Supplementary Information
Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HIP
Benjamin Bishop1, A. Radu Aricescu1, Karl Harlos1, Chris A. O'Callaghan2, E. Yvonne Jones1, Christian Siebold1
1
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of
Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom. 2
Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive,
Oxford OX3 7BN, United Kingdom.
Correspondence should be addressed to C.S. (
[email protected]).
Supplementary Figure 1. Measurements of the molecular weight of HIP ectodomain constructs by multiangle laser light scattering (MALS). (a) Schematic representation of the HIP domain organisation. Color coding and symbols are as in Fig. 1a. Theoretical molecular weights of glycosylated proteins were calculated using the PROTPARAM program at the EXPASY server (www.expasy.ch). (b,c).The measured 164 kDa molecular weight for the glycosylated, proteolytically stabilised full-length HIP ectodomain (with Arg189, Arg210 and Lys211 mutated to alanine, eHIPS) is consistent with a dimer, whereas the observed mass for the N-terminal truncated eHIP∆N (63 kDa) corresponds to a monomer. These results are consistent with size exclusion chromatography data (not shown).
Supplementary Figure 2. Superposition of the two copies of eHIP∆N in the asymmetric unit. One copy is colored in grey and the other one is color-coded (red where there are significant differences between the structures and blue where parts are superimposable) using the significance level defined by default criteria of program ESCET (http://shelx.uni-ac.gwdg.de/~trs/escet/). The rigid body movement of the second EGF domain and the differences of blade 4 and 5 of the β-propeller domain are indicated with dotted circles. Orientation of the left panel is as in Fig. 1d. The right panel is rotated by 90º counter clockwise around the x axis.
Supplementary Figure 3. Sequence alignment of the HIP family members. The HIP sequences were aligned using MULTALIN (bioinfo.genotoul.fr/multalin/multalin.html) and formatted with ESPRIPT (espript.ibcp.fr/ESPript/ESPript/). Numbering corresponds to the full length human HIP (including the secretion signal). Domain organisation and secondary structure assignments for human HIP are displayed above the alignment. Following refinement, clear electron density corresponding to the N-linked Nacetylglucosamine (GlcNAc) could be seen for only one out of the 4 glycosylation sites predicted by the NetNGlyc server (http://www.cbs.dtu.dk/services/NetNGlyc/): Asn99, Asn416, Asn447 and Asn459 (marked by filled orange pentagons). Residues forming hydrophilic interactions with DhhN and ShhN are highlighted in blue, residues exclusively forming hydrogen bonds with DhhN are colored in green. Asp383 which participates in the zinc-binding site is colored cyan. Residues forming hydophobic interactions with DhhN and ShhN are marked below the sequences with an asterisk (*), with exclusively DhhN with a hash (#) and with exclusively ShhN with a section sign (§). Disulfide bridges are numbered in green.
a
b
c
Supplementary Figure 4. Size-exclusion purification of Hh-HIP complexes. (a) The elution profiles of eHIP∆N (orange), ShhN-eHIP∆N (blue) and DhhN-eHIP∆N (green) are overlaid. Prior to complex formation, eHIP∆N was deglycosylated using endoglycosidase F1 (see methods for details). Fractions analysed by SDS-PAGE are marked below the x axis. (b,c), SDS-PAGE analysis of the fractions of the ShhN-eHIP∆N (b) and DhhN-eHIP∆N (c) complexes. eHIP∆N The first peak corresponds to the HheHIP∆N complexes, the second to the uncomplexed Hh ligands, which were added in a 1.5 molar excess prior to size exclusion chromatography. MW: Sigma Molecular Weight Marker S8445.
a
b
Supplementary Figure 5. Sequence analysis of the N-terminal signalling domains of Hedgehog family members. (a) The sequence alignment is prepared as in Supplementary Fig. 2. Numbering is that of the full length human Sonic hedgehog. Secondary structure assignments for mouse ShhN (pdb accession code 1VHH) and human DhhN are displayed above the alignment. Residues forming hydrophilic interactions with HIP are highlighted in violet and exclusively formed by ShhN in green. Zinc coordinating residues are in grey. Calcium-coordinating residues are in yellow. Residues forming hydophobic interactions with HIP are marked below the sequences with an asterisk (*), exclusively formed by DhhN with a hash (#) and exclusively formed by ShhN with a section sign (§). The black boxes highlight sequence variations of the Drosophila family in the otherwise highly conserved zinc-binding site residues. (b) Phylogenetic analysis of the sequences aligned in (a). Hh proteins only appear in cnidarians, sporadically in other invertebrate phyla (annelida, mollusca and nematoda) and in all arthropod species for which we could access sequence data. Vertebrate Hh proteins are typically encoded by three genes: Sonic Hh, Indian Hh and Desert Hh (plus Tiggy-winkle, a fourth gene in zebrafish). The abbreviations used are: BOS: Bos tauris; XENLA: Xenopus laevis; DANRE: Danio rerio; CAPSP: Capitella sp.; BIOGL: Biomphalaria glabrata; TRICA: Tribolium castaneum; GLOMA: Glomeris marginata; PEDHU: Pediculus humanus corporis; NASVI: Nasonia vitripennis; GRYBI: Gryllus bimaculatus; ACHTE: Achaearanea tepidariorum; AEDAE: Aedes aegypti; CULQU: Culex quinquefasciatus; ANOGA: Anopheles gambiae; EUPSC: Euprymna scolopes; OCTBI: Octopus bimaculoides; PATVU: Patella vulgata; HELRO: Helobdella robusta; DROME: Drosophila melanogaster; DROSI: Drosophila simulans; DROSE: Drosophila sechellia; DROYA: Drosophila yakuba; DROER: Drosophila erecta; DROWI: Drosophila willistoni; TRISP: Trichinella spiralis; NEMVE: Nematostella vectensis.
Supplementary Figure 6. Biophysical validation of the presence of zinc in Hh ligand-HIP complex crystals. (a) Fluorescence scan of a ShhN-eHIP∆N crystal around the Zn K-edge performed at ESRF beamline BM14.Upper panel is the X-ray absorption spectrum, lower panel shows the anomalous scattering factors f' and f''. The spectra clearly demonstrate the presence of zinc in the crystal. (b) X-ray fluorescence emission analysis of a DhhN-eHIP∆N complex crystal collected at ESRF beamline ID29. The peak at the Zn-Kα egde cleary indicates the presence of zinc. In both cases, no zinc was added at any stage of protein purification or crystallization. (c) Close-up view of the HIP-complexed ShhN zinc-binding site. The anomalous difference Fourier map based on the ShhN- eHIP∆N complex calculated to 3.2 Å and contoured at 6 σ is shown in green. (d) Close-up view of the HIP-complexed ShhN calcium-binding site. The 3.2 Å Fo-Fc simulated annealed omit electron density map calculated without the two calcium ions at 4 σ is shown in yellow.
Supplementary Figure 7. SPR equilibrium binding data of different HIP constructs with ShhN or DhhN, respectively. Sensorgrams (insets) and plots of the equilibrium binding response (RU) from the sensorgrams as a function of ShhN or DhhN concentration (0.5 to 5000 nM) are displayed.
Supplementary Figure 8. Comparison of the metal-binding properties of Hh ligands. Close up views of (a) HIP-complexed ShhN (orange), (b) CDO-complexed ShhN (slate) and (c) DhhN in the calcium bound form (salmon) shown as shown as coils. Residues involved in metal interactions are drawn in stick representation (oxygen: red, nitrogen: blue). Asp383 from the HIP-BL1 loop is shown in green. The zinc ion is shown as grey sphere, the calcium ions as violet spheres. Interactions with the zinc ion as depicted as grey dotted lines and with calcium as violet dotted lines. Asp132, which is coordinating calcium only in the CDOcomplexed ShhN, is marked with a yellow asterisk. (d) Superposition of the three binding sites depicted in a-c.
Supplementary Figure 9. Comparison of ShhN-HIP and DhhN-HIP complexes. (a) Superposition of ShhN-HIP with calcium (orange/green) and without calcium (blue), and DhhN-HIP asymmetric unit molecule 1 (yellow) and asymmetric unit molecule 2 (cyan), respectively, are calculated using the HIP receptor as template. ShhN-HIP with calcium is highlighted as solvent accessible surface (probe radius 1.4 Å). HIP loops BL1, BL2 and BL3 interacting with the Hh ligand are depicted. Zinc and calcium ions are shown as spheres. In the DhhN-eHIP∆N crystal there are two copies of the complex in the asymmetric unit, one of which reveals additional interactions that involve a third ligand binding loop (BL3) of HIP (see also Fig. 4c). BL3 is disordered in all other complexes. Since in SPR assays both ShhN and DhhN bind HIP with similar affinities and mutation of the main interface residue (Trp310 to alanine) has no significant effect (Fig. 3a, Supplementary Fig. 7), it appears that the BL3 interaction is caused by crystal packing. (b) Close-up view of the Hh-HIP interface. Residues coordinating the zinc are shown in stick representation in atomic coloring (oxygen: red, nitrogen: blue). The zinc is depicted as grey sphere. (c,d) Tables of root mean square deviations (rmsd) of the different Hh ligands (c) and HIP recptors (d) of the Hh-HIP complexes. Brackets indicate the number of Cα atoms used for rmsd calculations. Color coding is as in a.
