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• W1987325953 abstract "The crystal structure of a Wnt morphogen bound to its Frizzled receptor ectodomain provides insights into the evolutionary provenance of this complex fold and offers an explanation for why Wnts utilize both lipid- and protein-mediated contacts to engage Frizzleds. The crystal structure of a Wnt morphogen bound to its Frizzled receptor ectodomain provides insights into the evolutionary provenance of this complex fold and offers an explanation for why Wnts utilize both lipid- and protein-mediated contacts to engage Frizzleds. The core repertoire of cell signaling pathways that guide the development of metazoan organisms notably includes two systems, Wnt and Hedgehog, that appear to have an enigmatically intertwined evolutionary history (Nusse, 2003Nusse R. Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface.Development. 2003; 130: 5297-5305Crossref PubMed Scopus (269) Google Scholar; Buechling and Boutros, 2011Buechling T. Boutros M. Wnt signaling signaling at and above the receptor level.Curr. Top. Dev. Biol. 2011; 97: 21-53Crossref PubMed Scopus (43) Google Scholar). The molecular parallels center on their respective signaling receptor types, Frizzled and Smoothened (Smo), that are related heptahelical transmembrane molecules capped by N-terminal cysteine-rich Frizzled (Fz) ectodomains (Nusse, 2003Nusse R. Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface.Development. 2003; 130: 5297-5305Crossref PubMed Scopus (269) Google Scholar; Krishnan et al., 2012Krishnan A. Almén M.S. Fredriksson R. Schiöth H.B. The origin of GPCRs: identification of mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in fungi.PLoS ONE. 2012; 7: e29817Crossref PubMed Scopus (126) Google Scholar). These latter Fz modules were conjectured to have an ancient sterol or lipid-binding function by their distant structural homology to the cholesterol-carrying domain of the Niemann-Pick type C (NPC) transporter NPC1, and the riboflavin-bearing module of a folate receptor-like protein (Kwon et al., 2009Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar; Bazan and de Sauvage, 2009Bazan J.F. de Sauvage F.J. Structural ties between cholesterol transport and morphogen signaling.Cell. 2009; 138: 1055-1056Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). While Wnt and Hedgehog morphogens are unrelated proteins, similar enzymatic machinery perform critical lipid modifications that dictate their engagement to the Fz domains of Frizzled receptors––in the case of Wnts––or indirectly activate Smo, perhaps through its binding of a sterol-like molecule, by Hedgehog interaction with 12-transmembrane Patched transporters (Hausmann et al., 2009Hausmann G. von Mering C. Basler K. The hedgehog signaling pathway: where did it come from?.PLoS Biol. 2009; 7: e1000146Crossref PubMed Scopus (55) Google Scholar; Nachtergaele et al., 2012Nachtergaele S. Mydock L.K. Krishnan K. Rammohan J. Schlesinger P.H. Covey D.F. Rohatgi R. Oxysterols are allosteric activators of the oncoprotein Smoothened.Nat. Chem. Biol. 2012; 8: 211-220Crossref PubMed Scopus (212) Google Scholar). While the structure of unlipidated Hedgehog has been captured both free and in complex with several cell surface molecules (Beachy et al., 2010Beachy P.A. Hymowitz S.G. Lazarus R.A. Leahy D.J. Siebold C. Interactions between Hedgehog proteins and their binding partners come into view.Genes Dev. 2010; 24: 2001-2012Crossref PubMed Scopus (156) Google Scholar), and partly discloses the nature of its affinity for Patched (Bosanac et al., 2009Bosanac I. Maun H.R. Scales S.J. Wen X. Lingel A. Bazan J.F. de Sauvage F.J. Hymowitz S.G. Lazarus R.A. The structure of SHH in complex with HHIP reveals a recognition role for the Shh pseudo active site in signaling.Nat. Struct. Mol. Biol. 2009; 16: 691-697Crossref PubMed Scopus (109) Google Scholar), Wnts have steadfastly eluded crystallographic analysis because of the inherent difficulties in producing recombinant versions of the lipidated and glycosylated, cysteine-rich 350–400 amino acid chains. By coexpressing the Xenopus Wnt8 protein with the compact Fz domain of the human Fz8 receptor, Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar succeeded in capturing a stable complex in solution