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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.
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