Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling Min-Sung Kima, Adam M. Saundersb, Brent Y. Hamaokaa,1, Philip A. Beachyb,2, and Daniel J. Leahya,2 aDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and bDepartment of Developmental Biology, Institute for Stem Cell Biology and Regenerative Medicine, and Howard Hughes Medical Institute, Stanford University Schoolof Medicine, Stanford, CA 94305 Contributed by Philip A. Beachy, June 23, 2011 (sent for review April 25, 2011). Glypicans are heparan sulfate proteoglycans that modulate the to these factors as well as to establish their proper distribution signaling of multiple growth factors active during animal develop- (9, 10, 12, 16–21). The heparan sulfate attachments of glypicans ment, and loss of glypican function is associated with widespread are clearly important for mediating interactions with these growth developmental abnormalities. Glypicans consist of a conserved, factors and downstream signaling components (22, 23), but approximately 45-kDa N-terminal protein core region followed recent work has demonstrated a role for the N-terminal protein by a stalk region that is tethered to the cell membrane by a glyco- domain, which lacks heparan sulfate modifications, in mediating syl-phosphatidylinositol anchor. The stalk regions are predicted to responsiveness to at least Wnt and Hh signals (23–26). be random coil but contain a variable number of attachment sites Curiously, glypicans appear able to play both positive and for heparan sulfate chains. Both the N-terminal protein core and negative roles in mediating Hh signaling. The protein region the heparan sulfate attachments are important for glypican func- of Dally-like contributes positively to Drosophila Hh responsive- tion. We report here the 2.4-Å crystal structure of the N-terminal ness, and the developmental defects in omodysplasia, particularly protein core region of the Drosophila glypican Dally-like (Dlp). This the bone growth defects, are suggestive of a positive role for structure reveals an elongated, α-helical fold for glypican core glypican-6 function in response to Indian hedgehog (7). Notably, regions that does not appear homologous to any known structure. glypican-4 and glypican-6 are most similar to Dlp (vs. Dally) and The Dlp core protein is required for normal responsiveness to complement Dlp function in a Drosophila cultured cell-based Hh Hedgehog (Hh) signals, and we identify a localized region on signaling assay (25). In contrast, the protein region of glypican-3, the Dlp surface important for mediating its function in Hh signal- which is more similar to Dally than Dally-like, is a negative reg- ing. Purified Dlp protein core does not, however, interact appreci- ulator of Hh responsiveness in the mouse (24, 25, 27, 28). Based ably with either Hh or an Hh:Ihog complex. on sequence homology and functional phenotypes, it has thus been speculated that the two major subfamilies of glypicans have lypicans are heparan sulfate proteoglycans (HSPGs) that evolved opposing activities in Hh signal responsiveness (25). Gconsist of an approximately 450 amino acid N-terminal pro- To investigate the molecular basis for glypican function, we tein domain followed by an approximately 100 amino acid stalk have undertaken structural and functional characterization of region that is attached to the outer cell membrane via a glycosyl- the N-terminal protein domain of Dlp and report here its 2.4-Å phosphatidylinositol anchor (1). The N-terminal domain of most crystal structure. We show that the N-terminal protein domains of glypicans is proteolytically processed by a furin-like convertase glypicans adopt an elongated α-helical structure with no evident to produce two chains that remain connected by disulfide bonds homology to any known structure. We have used structure-guided (2). This processing appears required for some but not all glypi- mutagenesis to identify a localized region on the Dlp surface im- can activity (2, 3). The stalk regions of glypicans are predicted portant for the ability of Dlp to mediate Hh signal response. – to be largely random coil and typically contain 1 5 heparan sul- These results are most consistent with Dlp functioning as a bind- fate attachment sites (1, 4). Six glypicans are present in humans ing protein in Hh signaling, but we are unable to detect high- and mice (glypican-1, -2, -3, -4, -5, and -6); two are present in affinity interactions between Dlp and either Hh or an Hh:Ihog Drosophila [Dally and Dally-like (Dlp)] (1). Based on sequence complex. These results establish a molecular basis for mapping similarity, glypicans assort into two subfamilies with glypican-1, and