Crystal Structure of the Human Laminin Receptor Precursor

The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (–)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR. The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (–)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR. The human LamR 3The abbreviations used are: LamRlaminin receptorMES4-morpholineethanesulfonic acidBSAbovine serum albuminPDBProtein Data BankEGCG(–)-epigallocatechin-3-gallate. precursor protein and the p40 ribosomal protein are encoded by the same gene (37LRP/p40) (1Jackers P. Minoletti F. Belotti D. Clausse N. Sozzi G. Sobel M.E. Castronovo V. Oncogene. 1996; 13: 495-503PubMed Google Scholar), demonstrating that human LamR is a protein that has acquired dual function through evolution, acting as both a cell surface receptor and a ribosomal protein (2Ardini E. Pesole G. Tagliabue E. Magnifico A. Castronovo V. Sobel M.E. Colnaghi M.I. Menard S. Mol. Biol. Evol. 1998; 15: 1017-1025Crossref PubMed Scopus (115) Google Scholar). In addition to its role as a ribosomal protein, LamR is a nonintegrin cell surface protein that has been identified as the receptor for the extracellular matrix molecule laminin-1 (3Rao N.C. Barsky S.H. Terranova V.P. Liotta L.A. Biochem. Biophys. Res. Commun. 1983; 111: 804-808Crossref PubMed Scopus (306) Google Scholar), pathogenic prion protein (4Gauczynski S. Peyrin J.M. Haik S. Leucht C. Hundt C. Rieger R. Krasemann S. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5863-5875Crossref PubMed Scopus (365) Google Scholar), Sindbis virus (5Wang K.S. Kuhn R.J. Strauss E.G. Ou S. Strauss J.H. J. Virol. 1992; 66: 4992-5001Crossref PubMed Google Scholar), Venezuelan equine encephalitis virus (6Ludwig G.V. Kondig J.P. Smith J.F. J. Virol. 1996; 70: 5592-5599Crossref PubMed Google Scholar), cytotoxic necrotizing factor types I and II (7McNichol B.A. Rasmussen S.B. Carvalho H.M. Meysick K.C. O'Brien A.D. Infect. Immun. 2007; 75: 5095-5104Crossref PubMed Scopus (24) Google Scholar), and adeno-associated virus serotypes 2, 3, 8, and 9 (8Akache B. Grimm D. Pandey K. Yant S.R. Xu H. Kay M.A. J. Virol. 2006; 80: 9831-9836Crossref PubMed Scopus (317) Google Scholar). At the cell surface, LamR exists as a monomer (37 kDa) and dimer (67 kDa) (4Gauczynski S. Peyrin J.M. Haik S. Leucht C. Hundt C. Rieger R. Krasemann S. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5863-5875Crossref PubMed Scopus (365) Google Scholar). The homo- or heterodimeric state of 67-kDa LamR has yet to be resolved, but its association with the cell surface is mediated by fatty acid acylation (9Landowski T.H. Dratz E.A. Starkey J.R. Biochemistry. 1995; 34: 11276-11287Crossref PubMed Scopus (102) Google Scholar). LamR expression acts as a prognostic factor in determining the degree of malignancy of human cancer patients (10Menard S. Tagliabue E. Colnaghi M.I. Breast Cancer Res. Treat. 1998; 52: 137-145Crossref PubMed Scopus (136) Google Scholar). Overexpression of LamR correlates with a highly invasive cell phenotype and increased metastatic ability (10Menard S. Tagliabue E. Colnaghi M.I. Breast Cancer Res. Treat. 1998; 52: 137-145Crossref PubMed Scopus (136) Google Scholar), mediated by the high affinity binding between LamR and laminin in the extracellular matrix. The dual function of human LamR as a receptor at the cell surface and as a component of the translational machinery may be important for understanding how overexpression of LamR in cancer affects disease pathogenesis. laminin receptor 4-morpholineethanesulfonic acid bovine serum albumin Protein Data Bank (–)-epigallocatechin-3-gallate. The role of LamR as a ribosomal protein is also of significant interest. Intracellular LamR is localized on the 40 S ribosome (11Auth D. Brawerman G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4368-4372Crossref PubMed Scopus (113) Google Scholar) and in the nucleus (12Sato M. Kinoshita K. Kaneda Y. Saeki Y. Iwamatsu A. Tanaka K. Biochem. Biophys. Res. Commun. 