PDBsum entry 3ho5

Signaling protein

PDB id: 3ho5

Contents

Protein chains

446 a.a.

154 a.a.

Metals

_ZN ×2

_CA ×2

References listed in PDB file

Key reference

Title

The structure of shh in complex with hhip reveals a recognition role for the shh pseudo active site in signaling.

Authors

I.Bosanac, H.R.Maun, S.J.Scales, X.Wen, A.Lingel, J.F.Bazan, F.J.De sauvage, S.G.Hymowitz, R.A.Lazarus.

Ref.

Nat Struct Biol, 2009, 16, 691-697. [DOI no: 10.1038/nsmb.1632]

PubMed id

19561609

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a perfect match.

Abstract

Hedgehog (Hh) signaling is crucial for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hedgehog-interacting protein (Hhip) is a surface receptor antagonist that is equipotent against all three mammalian Hh homologs. The crystal structures of human HHIP alone and bound to Sonic hedgehog (SHH) now reveal that HHIP is comprised of two EGF domains and a six-bladed beta-propeller domain. In the complex structure, a critical loop from HHIP binds the pseudo active site groove of SHH and directly coordinates its Zn2+ cation. Notably, sequence comparisons of this SHH binding loop with the Hh receptor Patched (Ptc1) ectodomains and HHIP- and PTC1-peptide binding studies suggest a 'patch for Patched' at the Shh pseudo active site; thus, we propose a role for Hhip as a structural decoy receptor for vertebrate Hh.

Figure 2.
(a) Cartoon representation of the complex of HHIP[ 12] (green) and SHH (pink). The three loops from HHIP[ 12] contacting SHH are labeled L1–L3. Zn^2+ and Ca^2+ cations are shown as gray and cyan spheres, respectively. The N and C termini of SHH, and the C terminus of HHIP, are all on the same side of the complex, suggesting that both components could be anchored to the same cell membrane. See Supplementary Figure 3a for a 90°-rotated version. (b) Alanine mutants in HHIP loops that contact SHH. Residues that were mutated to alanine are shown as spheres, with those that abolished SHH binding shown in red, those that had a notable impact shown in orange and those with minimal consequence shown in yellow. (c) Coordination of the Zn^2+ cation by residues from SHH and HHIP[ 12]. Key residues are shown as sticks, with nitrogen atoms colored blue and oxygen atoms colored red. Zn^2+ (gray sphere) is coordinated by residues His140, Asp147 and His182 from SHH (pink) and Asp383 from HHIP[ 12] (green). (d) The SHH Zn^2+-containing groove and the Ca^2+ binding site are distinct. SHH and HHIP are colored pink and green, respectively, with a transparent surface shown for the HHIP L2 loop. Zn^2+ and Ca^2+ cations are shown as gray and cyan spheres, respectively. SHH residues, which in IHH are genetically associated with brachydactyly type A1, are shown as sticks, and carbon atoms are colored either yellow (Zn^2+-containing groove) or orange (Ca^2+-coordinating) and are numbered according to human SHH. (e) Inhibition of SHH signaling in Gli-luciferase co-culture assays by HHIP[ 12] mutants. Assays were carried out as in Figure 1b. Results are plotted as the average of three independent triplicates normalized to 100% for the 'no inhibitor' data point, s.d.

Figure 3.
(a) HHIP, CDON and Ptc1 binding sites on SHH. Surface representation of SHH, with residues within 4.5 Å of HHIP (left, blue) and the third fibronectin type III (Fn) domain of CDON (center, blue). The Ihog binding site on Hh is mapped to the surface of SHH (center, cyan). Despite the functional similarity between Cdon and Ihog, Ihog binds to a distinct surface near the second Hh helix and the interaction requires heparin, which not only bridges the two binding partners but also facilitates Ihog dimerization^22. Right, the SHH surface is shown, where groups of mutated residues had some (blue) or no (yellow) effect on Ptc1 binding; numbering refers to human SHH^10, ^23, ^56. As Cdon and Boc can each directly bind to Shh and enhance signaling through Ptc1 (refs. 16,20), Shh mutants that abrogate Cdon and Boc binding may have indirect consequences on Ptc1 interaction with Shh and downstream signaling. (b) Sequence alignment of Hhip and Ptc1 in the region corresponding to the Hhip L2 loop. Residue conservation within the L2 loop is plotted below. The plot was generated from the alignment of 15 vertebrate Hhip type 1 sequences (Supplementary Fig. 8) and 8 Ptc1 sequences shown; for brevity, only human HHIP is shown. Asp383 of Hhip was arbitrarily chosen as a position 0. Residues of interest that contact SHH, are highly conserved or seem to be important for loop conformation are indicated (diamonds). Colors for shading and residues: red, acidic; blue, basic; green, polar; black, hydrophobic. The plot was created using WebLogo (http://weblogo.berkeley.edu/). (c) Competition ELISA of HHIP L2 peptide for soluble HHIP[ 1] binding to plate-bound SHH. The PTC1 L2–like peptide had lower affinity than the HHIP L2 peptide (IC[50] = 150 M) and thus was unable to compete in an accessible concentration range.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2009, 16, 691-697) copyright 2009.