and reveal for the first time the X-ray crystal structure of a Fz domain in the embrace of a Wnt morphogen. The overall structure of the Wnt is remarkable, having the appearance of a grasping right hand with thumb and forefinger jutting out from a thicker palm and knuckles base, pinching the globular Fz8 ectodomain (Figure 1A ). Both thumb and finger extensions in Wnt are long β-strand hairpin loops cross-braced by disulfide bridges; from Ser187 at the tip of the thumb, a palmitoleic acid (PAM) lipid group projects into a hydrophobic groove in the captive Fz domain, a binding site 1 coincident with the predicted lipid or sterol pocket in Fz folds (Bazan and de Sauvage, 2009Bazan J.F. de Sauvage F.J. Structural ties between cholesterol transport and morphogen signaling.Cell. 2009; 138: 1055-1056Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In contrast, the Wnt finger β-hairpin makes a protein-protein site 2 contact with a shallow pocket on the reverse face of the Fz domain, utilizing a rare vicinal disulfide link (Carugo et al., 2003Carugo O. Cemazar M. Zahariev S. Hudáky I. Gáspári Z. Perczel A. Pongor S. Vicinal disulfide turns.Protein Eng. 2003; 16: 637-639Crossref PubMed Scopus (101) Google Scholar) between Cys320-Cys321 as part of the recognition probe (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar). The evolutionary origin of these unusual, dual Fz recognition heads in Wnt was not immediately obvious. However, upon deeper inspection, we find that it can best be read from their supporting scaffolds in the palm and knuckle regions of the structure that form two distinct protein domains (D1 and D2) separated naturally in the protein chains of Wnts by a length and sequence-variable linker (Figures 1A and 2A ). N-terminal domain D1 is largely a helical bundle (the “palm”) with two long β-hairpin loop excursions (and their respective disulfide link rungs) packed alongside each other, the second or β-hairpin 2 bearing the lipidated Ser187, while β-hairpin 1 is less well structured, partly collapsed and more degenerate in sequence. The four helices supporting these β-hairpins form a core αC-αF antiparallel bundle with a faint internal repeat of a foundational helix-helix unit (Figure S1A available online). Using the PDBeFold server (Krissinel and Henrick, 2004Krissinel E. Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3150) Google Scholar), the four-helical core structure of D1 is significantly superposed with saposin-like proteins, an ancient class of multipurpose lipid-interacting and carrier helical folds (Bruhn, 2005Bruhn H. A short guided tour through functional and structural features of saposin-like proteins.Biochem. J. 2005; 389: 249-257Crossref PubMed Scopus (151) Google Scholar) (Figures 1B and S1B); lacking the interhelical disulfide bridges common to mammalian saposins, the Wnt D1 helical module is more closely set in the mold of atypical saposin-like proteins found in bacteria (Lee et al., 2006Lee J.H. Yang S.T. Rho S.H. Im Y.J. Kim S.Y. Kim Y.R. Kim M.K. Kang G.B. Kim J.I. Rhee J.H. Eom S.H. Crystal structure and functional studies reveal that PAS factor from Vibrio vulnificus is a novel member of the saposin-fold family.J. Mol. Biol. 2006; 355: 491-500Crossref PubMed Scopus (8) Google Scholar). Instructively, saposins can serve as the loading machinery for the lipid cargo presented by the nonclassical Major Histocompatibility Complex (MHC) molecule CD1 (Darmoise et al., 2010Darmoise A. Maschmeyer P. Winau F. The immunological functions of saposins.Adv. Immunol. 2010; 105: 25-62Crossref PubMed Scopus (35) Google Scholar; León et al., 2012León L. Tatituri R.V. Grenha R. Sun Y. Barral D.C. Minnaard A.J. Bhowruth V. Veerapen N. Besra G.S. Kasmar A. et al.Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.Proc. Natl. Acad. Sci. USA. 2012; 109: 4357-4364Crossref PubMed Scopus (37) Google Scholar). Thus, a plausible evolutionary path for the Wnt D1 domain involves the covalent acquisition of a lipid that was previously engaged noncovalently. In this way, Wnts could have exploited the inherent lipid affinity of the saposin-like fold to fulfill a functional requirement to associate with the plasma membrane and act as a locally acting morphogen for a primitive, lipid-interacting Fz receptor. As saposins have been captured in two different configurations, it is important to note that the Wnt D1 saposin-like domain resembles the closed or apo helical form (with an innate membrane-targeting ability), in contrast to the open or lipid-bound state that features V-shaped monomers clasped as dimers, or forming larger lipoprotein aggregates (Ahn et al., 2003Ahn V.E. Faull K.F. Whitelegge J.P. Fluharty A.L. Privé G.G. Crystal structure of saposin B reveals a dimeric shell for lipid binding.Proc. Natl. Acad. Sci. USA. 2003; 100: 38-43Crossref PubMed Scopus (162) Google Scholar; Rossmann et al., 2008Rossmann M. Schultz-Heienbrok R. Behlke J. Remmel N. Alings C. Sandhoff K. Saenger W. Maier T. Crystal structures of human saposins C and D: implications for lipid recognition and membrane interactions.Structure. 2008; 16: 809-817Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar; Popovic et al., 2012Popovic K. Holyoake J. Pomès R. Privé G.G. Structure of saposin A lipoprotein discs.Proc. Natl. Acad. Sci. USA. 2012; 109: 2908-2912Crossref PubMed Scopus (61) Google Scholar). All together, the Wnt D1 domain retains an ancestral saposin-like fold but is no longer capable of sequestering the covalently linked lipid due to the packing of additional N-terminal helices, inserted β-hairpins, and fused D2 domain. A dominant-negative variant of XWnt8 (DN-XWnt8) that was earlier characterized by Hoppler et al., 1996Hoppler S. Brown J.D. Moon R.T. Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.Genes Dev. 1996; 10: 2805-2817Crossref PubMed Scopus (306) Google Scholar acts as a receptor inhibitor, and its deduced chain boundaries suggest that a free Wnt D1 domain retains site 1 Fz-binding activity. The DN-XWnt8 is effectively truncated at the N-terminal boundary of the D2 domain, critically preserving the second interface motif strand that docks against the D1 saposin-like helical bundle (Figures 2A and 2C); the absence of this motif (in a 30-residue-shorter truncation) destabilizes the D1 fold and results in a much weaker Fz inhibitor (Hoppler et al., 1996Hoppler S. Brown J.D. Moon R.T. Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.Genes Dev. 1996; 10: 2805-2817Crossref PubMed Scopus (306) Google Scholar). This DN form of Wnt D1, analogously to the mini-Wnt construct of Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar that captures a free D2 domain, could be used to probe the looser protein-protein specificity determinants of site 1, which is dominated by the lipid contacts. Another alteration of the Wnt D1 chain that results in a loss of Ser187 lipidation and Fz binding activity is the cleavage of an N-terminal helix (that packs against the saposin-like helical bundle; Figure 1A) by the Tiki1 metalloprotease (Zhang et al., 2012Zhang X. Abreu J.G. Yokota C. Macdonald B.T. Singh S. Coburn K.L. Cheong S.M. Zhang M.M. Ye Q.Z. Hang H.C. et al.Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.Cell. 2012; 149: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This N-terminal Wnt processing may disturb the folding of the D1 domain, and/or affect a particular Wnt surface epitope necessary for Porc membrane-bound O-acyltransferase (MBOAT) recognition and activity, resulting in the secretion of a nonlipidated, nonfunctional Wnt. Yet the Ser187 position, while highly conserved, is not invariant in Wnts (Figure 2); for instance, a Gln residue at this position enables the Drosophila WntD to be secreted in a lipid-independent manner (Ching et al., 2008Ching W. Hang H.C. Nusse R. Lipid-independent secretion of a Drosophila Wnt protein.J. Biol. Chem. 2008; 283: 17092-17098Crossref PubMed Scopus (90) Google Scholar), where it retains a biological role in primordial germ cell migration through engagement of the Fz4 receptor (McElwain et al., 2011McElwain M.A. Ko D.C. Gordon M.D. Fyrst H. Saba J.D. Nusse R. A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration.PLoS ONE. 2011; 6: e26993Crossref PubMed Scopus (16) Google Scholar). We speculate that without a lipid anchor, an enlarged β-hairpin 2 loop in WntD may drive binding to the Fz4 hydrophobic groove with sufficient affinity to cement an altered site 1 contact (Figure 2). The β-hairpin extrusions of the Wnt D1 domain independently exhibit a structural resemblance (not shown) to an ancient class of antimicrobial peptides that share a common β-hairpin fold, frequently stitched together by multiple disulfide bridges; these β-hairpin molecules are typically amphipathic, membrane-active effector proteins that function in innate host defense (Yeaman and Yount, 2007Yeaman M.R. Yount N.Y. Unifying themes in host defence effector polypeptides.Nat. Rev. Microbiol. 2007; 5: 727-740Crossref PubMed Scopus (149) Google Scholar). Speculatively, the ancestral, duplicated β-hairpin/helical hairpin motif of Wnt D1 (Figure S1A) is doubly endowed with protein motifs that are known to engage membrane lipids. These embedded β-hairpins may indicate a deep link to an ancient host defense role for the progenitor Wnt D1 molecule. Intriguingly, immune defense functions are cataloged for saposin-like proteins, from humans to primitive eukaryotes (Darmoise et al., 2010Darmoise A. Maschmeyer P. Winau F. The immunological functions of saposins.Adv. Immunol. 2010; 105: 25-62Crossref PubMed Scopus (35) Google Scholar), with direct membrane-permeabilizing and pore-forming activities observed against pathogenic bacteria (Hoeckendorf et al., 2012Hoeckendorf A. Stanisak M. Leippe M. The saposin-like protein SPP-12 is an antimicrobial polypeptide in the pharyngeal neurons of Caenorhabditis elegans and participates in defence against a natural bacterial pathogen.Biochem. J. 2012; 445: 205-212PubMed Google Scholar). The most striking feature of the Wnt D2 domain is its exposed finger fold formed by a long and curled, antiparallel β strand column crowned by a β-hairpin 3 structure pinned together by two disulfide bridges, and presenting a third, vicinal disulfide link at the tip (Figure 1C). The Wnt D2 hairpin and its “knuckle” base intriguingly superpose with a diverse class of cystine-knot cytokines like platelet-derived growth factor (PDGF) and interleukin-17 (IL-17) that typically form side-by-side homo- or heterodimers (Hymowitz et al., 2001Hymowitz S.G. Filvaroff E.H. Yin J.P. Lee J. Cai L. Risser P. Maruoka M. Mao W. Foster J. Kelley R.F. et al.IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding.EMBO J. 2001; 20: 5332-5341Crossref PubMed Scopus (436) Google Scholar) (Figures S1C–S1E); nonetheless, the most primitive examples of the cystine-knot architecture––captured in the folds of horseshoe crab coagulogen and the mammalian Wnt signaling regulator sclerostin (Bergner et al., 1996Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (69) Google Scholar; Veverka et al., 2009Veverka V. Henry A.J. Slocombe P.M. Ventom A. Mulloy B. Muskett F.W. Muzylak M. Greenslade K. Moore A. Zhang L. et al.Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation.J. Biol. Chem. 2009; 284: 10890-10900Crossref PubMed Scopus (191) Google Scholar)––are monomers in solution, while the BMP inhibitor Noggin forms an unusual back-to-back structure (Groppe et al., 2002Groppe J. Greenwald J. Wiater E. Rodriguez-Leon J. Economides A.N. Kwiatkowski W. Affolter M. Vale W.W. Belmonte J.C. Choe S. Structural basis of BMP signalling inhibition by the cystine knot protein Noggin.Nature. 2002; 420: 636-642Crossref PubMed Scopus (429) Google Scholar). The alignment of the long β-hairpins of representative cystine-knot factors with the equivalent Wnt D2 structure also brings into proximity the eponymous, compact disulfide network of cystine-knots with the constellation formed by Cys260-Cys298, Cys276-Cys291, and Cys295-Cys337 in Wnt D2 (Figure S1C). The structure of IL-17F showed that only two of the three disulfide bridges of cystine-knot factors are conserved in the superfamily (Hymowitz et al., 2001Hymowitz S.G. Filvaroff E.H. Yin J.P. Lee J. Cai L. Risser P. Maruoka M. Mao W. Foster J. Kelley R.F. et al.IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding.EMBO J. 2001; 20: 5332-5341Crossref PubMed Scopus (436) Google Scholar), and one link of this pair is a topological match with the Cys295-Cys337 disulfide bridge in Wnt D2 (Figure S1C). Overall, we suggest that the Wnt D2 domain is a degenerate cystine-knot cytokine, with the more variable, N-terminal β-hairpin of cystine-knot structures replaced by the underlying loop and short helix architecture of the Wnt D2 “knuckle”; the conserved β strand-like interface motif that docks to the saposin-like domain could also have been repurposed from the first β-strand of the unraveled cystine-knot (Figures 1C and 2A). The manner in which the long β-hairpin finger tip of the D2 domain makes a discriminating contact with a slight pocket on the surface of the bound Fz domain is reminiscent of cytokine-receptor interfaces seen in the artemin-GFRα3 complex (Wang et al., 2006Wang X. Baloh R.H. Milbrandt J. Garcia K.C. Structure of artemin complexed with its receptor GFRα3: convergent recognition of glial cell line-derived neurotrophic factors.Structure. 2006; 14: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and a TGFβ-TGFβR2 assembly (Hart et al., 2002Hart P.J. Deep S. Taylor A.B. Shu Z. Hinck C.S. Hinck A.P. Crystal structure of the human TβR2 ectodomain—TGF-β3 complex.Nat. Struct. Biol. 2002; 9: 203-208PubMed Google Scholar) (Figure S1D). The architectural resemblance of the nonlipidic Fz recognition fragment of Wnt to a receptor-binding cytokine fold is suggestive of an ancient relationship to a monomeric cystine-knot factor, and the ability to express this compact D2 domain as a selective “mini-Wnt” Fz binder by Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar further supports this notion (Figures 2A and S2). Intriguingly, Norrin, a cystine-knot factor of the sclerostin family, has been experimentally defined as an agonist ligand of the Fz4 receptor (Xu et al., 2004Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar; Smallwood et al., 2007Smallwood P.M. Williams J. Xu Q. Leahy D.J. Nathans J. Mutational analysis of Norrin-Frizzled4 recognition.J. Biol. Chem. 2007; 282: 4057-4068Crossref PubMed Scopus (86) Google Scholar), and the present analysis posits that it may mimic the Wnt D2 structure and mode of Fz engagement. In particular, the mutagenic screen of the Norrin chain paints a potential Fz4 interaction patch in the adjacent β-hairpins of a cystine-knot fold (Smallwood et al., 2007Smallwood P.M. Williams J. Xu Q. Leahy D.J. Nathans J. Mutational analysis of Norrin-Frizzled4 recognition.J. Biol. Chem. 2007; 282: 4057-4068Crossref PubMed Scopus (86) Google Scholar; our modeling, not shown), similar to the conserved Wnt hairpin 3 surfaces (Figure S1B). The binary complex of a Wnt bound to a Fz domain is the organizing element of the three main signaling modes cataloged for Wnts (the canonical or β-catenin pathway, the noncanonical or planar cell polarity/PCP pathway, and the G protein-coupled receptor/GPCR or Ca2+ pathway) that diverge at the receptor level by the nature of the accessory proteins (Buechling and Boutros, 2011Buechling T. Boutros M. Wnt signaling signaling at and above the receptor level.Curr. Top. Dev. Biol. 2011; 97: 21-53Crossref PubMed Scopus (43) Google Scholar). The best understood Wnt signaling complex (that drives β-catenin stabilization in the cytoplasm) utilizes LDL-receptor-related proteins LRP5 or 6 to complete a ternary assembly (Bourhis et al., 2011Bourhis E. Wang W. Tam C. Hwang J. Zhang Y. Spittler D. Huang O.W. Gong Y. Estevez A. Zilberleyb I. et al.Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6.Structure. 2011; 19: 1433-1442Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar; Bao et al., 2012Bao J. Zheng J.J. Wu D. The structural basis of DKK-mediated inhibition of Wnt/LRP signaling.Sci. Signal. 2012; 5: pe22Crossref PubMed Scopus (48) Google Scholar). A conservation analysis of the present structure of XWnt8 suggests two potential LRP5/6 interaction sites that lie adjacent to each other on the top and rear faces of the Wnt D2 “knuckle” domain, one concave pocket (described by Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar) and another convex feature formed by two stacked loops at the base of the cystine-knot-like β-hairpin structure (Figure 2D). The available structures of Wnt antagonist fragments bound to ectodomain repeats of LRP5/6 point to a preferred Asn-X-Ile loop peptide as a docking motif for central toroidal pockets in LRP5/6 β-propellers (and which is also employed in Laminin-Nidogen and Agrin-LRP4 complexes) (Ahn et al., 2011Ahn V.E. Chu M.L. Choi H.J. Tran D. Abo A. Weis W.I. Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6.Dev. Cell. 2011; 21: 862-873Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar; Bourhis et al., 2011Bourhis E. Wang W. Tam C. Hwang J. Zhang Y. Spittler D. Huang O.W. Gong Y. Estevez A. Zilberleyb I. et al.Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6.Structure. 2011; 19: 1433-1442Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar; Chen et al., 2011Chen S. Bubeck D. MacDonald B.T. Liang W.X. Mao J.H. Malinauskas T. Llorca O. Aricescu A.R. Siebold C. He X. Jones E.Y. Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling.Dev. Cell. 2011; 21: 848-861Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar; Cheng et al., 2011Cheng Z. Biechele T. Wei Z. Morrone S. Moon R.T. Wang L. Xu W. Crystal structures of the extracellular domain of LRP6 and its complex with DKK1.Nat. Struct. Mol. Biol. 2011; 18: 1204-1210Crossref PubMed Scopus (140) Google Scholar; Bao et al., 2012Bao J. Zheng J.J. Wu D. The structural basis of DKK-mediated inhibition of Wnt/LRP signaling.Sci. Signal. 2012; 5: pe22Crossref PubMed Scopus (48) Google Scholar). The cystine-knot factor sclerostin notably bears this distinctive Asn-X-Ile LRP5/6-binding motif in a long loop at the base of the characteristic β-hairpin “finger” of the fold (Bourhis et al., 2011Bourhis E. Wang W. Tam C. Hwang J. Zhang Y. Spittler D. Huang O.W. Gong Y. Estevez A. Zilberleyb I. et al.Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6.Structure. 2011; 19: 1433-1442Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar; Veverka et al., 2009Veverka V. Henry A.J. Slocombe P.M. Ventom A. Mulloy B. Muskett F.W. Muzylak M. Greenslade K. Moore A. Zhang L. et al.Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation.J. Biol. Chem. 2009; 284: 10890-10900Crossref PubMed Scopus (191) Google Scholar), which is coincident with the proposed approach of the LRP chain to the conserved surface patches of the present Wnt D2 domain in the binary Fz complex (Figures 2A and 3). However, the Asn-X-Ile motif is wholly absent from the Wnt D2 conserved patches (or any other preserved sequence stretches in Wnts; Figure 2A) and instead suggests that the Wnt-LRP5/6 interaction will employ a very distinct recognition mode, akin to the alternative interface captured for the C-terminal globular domain of the Dickkopf inhibitor DKK1 by the E3 β-propeller of LRP6, versus the linchpin Asn-X-Ile interaction from an unstructured N-terminal segment of DKK1 (Cheng et al., 2011Cheng Z. Biechele T. Wei Z. Morrone S. Moon R.T. Wang L. Xu W. Crystal structures of the extracellular domain of LRP6 and its complex with DKK1.Nat. Struct. Mol. Biol. 2011; 18: 1204-1210Crossref PubMed Scopus (140) Google Scholar; Bao et al., 2012Bao J. Zheng J.J. Wu D. The structural basis of DKK-mediated inhibition of Wnt/LRP signaling.Sci. Signal. 2012; 5: pe22Crossref PubMed Scopus (48) Google Scholar). The architectural division of the Wnt into fused saposin- and cytokine-like halves advances an evolutionary schema for the structural and functional provenance of this complex fold and its dual mechanism for Fz recognition. In the present configuration, the lipidated “thumb” of the saposin-like D1 domain provides a generic targeting function for the specificity-determining, cytokine-like D2 domain of Wnts, allowing it to potentially couple to a range of Fz receptors, accessory receptors (like Ror and MuSK), presentation proteins (glypicans), and carrier proteins (soluble Fz-related proteins or sFRPs) that carry embedded, lipid-binding Fz domains (Figures 3 and S3) (Bazan and de Sauvage, 2009Bazan J.F. de Sauvage F.J. Structural ties between cholesterol transport and morphogen signaling.Cell. 2009; 138: 1055-1056Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This lipidated-Wnt interaction role is recapitulated by the fold of the Wnt inhibitory factor (WIF) that, distinctly from the helical Fz fold (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar), uses the hydrophobic interior of an immunoglobulin domain to sequester phospholipid acyl chains (Malinauskas et al., 2011Malinauskas T. Aricescu A.R. Lu W. Siebold C. Jones E.Y. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1.Nat. Struct. Mol. Biol. 2011; 18: 886-893Crossref PubMed Scopus (112) Google Scholar). However, while the C-terminal EGF repeats of WIF1 aid in fully capturing Wnts, the spartan WIF ectodomain of the Ryk tyrosine kinase may bind the lipidated thumb of Wnts in a similar manner to WIF1 (Malinauskas et al., 2011Malinauskas T. Aricescu A.R. Lu W. Siebold C. Jones E.Y. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1.Nat. Struct. Mol. Biol. 2011; 18: 886-893Crossref PubMed Scopus (112) Google Scholar) without perhaps fully engaging the cytokine-like D2 portion of Wnt. For noncanonical Wnt receptors Ryk and Ror1/2, and probably MuSK, there is evidence to suggest that their WIF and Fz domains––dedicated binders of the Wnt lipid moiety––allow them to form both Fz-independent as well as inclusive Wnt signaling complexes (Buechling and Boutros, 2011Buechling T. Boutros M. Wnt signaling signaling at and above the receptor level.Curr. Top. Dev. Biol. 2011; 97: 21-53Crossref PubMed Scopus (43) Google Scholar; Clark et al., 2012Clark C.E. Nourse C.C. Cooper H.M. The tangled web of non-canonical Wnt signaling in neural migration.Neurosignals. 2012; 20: 202-220Crossref PubMed Scopus (55) Google Scholar). As mentioned, saposins not only interact with lipid membranes, but also serve as lipid presentation or loading molecules for receptors (León et al., 2012León L. Tatituri R.V. Grenha R. Sun Y. Barral D.C. Minnaard A.J. Bhowruth V. Veerapen N. Besra G.S. Kasmar A. et al.Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.Proc. Natl. Acad. Sci. USA. 2012; 109: 4357-4364Crossref PubMed Scopus (37) Google Scholar) or drive the membrane association of bound enzymes (Atrian et al., 2008Atrian S. López-Viñas E. Gómez-Puertas P. Chabás A. Vilageliu L. Grinberg D. An evolutionary and structure-based docking model for glucocerebrosidase-saposin C and glucocerebrosidase-substrate interactions - relevance for Gaucher disease.Proteins. 2008; 70: 882-891Crossref PubMed Scopus (35) Google Scholar), and we suggest that a critical event in the early evolution of Wnts was the functionally advantageous gain of a Ser-acylation motif in the exposed hairpin 2 of a D1 domain progenitor that was a saposin-like lipid carrier protein for an ancestral Fz receptor (Figure 3). The closer homology of Porc and Hhat within the MBOAT family, the two enzymes that are respectively charged with modifying unrelated Wnt and Hedgehog morphogens, is one of the intriguing mechanistic parallels between the two signaling pathways (Nusse, 2003Nusse R. Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface.Development. 2003; 130: 5297-5305Crossref PubMed Scopus (269) Google Scholar; Buglino and Resh, 2010Buglino J.A. Resh M.D. Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.PLoS ONE. 2010; 5: e11195Crossref PubMed Scopus (42) Google Scholar; Chang et al., 2011Chang C.C.Y. Sun J. Chang T.Y. Membrane-bound O-acyltransferases (MBOATs).Front. Biol. 2011; 6: 177-182Crossref Google Scholar). However, the Ser-acylation motif of Wnt in a well-ordered loop of the folded D1 domain, is structurally and evolutionarily distinct from the N-terminal Cys acylation site of Hedgehog, in disordered chain tethered to the globular protein (Nusse, 2003Nusse R. Wnts and Hedgehogs: lipid-modified proteins and similarities in signaling mechanisms at the cell surface.Development. 2003; 130: 5297-5305Crossref PubMed Scopus (269) Google Scholar; Buglino and Resh, 2010Buglino J.A. Resh M.D. Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.PLoS ONE. 2010; 5: e11195Crossref PubMed Scopus (42) Google Scholar). As an example of a preexisting functional linkage like that proposed between primitive saposin-like proteins and Fz-like receptors, the NPC1 Fz domain utilizes a dedicated, secreted carrier protein called NPC2 (that has a WIF-like immunoglobulin fold) to transiently couple to the Fz domain and transfer loaded cholesterol molecules to its hydrophobic groove (Figure S3) (Kwon et al., 2009Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). The fortuitous fusion of this lipidated saposin-like protein with a primitive cystine-knot cytokine (or D2 domain progenitor) may have shackled the Fz-targeting function of a lipidated D1 domain to a primitive D2 cytokine with a binding allegiance to a different transmembrane receptor, prospectively an LRP-like molecule, through a sclerostin-like rear-mounted contact site (Figure 3). The evolution of this ancestral, full-length Wnt factor into the present form would then require the selective honing of a binding affinity of the exposed D2 hairpin 3 “forefinger” for lipid-coupled Fz domains; this opportunistic and late development of a Fz-binding ability is mirrored in the limited footprint and micromolar affinity of the Wnt D2 cytokine domain with Fz (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar). The retention of LRP-binding ability by the primitive D2 domain would have granted the ancestral Wnt the ability to bridge an LRP chain with the Fz GPCR and form an active ternary complex coupled to a pre-existing β-catenin pathway (Valenta et al., 2012Valenta T. Hausmann G. Basler K. The many faces and functions of β-catenin.EMBO J. 2012; 31: 2714-2736Crossref PubMed Scopus (1024) Google Scholar) (Figure 3). An analogous, piecemeal evolution of the composite Hedgehog morphogen precursor chain (a fusion of ancestral zinc protease and intein domains) and its activating interjection into a primitive Patched-Smo pathway has been likewise proposed (Hausmann et al., 2009Hausmann G. von Mering C. Basler K. The hedgehog signaling pathway: where did it come from?.PLoS Biol. 2009; 7: e1000146Crossref PubMed Scopus (55) Google Scholar; Ingham et al., 2011Ingham P.W. Nakano Y. Seger C. Mechanisms and functions of Hedgehog signalling across the metazoa.Nat. Rev. Genet. 2011; 12: 393-406Crossref PubMed Scopus (450) Google Scholar). The question that has bedeviled genomic prospectors in the Wnt field concerns the evolutionary origins of Wnt morphogens in the metazoan lineage. With remarkable consistency, Wnt genes can be classified into 13 subfamilies (12 of which encompass the 19 Wnts of the human repertoire) that persist from some of the earliest branches of the metazoan tree (Pang et al., 2010Pang K. Ryan J.F. Mullikin J.C. Baxevanis A.D. Martindale M.Q. Martindale M.Q. NISC Comparative Sequencing ProgramGenomic insights into Wnt signaling in an early diverging metazoan, the ctenophore Mnemiopsis leidyi.Evodevo. 2010; 1: 10Crossref PubMed Scopus (80) Google Scholar; Riddiford and Olson, 2011Riddiford N. Olson P.D. Wnt gene loss in flatworms.Dev. Genes Evol. 2011; 221: 187-197Crossref PubMed Scopus (49) Google Scholar). For instance, Cnidaria, represented by the genomes of the sea anemone Nematostella vectensis and the freshwater Hydra magnapapillata, surprisingly yield an almost complete deck of Wnts. The most primitive Wnts are the handful of unclassified molecules unearthed in Placozoa (Tricoplax adherens), Porifera (the sponges Oscarella lobularis and Amphimedon queenslandica), and Ctenophores (the comb jelly Mnemiopsis leidyi), and while these show a much greater divergence of sequence and frequent loss of disulfide bridges from bilaterian Wnts, they are still full-length, lipidated D1-D2 domain Wnt proteins (Figure 2A). A critical difference of these primitive Wnts with bilaterian Wnts is the lack of sequence and length conservation of the hairpin 3 extension that forms the present Fz contact site (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar), arguing that this particular, molecular attachment for D2 was the last to evolve. The presence of Fz- and Smo-like GPCRs in the genome of fungi and social amoeba Dictyostelium discoideum, without identifiable Wnt ligands, suggests that these lipid- and sterol-sensing receptors predate the emergence of Wnts (Harwood, 2008Harwood A.J. Dictyostelium development: a prototypic Wnt pathway?.Methods Mol. Biol. 2008; 469: 21-32Crossref PubMed Scopus (20) Google Scholar; Krishnan et al., 2012Krishnan A. Almén M.S. Fredriksson R. Schiöth H.B. The origin of GPCRs: identification of mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in fungi.PLoS ONE. 2012; 7: e29817Crossref PubMed Scopus (126) Google Scholar). The compelling structure of a Wnt clasping a Fz receptor domain (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (570) Google Scholar) allows us to draw a plausible molecular path for the evolution of this critical morphogen-receptor pairing. We thank the reviewers for constructive comments that greatly improved our manuscript. We acknowledge support from HHMI and NIH RO1-GM097015 to K.C.G. and from a Jane Coffin Childs Postdoctoral Fellowship to C.Y.J.; J.F.B. is a visiting scientist in the Garcia lab. Download .pdf (3.97 MB) Help with pdf files Document S1. Figures S1–S3 and Supplemental Experimental Procedures" @default.
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