1996; 229: 896-901Crossref PubMed Scopus (41) Google Scholar). Human LamR and Rps0, the homolog in yeast, which does not exhibit laminin binding activity, share 60% sequence identity (see Fig. 1). Rps0 is responsible for 20 S ribosomal RNA processing and maturation of the 40 S ribosomal protein subunit, directly affecting protein synthesis levels (13Ford C.L. Randal-Whitis L. Ellis S.R. Cancer Res. 1999; 59: 704-710PubMed Google Scholar). Both the localization of human LamR to more than one intracellular component and the multiple functions of its homolog in yeast suggest that human LamR may also have numerous functions at the intracellular level. Recombinant LamR Expression and PurificationResidues 1–220 of human 37-kDa LamR precursor protein (LamR220) were subcloned from a full-length LamR cDNA into an Escherichia coli expression vector that includes a tobacco etch virus-cleavable, N-terminal His6 tag. The construct was verified by automated DNA sequencing. The vector encoding LamR220 was transformed into E. coli strain BL21 (DE3*), and cultures were grown in Luria broth medium at 37 °C to an A600 of 0.6. Protein expression was induced by the addition of isopropyl-thiogalactopyranoside (0.1 mm) for 12 h at 20 °C. Cells were harvested, resuspended in lysis buffer (50 mm Tris (pH 8.0), 300 mm NaCl, 0.1% Triton X-100, 10% glycerol, EDTA-free protease inhibitor tablet (Roche Applied Science)), and lysed by French press. The lysate was centrifuged at 16,000 RPM for 30 min, and the supernatant was collected. The soluble fraction was purified by nickel-nitrilotriacetic acid chromatography (Qiagen) followed by gel filtration chromatography (Superdex 75, Amersham Biosciences). Protein was concentrated in spin concentrators (Amicon, Millipore). Crystallization and Data CollectionCrystals of LamR220 were grown at 17 °C by the hanging-drop vapor diffusion method in drops containing a 1:1 (v/v) ratio of protein solution at 10 mg/ml and reservoir solution containing 17% (w/v) polyethylene glycol 10,000, 120 mm sodium citrate, 100 mm MES (pH 6.0), and 5% (w/v) polyethylene glycol 1,500. Crystals belong to the tetragonal space group P43212 with unit cell dimensions of a = 75.69 Å, b = 75.69 Å, and c = 99.01 Å. Crystals were soaked briefly in crystallization buffer with 20% glycerol (v/v) and then flash-frozen in liquid nitrogen. There is one LamR220 molecule in the asymmetric unit with a solvent content of 56%. Diffraction data were collected to a resolution of 2.15 Å at X29 at the National Synchrotron Light Source at Brookhaven National Laboratory. Data were processed with HKL2000 (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). A molecular replacement solution was found with AMoRE (15Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using as a search model the structure of the Archaeoglobus fulgidus 30 S ribosomal protein S2p (PDB code 1VI6, chain A) (16Badger J. Sauder J.M. Adams J.M. Antonysamy S. Bain K. Bergseid M.G. Buchanan S.G. Buchanan M.D. Batiyenko Y. Christopher J.A. Emtage S. Eroshkina A. Feil I. Furlong E.B. Gajiwala K.S. Gao X. He D. Hendle J. Huber A. Hoda K. Kearins P. Kissinger C. Laubert B. Lewis H.A. Lin J. Loomis K. Lorimer D. Louie G. Maletic M. Marsh C.D. Miller I. Molinari J. Muller-Dieckmann H.J. Newman J.M. Noland B.W. Pagarigan B. Park F. Peat T.S. Post K.W. Radojicic S. Ramos A. Romero R. Rutter M.E. Sanderson W.E. Schwinn K.D. Tresser J. Winhoven J. Wright T.A. Wu L. Xu J. Harris T.J. Proteins. 2005; 60: 787-796Crossref PubMed Scopus (211) Google Scholar). Rigid body refinement, simulated annealing, and positional and B-factor refinement were performed with CNS (17Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (8) Google Scholar) and Refmac (18Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). Coot was used for model building (19Emsley P. Cowtan K. Acta Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). According to PROCHECK (20Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), 95.4% of the residues have backbone torsion angles in most favored regions, and 4.6% have backbone torsion angles in additional allowed regions. In Vitro Binding Affinity for laminin-1LamR220 and full-length LamR (LamR295) binding affinity for laminin-1 were tested in vitro. White polystyrene enzyme-linked immunosorbent assay 96-well microplates, precoated with murine laminin-1 (New England Biolabs), were blocked overnight at 4 °C with blocking buffer (3.0% BSA, 0.1% sodium azide in phosphate-buffered saline). Wells were incubated with increasing concentrations of LamR220 or LamR295, which was nickel affinity-purified as described above for 1 h at 37 °C. Each well was washed three times with blocking buffer. Penta-His horseradish peroxidase conjugate (1:500) (Qiagen) was incubated for 2 h at room temperature, and wells were washed three times with blocking buffer. Substrate solution was added and incubated for ∼15 min before