The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla.
Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.
McLellan
Jason S
JS
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Zheng
Xiaoyan
X
Hauk
Glenn
G
Ghirlando
Rodolfo
R
Beachy
Philip A
PA
Leahy
Daniel J
DJ
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry; D058851:GPI-Linked Proteins; D053823:Hedgehog Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism
C474334:BOC protein, human; C107286:CDON protein, human; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D005353:Fibronectins; D058851:GPI-Linked Proteins; C501180:Gas1 protein, mouse; D053823:Hedgehog Proteins; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C509239:SHH protein, human; C465570:Shh protein, mouse; D025521:Tumor Suppressor Proteins; C510880:iHog protein, Drosophila; C514061:ihh protein, mouse; D002118:Calcium
Genes Dev. 2004 Mar 15;18(6):641-59
15075292
Genes Dev. 2007 May 15;21(10):1244-57
17504941
Cell. 2006 Apr 21;125(2):343-57
16630821
J Cell Biol. 1997 Jul 14;138(1):203-13
9214393
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
J Clin Invest. 2005 Apr;115(4):900-9
15841179
EMBO J. 2002 Jan 15;21(1-2):114-24
11782431
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Nat Rev Mol Cell Biol. 2005 Apr;6(4):306-17
15803137
Genes Dev. 2001 Dec 1;15(23):3059-87
11731473
Biophys J. 2000 Feb;78(2):719-30
10653785
Nature. 1995 Nov 9;378(6553):212-6
7477329
Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
15299374
Biophys J. 2000 Mar;78(3):1606-19
10692345
Cell. 1996 Jun 14;85(6):841-51
8681379
Dev Cell. 2006 May;10(5):647-56
16647304
Nature. 2006 Nov 16;444(7117):369-73
17086203
Cell. 1992 Aug 21;70(4):595-607
1505026
Methods Enzymol. 1997;276:307-26
27754618
J Mol Biol. 1999 Feb 5;285(5):2177-98
9925793
Cell. 2002 Oct 4;111(1):63-75
12372301
Protein Sci. 2002 Sep;11(9):2067-79
12192063
Acta Crystallogr D Biol Crystallogr. 1999 Aug;55(Pt 8):1432-43
10417412
Nature. 1999 Feb 18;397(6720):617-21
10050855
Annu Rev Biochem. 2004;73:891-923
15189162
Nature. 2007 Dec 13;450(7172):983-90
18075577
Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17208-13
17077139
Sci STKE. 2007 Sep 11;2007(403):pe50
17848687
Protein Expr Purif. 2006 Mar;46(1):23-32
16260150
J Mol Biol. 1993 Dec 20;234(4):946-50
8263940
Nature. 2005 May 5;435(7038):58-65
15875013
Genes Dev. 2007 May 15;21(10):1231-43
17504940
Arch Dis Child. 2002 Apr;86(4):293-6
11919111
Cell Res. 2004 Jun;14(3):179-87
15225411
Nat Genet. 1996 Nov;14(3):357-60
8896572
Nat Genet. 1996 Nov;14(3):353-6
8896571
Science. 1996 Jun 14;272(5268):1668-71
8658145
Nat Struct Biol. 2003 May;10(5):394-401
12652322