PDBsum entry 2wg3

Signaling protein

PDB id: 2wg3

Contents

Protein chains

155 a.a.

406 a.a.

Ligands

PO4 ×2

NAG ×2

Metals

_CL ×6

_ZN ×2

Waters ×123

References listed in PDB file

Key reference

Title

Structural insights into hedgehog ligand sequestration by the human hedgehog-Interacting protein hhip.

Authors

B.Bishop, A.R.Aricescu, K.Harlos, C.A.O'Callaghan, E.Y.Jones, C.Siebold.

Ref.

Nat Struct Biol, 2009, 16, 698-703. [DOI no: 10.1038/nsmb.1607]

PubMed id

19561611

Abstract

Hedgehog (Hh) morphogens have fundamental roles in development, whereas dysregulation of Hh signaling leads to disease. Multiple cell-surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the Hedgehog-interacting protein (Hhip) is a highly conserved, vertebrate-specific inhibitor of Hh signaling. We have solved a series of crystal structures for the human HHIP ectodomain and Desert hedgehog (DHH) in isolation, as well as HHIP in complex with DHH (HHIP-DHH) and Sonic hedgehog (Shh) (HHIP-Shh), with and without Ca2+. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn2+ and Ca2+ binding sites--functions that may be common to all vertebrate Hh proteins. Zn2+ makes a key contribution to the Hh-HHIP interface, whereas Ca2+ is likely to prevent electrostatic repulsion between the two proteins, suggesting an important modulatory role. This interplay of several metal binding sites suggests a tuneable mechanism for regulation of Hh signaling.

Figure 1.
(a) Schematic domain organization of the HHIP receptor. SP, signal peptide; L1 and L2, interdomain linker regions; EGF1 and EGF2, epidermal growth factor repeat domains; Hx, membrane-attachment helix. -propeller blades are color coded and numbered. Proteolytic cleavage site residues Arg189 and Arg210, identified by N-terminal sequencing, are highlighted. The crystallization construct (eHHIP N) and the stabilized full-length ectodomain construct (eHHIPS) are shown. (b) SeMet SAD-phased and phase-extended electron density map (calculated to 2.8 Å, contoured at 1 ) with a rainbow-colored C trace of eHHIP N. (c) Ribbon diagram of eHHIP N with color coding as in a and b. The six blades of the -propeller domain (each consisting of a four-stranded -sheet) are numbered as in a. The 11 disulfide bridges are shown in black stick representation and marked with Roman numerals. (d) Electrostatic properties. eHHIP N is shown as solvent-accessible surface colored by electrostatic potential contoured at 10 kT (red, acidic; blue, basic). The prominent negatively charged patch that interacts with Hh ligands is marked with a dotted circle.

Figure 4.
(a) Structural superpositions. DHHN with Ca^2+ (slate) and without (magenta), and ShhN with Ca^2+ (orange) and without (yellow; PDB 1VHH^24) are shown as coils. Zn^2+ and Ca^2+ ions are depicted as spheres. The close-up highlights loop Lys88–Gly94. Glu90 and Glu91 (stick representation) change conformation upon Ca^2+ binding. (b) The ShhN solvent-accessible surface colored by residue conservation (Supplementary Fig. 5). Hh binding loops of HHIP are shown in cyan. (c) Comparison of ShhN–HHIP and DHHN–HHIP complexes. The ShhN surface is colored orange. The Shh loop Lys88–Gly94, which is ordered only in the Ca^2+-bound ShhN–HHIP, is highlighted in red. Hh binding loops of HHIP are shown as coils (ShhN–eHHIP N with Ca^2+ in green; ShhN–eHHIP N without Ca^2+ in blue; DHHN–eHHIP N without Ca^2+: molecule 1 in yellow and molecule 2 in cyan). A third ligand binding loop of HHIP (BL3) is observed in only one copy of the DHHN–eHHIP N asymmetric unit (dotted ellipse; see also Supplementary Fig. 9). (d) Effects of Ca^2+ binding. ShhN–eHHIP N complexes are shown as coils (with Ca^2+, in orange and green, and without Ca^2+, in violet and blue). Spheres highlight HHIP-Glu381 and Shh-Glu90. Shh loop Lys88–Gly94 is depicted as a dotted line. (e) Comparison of the Shh–HHIP and Shh-CDON (PDB 3D1M^26) complexes. Shh ligands are superimposed (HHIP, green; CDON, blue; HHIP-complexed ShhN, orange; CDON-complexed ShhN, red). Metal ions are depicted as spheres.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: Nat Struct Biol (2009, 16, 698-703) copyright 2009.