reading, and fluorescent absorbance was detected at 490 nm on an enzyme-linked immunosorbent assay plate reader (ELX800, Biotek Instruments, Inc.). Controls for buffer alone and nonspecific protein, BSA (New England Biolabs), were also tested. Experiments were performed in triplicate. A binding curve and Kd were generated for LamR220 and LamR295. The Kd was calculated using a one-site binding hyperbola and the equation Y = Bmax × X/(Kd + X). Each group was tested in triplicate, and binding affinity was determined by normalizing to background fluorescence. In Vitro Sindbis Virus Vector Inhibition3 × 106 BHK-55 cells were plated in a 12-well plate for 24 h in αMEM (Cellgro) supplemented with 10% fetal bovine serum. Sindbis virus vector that expressed a luciferin reporter upon replication (multiplicity of infection = 100) was incubated with control protein (BSA, New England Biolabs), LamR220, or alone and rotated at room temperature for 1 h. Cells were washed with Optimem (Invitrogen), and 300 μl of sample was added. Samples were incubated, rotating gently for 1 h at room temperature to allow Sindbis viral vector attachment. The supernatant was aspirated, cells were washed with phosphate-buffered saline, and 1 ml of medium was added. The cells were incubated overnight at 37 °C, and medium was aspirated the next day. 200 μl of unsupplemented medium and 200 μl of luciferin substrate (SteadyGlo luciferase assay, Promega) were added. The cells were shaken vigorously for 15 min. The relative luciferase units, which correspond to infectivity of Sindbis virus vector, of each sample were read using a luminometer (Glomax 20/20, Promega). Experiments were performed in triplicate. A two-tailed Student's t test was performed (p < 0.05) to determine statistical significance. LamR Expression, Purification, Crystallization, and Structural DeterminationTo better understand the function of LamR and its interaction with binding partners, we sought to crystallize the human LamR precursor protein. To this end, several different-length constructs were overexpressed in E. coli, including residues 1–295 (full-length), residues 1–220, and residues 1–195. Of these, only the 220-residue version of LamR (abbreviated LamR220) could be purified in amounts suitable for crystallization trials. LamR220 binds laminin-1 in vitro (supplemental Fig. 1a) and inhibits Sindbis virus vector infection of baby hamster kidney (BHK) cells (supplemental Fig. 1b). These data demonstrate that the first 220 residues of LamR are sufficient for interacting with key binding partners. Crystals of LamR220 were obtained at pH 6.0 and belong to tetragonal space group P43212 with one molecule in the asymmetric unit. The crystal structure of LamR220 was determined by molecular replacement, using the crystal structure of the 30 S ribosomal protein S2p from A. fulgidus (PDB code 1VI6) as a search model (16Badger J. Sauder J.M. Adams J.M. Antonysamy S. Bain K. Bergseid M.G. Buchanan S.G. Buchanan M.D. Batiyenko Y. Christopher J.A. Emtage S. Eroshkina A. Feil I. Furlong E.B. Gajiwala K.S. Gao X. He D. Hendle J. Huber A. Hoda K. Kearins P. Kissinger C. Laubert B. Lewis H.A. Lin J. Loomis K. Lorimer D. Louie G. Maletic M. Marsh C.D. Miller I. Molinari J. Muller-Dieckmann H.J. Newman J.M. Noland B.W. Pagarigan B. Park F. Peat T.S. Post K.W. Radojicic S. Ramos A. Romero R. Rutter M.E. Sanderson W.E. Schwinn K.D. Tresser J. Winhoven J. Wright T.A. Wu L. Xu J. Harris T.J. Proteins. 2005; 60: 787-796Crossref PubMed Scopus (211) Google Scholar). Data collection and refinement statistics at 2.15 Å resolution are given in Table 1.TABLE 1LamR220 data collection and refinement statistics (molecular replacement)Data collectionSpace groupP43212Cell dimensionsa, b, c (Å)75.69, 75.69, 99.01α, β, γ (°)90.00, 90.00, 90.00Resolution (Å)50.0 (2.15)aValues in parentheses are for highest resolution shell (2.25-2.15 Å).Rsym or Rmerge (%)14.4 (47.7)I/δI5.0 (4.0)Completeness (%)100 (100)Redundancy9.4 (9.7)RefinementResolution (Å)50.0-2.15No. of reflections14762Rwork/Rfree (%)18.5/22.4No. of atoms1712Protein1563Ligand/ion0Water149Average B-factor21.8r.m.s.br.m.s., root mean square. deviationsBond lengths (Å)0.009Bond angles (°)1.22a Values in parentheses are for highest resolution shell (2.25-2.15 Å).b r.m.s., root mean square. Open table in a new tab Overall Structure of Human LamRConsistent with a sequence identity of 32% between LamR and A. fulgidus S2p (residues 