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.
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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.
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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.).
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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.
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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. c–f, 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 c–f, 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.
a–d, 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.
Supplementary information
Supplementary Information
This file contains Supplementary Discussion, Supplementary Fig. 1, and legends of Supplementary Videos 1–4.
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.
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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
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DOI: https://doi.org/10.1038/s41586-021-03996-0
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