comparing functional regions of different glypicans. -2, -4, -6, and Dlp in one family and glypican-3, -5, and Dally in another (1). Results Glypicans are active in development in both vertebrates A fragment of the Drosophila melanogaster Dally-like protein that and invertebrates. Loss of Dally in fruit flies results in defects encompasses its N-terminal globular region and is fully functional in brain, eye, wings, antennae, and genitalia (5). Loss of in assays of Hh responsiveness (DlpΔNCF) (25) was expressed in glypican-3 in humans is responsible for Simpson–Golabi–Behmel overgrowth syndrome, in which widespread visceral and skeletal abnormalities are present along with a predisposition to tumor Author contributions: M.-S.K., A.M.S., B.Y.H., P.A.B., and D.J.L. designed research; M.-S.K., A.M.S., and B.Y.H. performed research; M.-S.K., A.M.S., B.Y.H., P.A.B., and D.J.L. formation (6). Loss of glypican-6 has recently been shown to analyzed data; and M.-S.K., P.A.B., and D.J.L. wrote the paper. cause omodysplasia, a genetic disorder characterized by variable The authors declare no conflict of interest. heart defects, cognitive delay, skeletal and facial abnormalities, Freely available online through the PNAS open access option. and shortness of stature (7). Much of the function of glypicans Data deposition: The atomic coordinates and structure factors have been deposited in is attributable to modulation of signaling by several heparin- the Protein Data Bank, www.pdb.org (PDB ID code 3ODN). binding growth factors active during development including 1Present address: Department of Chemistry and Biochemistry, University of California, members of the fibroblast growth factor, Hedgehog (Hh), San Diego, La Jolla, CA 92093. β – Wnt, and transforming growth factor- families (8 15). Each 2To whom correspondence may be addressed. E-mail: [email protected] or pbeachy@ of these factors functions as a morphogen to elicit distinct stanford.edu. concentration-dependent responses within target cells, and glypi- This article contains supporting information online at www.pnas.org/lookup/suppl/ cans have been shown to be required both for normal response doi:10.1073/pnas.1109877108/-/DCSupplemental. 13112–13117 ∣ PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1109877108 Downloaded by guest on September 30, 2021 Table 1. Data collection and refinement statistics Native SeMet Space group C2 C2 Cell dimensions, Å a ¼ 97.02, b ¼ 66.42, a ¼ 96.75, b ¼ 66.29, c ¼ 85.73, β ¼ 104.85° c ¼ 84.38, β ¼ 103.76° Peak Remote Wavelength, Å 0.97929 0.97929 0.96406 Resolution, Å 30–2.4 30–2.8 30–2.8 Rsym* 9.9 (63.6) 5.7 (26.2) 5.5 (26.1) Unique reflections 20,563 12,923 12,861 Mean I∕σðIÞ 13.42 (2.19) 18.5 (2.58) 19.61 (2.4) Completeness, % 99.1 (99.9) 89.8 (55.2) 92.2 (62.5) Refinement R ∕R † 24 69∕29 82 work free ,% . Number of atoms Protein 2,934 Water 69 RMSD Bond lengths, Å 0.01 Bond angles, ° 1.15 Ramachandran Most favored 313 (94.6%) Allowed 16 (4.8%) Generously allowed 2 (0.6%) Disallowed 0 The values in parentheses are for highest-resolution shell. ¼ ∑ jIð Þ − hIð Þij∕∑ ð Þ *Rsym hkl hkl hkl hkl hkl . † ¼ ∑ j − j∕∑ ¼ 5% Rcrys hkl Fobs Fcalc hklFobs; Rfree test set . dhfr−∕− CHO cells (29), purified, and crystallized. The structure are apparent. A region of positive electrostatic surface potential of DlpΔNCF was determined by multiwavelength anomalous dif- is present on the M lobe (Fig. S2), but DlpΔNCF binds only weakly fraction using crystals of selenomethionyl-substituted DlpΔNCF. to heparin agarose, from which it elutes in approximately The native DlpΔNCF structure was refined with diffraction data 300 mM NaCl. extending to 2.4 Å (Table 1). α DlpΔNCF adopts a cylindrical, all -helical structure approxi- mately 110 Å in length and 30 Å in diameter for which automated homology searches find no structural homologs (Fig. 1A) (30). A B C309 Although three stretches of polypeptide traverse the long axis N N C317:C553 S-S bond of DlpΔNCF, concerted kinks or breaks in long helices and asso- ciations with shorter helices define three lobes within the α C306 DlpΔNCF structure. We term these lobes the N lobe (N-terminal α segment of α1, α6, α7, and α13), M lobe (middle segment of α1; N-lobe α C-terminal segment of α5, α8, α12, and α14), and C lobe (C-term- inal segment of α1, α2, α3, α4; N-terminal segment of α5, α9, α10, α C296:C328 C321:C538 α α BIOPHYSICS AND and 11) based on the region of 1 contained in the lobe (Fig. 1A). S-S bond S-S bond Electron density for much of the N lobe is poor, and residues COMPUTATIONAL BIOLOGY 74–119, 570–571, and 577–588 in this region are not modeled, R α α which may contribute to a higher than desirable free (Table 1).