15–183) (Fig. 1), the two proteins share a similar overall architecture, classified (Structural Classification of Proteins (SCOP)) as an α/β protein with a flavodoxin-like fold (Fig. 2a). A central β sheet composed of five parallel β strands (β3-β7) is flanked by three α helices on one side (αB, αC, and αE) and a single α helix (αD) on the other side. An N-terminal α helix (αA) and two anti-parallel β strands (β1-β2) pack against the α/β core of the protein. Residues 1–8 and 206–220 of LamR220 are disordered in the structure. Structural Comparison of Human LamR and S2 Protein from Other SpeciesSuperimposition of the structures of LamR220 and A. fulgidus S2p yields a root-mean-square deviation in Cα positions of just 0.9 Å (174 atoms) and reveals two areas in which the structures are divergent (Fig. 2b): a segment between β4 and β5 (residues 111–118 in LamR) and a segment after the last α helix (αE) (residues 188–196 in LamR), in which LamR contains a five-residue insertion relative to A. fulgidis S2p. The segment between β4 and β5 contains an equal number of residues in the two proteins. In A. fulgidis S2p, the segment is stabilized in a folded-back conformation via a salt bridge between Arg-113 in this segment (Arg-117 in LamR) and Asp-93 (β4), the latter of which is not conserved in LamR (Thr-97). In the LamR220 structure, this segment instead projects away from the domain and packs against the same segment in a symmetry-related (two-fold) molecule (Fig. 2c). In this crystallographic dimer, Ala-114 packs into a tight pocket in the symmetry-related molecule formed by the β4-β5 segment and the end of αD, and Phe-116 is in van der Waals contact with Tyr-139 (αD) (Fig. 2c). Although Phe-116 is generally conserved from Saccharomyces cerevisiae through vertebrate species, Ala-114 is conserved only in vertebrates. The total surface area buried in this interface is a modest 832 Å2, and LamR220 runs as a monomer in solution, but in the context of a membrane attachment and a possible covalent dimerization linkage (9Landowski T.H. Dratz E.A. Starkey J.R. Biochemistry. 1995; 34: 11276-11287Crossref PubMed Scopus (102) Google Scholar), this crystallographic dimer could be functionally significant. Functional Domains of LamRThe structural differences noted between LamR220 and A. fulgidis S2p (Fig. 2b) could be important for ribosomal protein function or for the acquired function as the receptor for laminin. Analysis of the 3.0 Å resolution structure of the 30 S ribosomal subunit from Thermus thermophilus (PDB code 1J5E) (21Wimberly B.T. Brodersen D.E. Clemons Jr., W.M. Morgan-Warren R.J. Carter A.P. Vonrhein C. Hartsch T. Ramakrishnan V. Nature. 2000; 407: 327-339Crossref PubMed Scopus (1718) Google Scholar) indicates that the two major structural deviations between human LamR220 and A. fulgidis S2p (between β4 and β5 and after αE) would not appear to affect ribosomal function, since no RNA or protein contacts are present in these regions. This suggests that the structural differences in human LamR versus A. fulgidis S2p are important for laminin binding. Previously, of LamR in binding implicated a residues as as a binding for laminin V. G. Sobel M.E. J. Biol. PubMed Google Scholar). In the LamR220 crystal structure, this of residues the between and most of The only of this sequence that is are residues in the is that the of LamR220 a residues this the of of LamR to the laminin binding may to be In a residues was V. G. Sobel M.E. J. Biol. PubMed Google Scholar). This β4 and most of αC, is an of the protein fold and is to as a of LamR in has been implicated in a of including and In the of LamR function at the cell surface of tumor cells, by binding of which most with laminin, or by infection with Sindbis virus vector, which is by has been with M. K. M. M. Y. T. Y. T. K. Cancer 2000; PubMed Scopus (41) Google H. K. Y. K. Mol. Biol. 2004; PubMed Scopus Google B. A. H. I. M. P. R. A. D. 2004; PubMed Scopus Google Y. R. Nature. 1999; PubMed Scopus Google Scholar). Both and Sindbis virus through two the ability of LamR at the surface to with laminin. The structural of LamR to an understanding of how LamR interacts with its binding and in the of therapeutics that LamR in the of and viral and for of this and and for in data collection at and Brookhaven National for which from the of and and of of the U. S. of and from the National for of the National of with

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