–DISP1-A complex (transparent grey), overlaid on a ribbon diagram of ShhN (goldenrod). Right, cryo-EM density from the Drosophila HhNDisp complex in Cannac et al24. (transparent grey, EMD: 10464), overlaid on the ribbon diagram of HhN (orchid, model extracted from PDB ID: 6TD6). Middle, superimposition of the ShhN–DISP1-A and HhNDisp complex models reported here and in Cannac et al24., with mouse DISP1-A and ShhN in khaki and goldenrod, and Drosophila Disp and HhN in pink and orchid. The model of Drosophila HhN was based on docking of a Drosophila HhN structure56 within a 4.8 Å density map. Relative to Drosophila HhN, murine ShhN is translated upwards, away from the membrane, and rotated towards the right. Asterisks indicate corresponding positions near the N termini of ShhN and HhN proteins. This difference is somewhat puzzling in light of the ability of mammalian DISP1 to rescue Drosophila disp mutant function6, and we cannot definitively account for it. One functional difference is that mammalian DISP1 cooperates with SCUBE2 for its Hedgehog-releasing activity, whereas Drosophila lacks a Scube orthologue. The ECD domain split and the Hedgehog interaction with the furin-cleaved linker arm together help explain the requirement for furin cleavage22 in DISP1 function.

Extended Data Fig. 10 Conformational dynamics link intramembrane Na+ site occupancy to Hedgehog release.

a, Three-dimensional variability analysis (3DVA) of the ShhN–DISP1-A complex dataset reveals a conformational series with ShhN bound or absent at opposite ends of the first principal component (PC1). Front (first line) and top (second line) views of reconstructed densities from the extremes of PC1 (unbound, khaki; bound, salmon) are shown, with a superimposed view of these extremes in the centre. The overall changes in ECD position are illustrated by lines drawn atop the reconstructed densities and by schematized diagrams to the left. Reconstructed densities at the Na+ coordination sites in the transmembrane domain indicate that these sites change from fully occupied in the unbound state to site I only occupied in the bound state. See also Supplementary Video 4. b, 3DVA analysis of the apoprotein preparation shows a similar conformational series along PC1, along with similar shifts in Na+ site occupancy.

Extended Data Fig. 11 Na+ ion utilization by DISP1, PTCH1, and other members of the RND transporter family.

a, Close-up views of Na+ ion sites I, II, and III in DISP1, showing the locations of liganding oxygens from amino acid side chains and main-chain carbonyls (see main text), and of the corresponding locations in PTCH135 (PDB ID: 6RMG), based on structural alignment of the two proteins. Note the presence in both proteins of a charge-neutralizing acidic residue at each site, and the conservation of side-chain oxygens as ligands. b, Structural alignment, using Chimera Matchmaker, of RND family members, including DISP1, PTCH1, NPC1, and several prokaryotic RND transporters. c, Tabulated conservation of charge-neutralizing residues (3 total) or oxygen ligands from amino acid side-chains (10 total) in the indicated proteins, aligned from structure (b, see PDB IDs from the second right column) or, if possible without ambiguity, aligned from sequence. References to specific sequences, structures, or background information are given within the table, including refs .1,2,6,24,27,32,35,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71. Note the close conservation of Na+-liganding side-chains and charge-neutralizing residues in PTCH1, known to require Na+ for its activity, and in Disp from Drosophila melanogaster. The prokaryotic Na+-utilizing SecD1/SecF1 peptide translocator from Vibrio alginolyticus (encoded as two peptides; aligned by homology to Thermus thermophilus SecDF), in contrast, appears to have evolved a distinct mode of Na+ interaction. See Supplementary Discussion. Abbreviations: Mmus, Mus musculus; Dmel, Drosophila melanogaster; Hsap, Homo sapiens; Ecol, Escherichia coli; Paer, Pseudomonas aeruginosa; Cjej, Campylobacter jejuni; Abau, Acinetobacter baumannii; Msme, Mycolicibacterium smegmatis; Bmul, Burkholderia multivorans; Tthe2, Thermus thermophilus; Valg, Vibrio alginolyticus.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Supplementary Fig. 1, and legends of Supplementary Videos 1–4.

Reporting Summary

Supplementary Video 1

Conformational dynamics associated with R–T transition (PC2). The structural changes associated with the transition between DISP1-A R and T subclasses are visualized using PC2 of 3DVA in cryoSPARC. The inset histogram of PC scores (x-axis) for normalized number of particles (y-axis) assigned to R1, R2 or T1, T3 shows the quantitative relationship between this variance component and the standard 3D classification of particles presented in Extended Data Fig. 5.

Supplementary Video 2

Overview of the 28 lipids or detergent molecules associated with DISP1-A. A movie displaying DISP1-A (pale ribbon) with 26 CHS (blue or cyan) and 2 LMNG molecules (magenta) resolved in the structure. The front lifted CHS is coloured cyan (see text).

Supplementary Video 3

Identification of a Na+ permeation channel in DISP1-A. Using the CAVER 3 PyMOL plugin with a probe of radius 1.0 Å, we identified a continuous pathway through the transmembrane domain of DISP1-A (blue surface). This channel is occupied by resolved water molecules (red spheres), and provides access to the transmembrane cavity that contains the Na+ ions and water. Finally, inspection of the exterior solvent-excluded surface of DISP1-A, computed with 1.4 Å probe radius in ChimeraX and shown in khaki, shows the extra- and intracellular openings through which water (and Na+) may pass through the transmembrane domain.

Supplementary Video 4

DISP1 protein conformation and Hedgehog binding/release are linked to Na+ site occupancy. Animation of PC1 identified by 3DVA of ShhN–DISP1-A cryo-EM data. In synchronization, the left shows a wide-angle view from the front of DISP1 (khaki), with bound ShhN (goldenrod) appearing in coordination with tensing of DISP1 ECDs. The centre view highlights at a lower density threshold the DISP1 residues preceding the furin site, which form a one-armed embrace of ShhN upon binding (hot pink). At right, a tilted, cut-away top view illustrating loss of density in Na+ sites II & III, again coincident with bound ShhN.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, Q., Asarnow, D.E., Ding, K. et al. Dispatched uses Na+ flux to power release of lipid-modified Hedgehog. Nature 599, 320–324 (2021). https://doi.org/10.1038/s41586-021-03996-0

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing