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• W2035132495 abstract "Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200Å2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane. Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200Å2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane. Injured neurons in mature organisms are unable to effectively regrow their axons after central nervous system damage. One of the many factors restricting axonal regeneration after injury is the growth-inhibiting components associated with damaged myelin. At least three of these components, Nogo-66, myelin-associated glycoprotein (MAG), 3The abbreviations used are: MAG, myelin-associated glycoprotein; NgR, Nogo receptor; LRR, leucine-rich repeat; CHO, Chinese hamster ovary; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean square; NCAM, neural cell adhesion molecule; SIRAS, single isomorphous replacement with anomalous scattering.3The abbreviations used are: MAG, myelin-associated glycoprotein; NgR, Nogo receptor; LRR, leucine-rich repeat; CHO, Chinese hamster ovary; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; r.m.s., root mean square; NCAM, neural cell adhesion molecule; SIRAS, single isomorphous replacement with anomalous scattering. and oligodendrocyte myelin glycoprotein, either individually or collectively, have been shown to be potent inhibitors of neurite outgrowth (1He Z. Koprivica V. Annu. Rev. Neurosci. 2004; 27: 341-368Crossref PubMed Scopus (185) Google Scholar, 2Filbin M.T. Nat. Rev. Neurosci. 2003; 4: 703-713Crossref PubMed Scopus (701) Google Scholar). All three signal inhibition through the Nogo receptor complex, composed of the ligand-binding Nogo-66 receptor (NgR) and two complementary co-receptors p75 and Lingo-1 that act as a signal-transducing pair on an axon's cell membrane (3Barker P.A. Neuron. 2004; 42: 529-533Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 4Bandtlow C. Dechant G. Sci. STKE 2004,. 2004; : 24Google Scholar). Although both NgR and the p75 nerve growth factor receptor have well documented roles in the context of myelin inhibition, reports exploring the role of Lingo-1 are more recent. Human Lingo-1 is a central nervous system-specific transmembrane glycoprotein (Fig. 1) also known as LERN-1, which belongs to a larger family of LRR-Ig-containing proteins involved in central nervous system development and axonal growth (5Carim-Todd L. Escarceller M. Estivill X. Sumoy L. Euro. J. Neurosci. 2003; 18: 3167-3182Crossref PubMed Scopus (70) Google Scholar). Its large extracellular or ectodomain is thought to be of functional importance in protein-protein recognition and is characterized by a tandem array of multiple LRRs and one Ig-like domain. The first studies examining the role of Lingo-1 demonstrated that in cultured neurons Lingo-1 directly associates with NgR and p75 and that whenever myelin-NgR/p75-mediated growth inhibition is observed, Lingo-1 is present, and is essential to this process (6Mi S. Lee X. Shao Z. Thill G. Ji B. Relton J. Levesque M. Allaire N. Perrin S. Sands B. Crowell T. Cate R.L. McCoy J.M. Pepinsky R.B. Nat. Neurosci. 2004; 7: 221-228Crossref PubMed Scopus (697) Google Scholar). The functional capacity of the tripartite complex to launch the downstream RhoA-dependent signaling pathway that evokes the inhibition of neurite outgrowth has been reported. Of note is the finding that truncated Lingo-1 lacking the intracellular domain restores neurite outgrowth in vitro by interrupting the interaction of Lingo-1 with its binding partners. Direct physical interactions between the full-length Lingo-1 and either NgR or p75 have been reported, as have interactions of truncated soluble Lingo-1 with either NgR or p75 (6Mi S. Lee X. Shao Z. Thill G. Ji B. Relton J. Levesque M. Allaire N. Perrin S. Sands B. Crowell T. Cate R.L. McCoy J.M. Pepinsky R.B. Nat. Neurosci. 2004; 7: 221-228Crossref PubMed Scopus (697) Google Scholar). More recently, our understanding of this inhibitory system has changed with the identification of yet another member of the Nogo receptor complex, designated TROY (7Park J.B. Yiu G. Kaneko S. Wang J. Chang J. He Z. Neuron. 2005; 45: 345-351Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). The latter belongs to the same, tumor necrosis factor-receptor family as p75 but, unlike p75, is broadly expressed in adult neurons, where it can substitute for p75 in the signaling complex, allowing for RhoA activation and outgrowth inhibition in neurons lacking p75 (7Park J.B. Yiu G. Kaneko S. Wang J. Chang J. He Z. Neuron. 2005; 45: 345-351Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 8Shao Z. Browning J. Lee X. Scott M. Shulga-Morskaya S. Allaire N. Thill G. Sah Levesque M.D. McCoy J.M. Murray B. Jung V. Pepinsky R.B. Mi S. Neuron. 2005; 45: 353-359Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The current model for myelin-mediated inhibition includes therefore an alternative signaling complex that involves NgR, Lingo-1, and TROY. In addition to its expression on neurons, Lingo-1 is also detected in oligodendrocytes (9Mi S. Miller R.H. Lee X. Scott M.L. Shulag-Morskaya S. Shao Z. Chang J. Thill G. Levesque M. Zhang M. Hession C. Sah D. Trapp B. He Z. Jung V. McCoy J.M. Pepinsky R.B. Nat. Neurosci. 2005; 8: 745-751Crossref PubMed Scopus (512) Google Scholar). In this study, Lingo-1 was reported to be a negative regulator of oligodendrocyte maturation and myelination, exerting its function through a Fyn-RhoA pathway. Although a link to these signaling molecules is note-worthy in light of the fact that a role for Fyn/RhoA signaling has been reported in oligodendrocyte differentiation and myelination (10Liang X. Draghi N.A. Resh M.D. J. Neurosci. 2004; 24: 7140-7149Crossref PubMed Scopus (196) Google Scholar), the precise mechanism by which Lingo-1 signaling is initiated and the nature of protein-protein interactions involved remain largely unknown. Agonist and antagonist or null versions of Lingo-1 modulate the amount of functional Lingo-1 and hence its activity. Blockade of normal Lingo-1 activity, modeled either by introduction of exogenous Lingo-1 to oligodendrocyte-neuron cocultures or with knock-out mice lacking Lingo-1, not only permits outgrowth of oligodendrocyte processes, it also results in highly developed myelinated axons. These findings coupled with complementary gain-of-function results suggest that Lingo-1 inhibitory signaling could be one of the factors controlling central nervous system myelination. An interesting point in this respect is the recent evidence that myelin debris inhibits differentiation of oligodendrocyte precursors and that myelin could in fact be an underlying cause of impaired remyelination (11Kotter M.R. Li W.-W. Zhao C. Franklin R.J.M. J. Neurosci. 2006; 26: 328-332Crossref PubMed Scopus (510) Google Scholar). Whether this mechanism is relevant to the Lingo-1 inhibitory action in oligodendrocytes is not known. All these findings underline the importance and complexity of the molecular action of Lingo-1 and have stimulated much interest in this molecule as a promising therapeutic target, in particular for the treatment of diseases associated with myelin deficiencies, such as multiple sclerosis and leukodystrophies. Because development of therapeutics to block Lingo-1 specific interactions may lead to a new class of inhibitors, the atomic details of its ligand-recognition module should have an immediate impact on the discovery of such inhibitors. With this goal in view, we have produced recombinant soluble Lingo-1 protein, confirmed its biological and functional binding activity, and determined its ectodomain crystal structure to 2.7-Å resolution. The structure provides the first atomic insights into this new member of an apparently unique signaling protein family that contains both LRR and Ig-like domains in their extracellular region. Structural and solution characterization of this molecule reveal another important and previously not recognized concept, oligomerization of Lingo-1, which may relate to the proposed role of Lingo-1 in the central nervous system. The unique structure of this molecule defines its potential functional binding sites and should now provide a basis for further research that will address physiological relevance of these findings to central nervous system function. Protein Expression and Purification—An extracellular portion of human Lingo-1 was expressed in lectin-resistant CHO Lec 3.2.8.1 cells as a C-terminal 6-His-tagged protein (residues 1-549, signal sequence 1-33). The human Lingo-1-His was subcloned into pSMEG vector behind a murine cytomegalovirus promoter and verified by sequencing analysis. CHO cells were grown and maintained in a humidified incubator with 5% CO2 at 37 °C. DNA transfection and large-scale production of conditioned cell culture media for Lec3.2.8.1 cells were performed as described previously (12Zhong X. Kriz R. Kumar R. Guidotti G. Biochim. Biophys. Acta. 2005; 1723: 143-150Crossref PubMed Scopus (13) Google Scholar). The media expressing Lingo-1-His was exchanged into a buffer of 1 m Tris, 100 mm NaCl, pH 8.0, to which a mixture of protease inhibitors (complete inhibitors from Roche Applied Science) was added. The protein was captured by nickel-nitrilotriacetic acid resin and then purified by gel-filtration chromatography (Superdex-200). NgR 1D4 (residues 27-451) fused at the C terminus with the 10-amino acid 1D4 epitope tag was expressed in CHO-A2 cells using the honeybee meletin secretory leader, then purified from the media by anti-1D4 affinity chromatography followed by gel filtration. Neurite Outgrowth Assays—96-well plates were coated with a thin layer of nitrocellulose (Bio-Rad) before incubating with Lingo-1-His or control IgG-Fc (R&D Systems) proteins in the presence of 2.5 μg/ml MAG-Fc (R&D Systems) at 4 °C overnight. Wells were subsequently coated with 17 μg/ml of poly-d-lysine (Sigma), followed by an incubation in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Postnatal day 5 rat cerebella granule neurons were dissociated and seeded at a density of 1 × 104 cells per well. Cells were cultured for 18-20 h before being fixed with 4% paraformaldehyde and stained with a neuronal specific anti-β-tubulin antibody (Tuj1, Covance). The average of total neurite lengths from each neuron was quantitated by Cellomics' Neurite Outgrowth Bioapplication from at least 400 neurons per well, in triplicate wells per experiment. Results have been repeated independently for more than three times. Cell-based Binding Assays—25,000 CHO-DUKX cells stably expressing NgR and p75 were seeded overnight in 96-well plate and then incubated with various concentration of Lingo-1-His in Hanks' balanced salt buffer with 1%fetal bovine serum, 20 nm HEPES for 2 h at 37°C. Alkaline phosphatase-conjugated anti-His IgG was added, and the mixture was incubated for another hour. Bound Lingo-1-His was detected by incubation with AttoPhos substrate (Promega) at 0.6 mg/ml for half an hour and read on Flex Station (emission: 440 nm, excitation: 560 nm). Biacore Experiments—Surface plasmon resonance with BIA-core was used to determine the equilibrium dissociation constant (KD) between NgR 1D4 and Lingo-1-His. NgR 1D 4 was immobilized onto a CM5 chip using amine-coupling chemistry. A titration series using 2-fold serial dilutions was performed with the analyte Lingo-1-His ranging in concentration from 10 to 0.039 μm.A KD of ∼1 μm was determined for the NgR 1D4 and Lingo-1-His interaction by steady-state equilibrium analysis using BIAevaluation 3.0. Crystallization—To obtain diffraction quality crystals, the 6-His tag and stalk region were removed by proteolytic treatment with chymotrypsin for 2 h at 18°C. The cleaved Lingo-1 was further purified and analyzed by gel filtration, SDS-PAGE, and mass spectrometry (molecular mass ∼ 66.4 kDa compared with a value of ∼71.62 kDa obtained for Lingo-1-His). For crystallization, the protein was concentrated to 4-6 mg/ml in TBS (Tris-buffered saline, 50 mm Tris, pH 8.0, 150 mm NaCl). Crystals were obtained at 18 °C in hanging drops using 1.2-1.4 m (NH4)2SO4, 0.1 m sodium citrate, pH 5.0, as a precipitant. The protein crystallized in two forms, with both forms found in the same crystallization droplets: I222, with two molecules per asymmetric unit and 74% solvent content, and P21212, with four molecules per asymmetric unit and 73% solvent content. For data collection, crystals were gradually transferred from the mother liquor to the stabilizing cryoprotecting solution containing 2.9 m sodium malonate, pH 5.2. This solution, in which crystals were found to be stable over the period of several days and over the pH range 5-7, was used for crystal derivatization. A single derivative that allowed structure determination by the SIRAS method was obtained from crystals were soaked in 50 mm K2PtCl6 and 2.9 m sodium malonate at pH 7.0 for 24 h. Prior to data collection, all crystals were flush cooled under a nitrogen stream at 100 K. Data Collection, Phasing, and Refinement—Two data sets obtained from crystals of space group I222 were used for phase determination: the 3.5-Å native data set and the 3.6-Å data set for the platinum derivative, both measured in house with Saturn92 CCD mounted on an FR-E CuKα rotating anode source (Rigaku, Japan). The higher resolution native data set was collected to 2.7 Å at Advanced Photon Source beamline 22-ID of Southeast Regional Collaborative Access Team from a crystal that belongs to the P21212 space group. All data were integrated and scaled with HKL2000 (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38517) Google Scholar). The initial positions of platinum atoms in the derivative crystal were located with SHELXD (2001 Bruker-AXS, XM, version 6.12) using anomalous differences of platinum atoms at the CuKα edge. The input SAS coefficients were prepared with XPREP (2001 Bruker-AXS, version 6.12). Refinement of heavyatom parameters, phase calculation, and density modification by SOLOMON, all were performed with SHARP (14De La Fortelle E. Brigogne G. Methods Enzymol. 1997; 276: 472-493Crossref PubMed Scopus (1797) Google Scholar) at 20- to 3.6-Å resolution, using both anomalous and isomorphous differences from the native and derivative data sets. The final 3.6-Å SIRAS maps produced with SHARP were of interpretable quality and revealed two Lingo-1 molecules in the asymmetric unit. SHARP phases were further improved by 2-fold NCS averaging and phase extension to 3.5 Å in DM (15Cowtan K.D. Main P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 43-48Crossref PubMed Scopus (288) Google Scholar). The resulting maps allowed us to build an initial, 90% complete (∼855 residues) model with QUANTA. This model was then used for molecular replacement with the P21212 data set to utilize the higher 2.7-Å resolution data. A clear solution for four molecules in the asymmetric unit was identified with PHASER (16McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1599) Google Scholar). The I222 and P21212 crystal forms share the same tetrameric packing, in which the tetramer can be built by replicating a dimer around the 2-fold axis. Subsequent rounds of rebuilding and refinement against the 2.7-Å data set were done with COOT (17Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23207) Google Scholar) and REFMAC (18Winn M. Isupov M. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar). The final model contains 4 protein molecules (residues A1-477, B3-475, C2-477, and D3-476), 39 N-acetylglucosamine and 12 mannose residues, and 310 water molecules. Residues 1-2 at the N termini of B, C, and D, 476-477 at the C termini of B and D, and residues D32-34 were not modeled into the structure due to the lack of adequate electron density, presumably because of disordering. Geometric analysis of the final refined structure performed with MolProbity (19Davis I.W. Murray L.W. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2004; 32: W615-W619Crossref PubMed Scopus (812) Google Scholar) places 94% of all residues in favored regions and 0.16% as outliers. Statistics for data collection, phasing, and refinement are summarized in Table 1.TABLE 1Crystallographic statistics for data collection, phasing, and refinementData CollectionNative 1K2PtCI6Native 2Space GroupI222I222P21212Unit Cell Dimensionsa (Å)148.7149.6201.5b (Å)158.6157.3149.7c (Å)200.0200.3157.5SourceFR-E CuKαFR-E CuKαAPS ID-22Max. resolution (Å)3.5 (3.63-3.5)3.6 (3.73-3.6)2.7 (2.8-2.7)Reflections (total/unique)258,821/29,973201,963/27,406913,490/129,431Completness (%)98.2/95.398.9 (99.9)98.9 (94.7)RsymaRsym = ∑∑i|I(h)i- 〈I(h) 〉|/∑∑iI(h)i, where <I(h)> is the mean intensity. Numbers in parentheses reflect statistics for the highest resolution shells. (%)12.6 (62.2)11.9 (48.8)9.0 (61.9)I/σ(I)18.2 (3.2)21.1 (4.7)21.5 (1.3)PhasingSIRASModel RefinementNative 2Anom. I/σ(I) (4.5Å/3.6Å)2.3/1.2Resolution (Å)50.0-2.7RisobRiso = ∑|Fnat(h) - FPt(h)|/∑Fnat(h) and Rano is calculated for the amplitudes of the positive and negative counterparts of the Bijvoet pairs. (%)43.6Number of Reflections122,982RanobRiso = ∑|Fnat(h) - FPt(h)|/∑Fnat(h) and Rano is calculated for the amplitudes of the positive and negative counterparts of the Bijvoet pairs. (%)8.6Completness (%)98.7 (91.3)Number of Pt sites15Rfactor/Rfree*Rfree is calculated with 5% of the data. (%)21.5/25.5Phasing PowercPhasing power is defined by <|FH|>/<(lack-of-closure)>, where H represents heavy-atom. (4.5Å/3.6Å)anomalous isomorphousNo. of protein atoms15,1141.13/0.6 1.1/0.73No. of carbohydrate atoms700FOMdMean figure of merit is the estimated mean cosine of the phase error. (4.5Å/3.6Å)0.45/0.25r.m.s. deviationsbonds (Å) angles (deg)0.009 1.25a Rsym = ∑∑i|I(h)i- 〈I(h) 〉|/∑∑iI(h)i, where <I(h)> is the mean intensity. Numbers in parentheses reflect statistics for the highest resolution shells.b Riso = ∑|Fnat(h) - FPt(h)|/∑Fnat(h) and Rano is calculated for the amplitudes of the positive and negative counterparts of the Bijvoet pairs.c Phasing power is defined by <|FH|>/<(lack-of-closure)>, where H represents heavy-atom.d Mean figure of merit is the estimated mean cosine of the phase error.* Rfree is calculated with 5% of the data. Open table in a new tab Chemical Cross-linking—The solution state of Lingo-1 (residues 1-478) was analyzed by chemical cross-linking and dynamic light scattering, and of Lingo1-His by analytical centrifugation. To achieve a substantial level of cross-linking, 50 μm Lingo-1 in 50 mm Tris, pH 7.5, 150 mm NaCl was buffer exchanged with 50 mm Tris, pH 8.7, 150 mm NaCl using a desalting column. The buffer exchanged Lingo-1 at 50 μm was incubated with 75 μm glutaraldehyde at 18 °C for 2 h. Cross-linked Lingo-1 was purified away from glutaraldehyde by washing the mixture several times with 50 mm Tris; pH 7.5, 150 mm NaCl, followed by concentration of the protein using a Microcon. As a negative control, Lingo-1 in 50 mm Tris, pH 8.7, 150 mm NaCl was incubated at 18 °C for 2 h. Both these samples were run on 4-12% Bis-Tris SDS-PAGE gel and protein bands were visualized by Coomassie staining. Dynamic Light Scattering—The hydrodynamic radius of Lingo-1 was measured at various protein concentrations (ranged from 4 to 125 μm) using a DynaPro DLS instrument. Each protein sample was centrifuged at 13,000 rpm for 15 min to remove any particulates. The supernatant was transferred to a quartz cuvette, and 10-20 readings at 18 °C were averaged per sample. Analytical Ultracentrifugation—Sedimentation equilibrium experiments were performed on a Beckman XLI/XLA analytical ultracentrifuge at 20 °C at three different rotor speeds (9,000, 12,000, and 18,000 r.p.m.) and four concentrations (1.0, 5.8, 16.6, and 49.3 μm). Samples were loaded into six-channel (1.2-cm path length) carbon-Epon centerpieces in an An-50 Ti titanium rotor. Scans were recorded at 230, 250, and 280 nm with a 0.001-cm spacing and ten replicates per point, and equilibrium was judged to be achieved when there was no deviation between successive scans taken 3 h apart. Data were analyzed by nonlinear regression with WinNONLIN (20Yphantis D. Johnson M.L. Lary J.W. WinNONLIN program. National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, CT1997Google Scholar). The solvent density and viscosity were calculated with the program Sednterp (21Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). The data were fit to different associating models (monomer-dimer-trimer, monomer-dimer-trimer-tetramer, and monomer-dimer-tetramer). The residual, variance of the fit, and R2 were used to judge how well the data fit to the different models. Better fits were obtained for protein concentrations <50 μm, and the best fit was for a monomer-dimer-tetramer equilibrium model. Association constants for monomerdimer-tetramer equilibria obtained from WinNONLIN were converted from absorbance (Kn,abs) to molar units (Kn,M) with the equation, Kn,M = Kn,abs(ϵl)/n, where l is the path length of the cell (1.2 cm), ϵ is the molar extinction at the wavelength monitored (23,120 m-1 cm-1 at 250 nm), and n is the oligomer size. Sequence Analysis—The set of homologous Lingo-1 sequences was retrieved with a BLAST search from the publicly available protein data base UniProt (Swissprot, TrEMBL, and PIR). The ectodomain regions (amino acids 1-477) of Lingo-1 sequences (primary accession numbers: Q96FE5_Human, Q9N008_Macfa, Q5RDJ4_Ponpy, Q9D1T0_Mouse, Q50L44_Chick, andQ562A6_Rat) were extracted, subsequentlyanalyzed, and aligned by using FASTA (22Pearson W.R. Methods Enzymol. 1990; 183: 63-98Crossref PubMed Scopus (1646) Google Scholar). Graphics—The figures were generated using COOT (17Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23207) Google Scholar) and PyMol. 4W. L. DeLano (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA. Functional Characterization and Structure Determination of Recombinant Lingo-1—To obtain a homogeneous high mannose glycoform of the protein suitable for crystallography studies, the extracellular portion of glycosylated recombinant human Lingo-1 (amino acids 1-516, coding sequence 34-549, plus a C-terminal 6-histidine tag) was produced in lectin-resistant CHO Lec 3.2.8.1 cells and purified to homogeneity as described under “Experimental Procedures.” The obtained recombinant Lingo-1-His was evaluated in a neurite outgrowth assay and examined for binding to its partners NgR and p75. We first established that neurite outgrowth from cerebellar granule neurons can be inhibited by immobilized MAG-Fc but not control IgG-Fc and then showed that Lingo-1-His is able to completely reverse the MAG-induced neurite outgrowth inhibition (Fig. 2, A-C). The physical interaction of Lingo-1-His with NgR- and p75-expressing CHO cells was demonstrated in a separate experiment (Fig. 2D). We also examined the interaction of the Lingo-1-His protein with NgR in vitro, employing a surface plasmon resonance assay (Fig. 2E). The steady-state equilibrium analysis indicated that soluble Lingo-1-His maintained micromolar affinity binding (Kd ∼ 1 μm) to the immobilized soluble NgR. Although the use of immobilized and truncated proteins may not accurately reflect the binding affinities of these proteins on the cell surface, these data confirm that Lingo-1 structural determinants required for recognition of NgR are sufficiently contained within its ectodomain. Crystallization of the full-length ectodomain of Lingo-1-His produced small weakly diffracting crystals. Cleavage of the His tag and stalk region prior to crystallization, so that the resultant ectodomain extended from residues 1 to 478, yielded two new crystal forms, the best of which (crystal form P21212) diffracted to 2.7-Å resolution. The quaternary arrangement of the protein is the same in both crystal forms analyzed. In the I222 crystal form, the dimer of dimers is crystallographic in nature, whereas in the P21212 form, the same configuration is generated by four independent Lingo-1 molecules in the asymmetric unit. Experimental phases were derived by single isomorphous replacement with anomalous scattering (SIRAS) followed by molecular averaging and solvent flattening. A complete model for the four molecules in the crystallographic asymmetric unit was built iteratively and refined to an R-value of 21.5% (Rfree = 25.5%) at 2.7-Å resolution. A representative portion of the resultant electron density map is shown in Fig. 3A, and the summary of the data collection, phasing, and refinement statistics is given in Table 1. All four molecules adopt similar, but not identical, conformations; the root mean square (r.m.s.) deviations of 0.5-1.3 Å for Cα-atoms are mainly due to some flexibility of the interdomain tilt angle and to differences in loop regions. Protomer Architecture—Protomers of Lingo-1 fold into a two-module, kinked structure resembling a question mark (Fig. 3B). The N-terminal LRR module (residues 1-382) is an elongated, fairly deep arc with 15 parallel β-strands on the concave face and mostly irregular extended structures on the convex face. Overall, the canonical architecture of this module is most similar to that of the NgR ectodomain (24He X.L. Bazan J.F. McDermott G. Park J.B. Wang K. Tessier-Lavigne M. He Z. Garcia K.C. Neuron,. 2003; 38: 177-185Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 25Barton W.A. Liu B.P. Tzvetkova D. Jeffrey P.D. Fournier A.E. Sah D. Cate R. Strittmatter S.M. Nikolov D.B. EMBO J. 2003; 22: 3291-3302Crossref PubMed Scopus (193) Google Scholar), except for appreciable differences in arc curvature and arc length (Fig. 3C). In total, there are twelve complete LRRs, 23-25 residues each, plus one partial repeat, which together create a classic right-handed super-helical array. Each LRR begins with a β-strand and loops back by virtue of the consensus 24-residue sequence repeat motif: XL2XXL5XL7XXN10XL12XXL15XXXXF20XXL23X, where X can be any amino acid; L are hydrophobic residues, preferentially Leu, but also Ile, Val, Met, Phe, or Thr; N are less conserved in nature and include mostly Asn, but also Cys, Asp, Leu, or Trp; F represents Phe or Leu. The consensus residues at the indicated positions make up the interior of the LRR domain. As in a number of other LRR-containing proteins, the hydrophobic core of the LRR structure is sheltered on both ends by cysteine-rich capping regions. The “N-cap” (residues 3-32) has two anti-parallel β-strands, wi" @default.
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