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Crystal Structure and Cell Surface Anchorage Sites of Laminin α1LG4-5

2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês

10.1074/jbc.m610657200

1083-351X

David H. T. Harrison, Sadaf-Ahmahni Hussain, Ariana C. Combs, James M. Ervasti, Peter D. Yurchenco, Erhard Hohenester,

Signaling Pathways in Disease

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin α1LG4-5 domains and provide a mutational analysis of heparin, α-dystroglycan, and galactosylsulfatide binding. The two domains of α1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin α2 LG4-5 but with a substantially different interdomain angle. Recombinant α1LG4-5 binding to heparin, α-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to α-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands. The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin α1LG4-5 domains and provide a mutational analysis of heparin, α-dystroglycan, and galactosylsulfatide binding. The two domains of α1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin α2 LG4-5 but with a substantially different interdomain angle. Recombinant α1LG4-5 binding to heparin, α-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to α-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands. Laminin-111, recently renamed from laminin-1 to better reflect its α1β 1γ1 subunit composition, is one of the first two laminins to be expressed during embryonic development, appearing in the peri-implantation period in the basement membrane of the embryonic plate along with laminin-511 (laminin-10) and in the absence of other laminins in Reichert's membrane (1Miner J.H. Yurchenco P.D. Annu. Rev. Cell Dev. Biol. 2004; 20: 255-284Crossref PubMed Scopus (580) Google Scholar, 2Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (676) Google Scholar). Later in development, laminin-111 is strongly expressed in placenta, liver, kidney, and testis, where it is thought to play a role in organogenesis (3Sasaki T. Giltay R. Talts U. Timpl R. Talts J.F. Exp. Cell Res. 2002; 275: 185-199Crossref PubMed Scopus (93) Google Scholar). In the adult, the laminin α1 chain has very limited expression and is largely supplanted by the laminin α5 chain. Targeted inactivation of the LAMA1 gene coding for the α1 chain was found to result in a failure of Reichert's membrane with developmental arrest by embryonic day 6.5 in the mouse (4Miner J.H. Li C. Mudd J.L. Go G. Sutherland A.E. Development. 2004; 131: 2247-2256Crossref PubMed Scopus (241) Google Scholar). In-frame deletion of the mouse laminin α1 exons corresponding to laminin G-like (LG) 3The abbreviations used are: LG, laminin G-like domain; DG, dystroglycan; WT, wild type (native sequence); EWB, enzyme-linked immunosorbent assay wash buffer; GM1, Galβ3GalNAcβ4(NeuAcα3)Galβ4Glcβ1Cer. domains 4 and 5 was found to result in similar stage lethality accompanied by defective epiblast differentiation without loss of basement membrane (5Scheele S. Falk M. Franzen A. Ellin F. Ferletta M. Lonaio P. Andersson B. Timpl R. Forsberg E. Ekblom P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1502-1506Crossref PubMed Scopus (54) Google Scholar), the last possibly a result of the partially redundant expression of laminin-511 (4Miner J.H. Li C. Mudd J.L. Go G. Sutherland A.E. Development. 2004; 131: 2247-2256Crossref PubMed Scopus (241) Google Scholar). The α1 subunit provides most of the unique characteristics of laminin-111. The N-terminal LN domain participates in polymerization by interacting with the LN domains of the β1 and γ1 chains. The C-terminal LG domains, LG1-3, bind to the α6β1 integrin, whereas LG4-5 bind to heparin, sulfated glycolipids, and α-dystroglycan (α-DG). The polymerization and cell-anchoring activities are thought to act in concert to assemble a functional basement membrane on a cell surface (6Li S. Harrison D. Carbonetto S. Fassler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar). Our earlier understanding of the laminin α1 LG1-5 structure was based on crystal structures of LG4 and LG4-5 from the related α2 chain (∼40% sequence identity). The LG domain fold was revealed as a multistranded β-sandwich with one bound calcium ion. In the LG4-5 pair, the two domains are connected in a V-shaped arrangement, in which LG5 is disulfide-bonded to the linker preceding LG4 (7Hohenester E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 8Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar). Although the structure of LG1-3 remains to be elucidated, it is thought that these three domains form a closed arrangement with similar angles between domains and separated from the LG4-5 pair by a hinge-like region (9Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (258) Google Scholar). Analysis of contributions of α1LG4-5 to laminin interactions with Schwann cells, myotubes, and developing epithelia has led to a model in which these domains provide the major anchoring activity of laminin, an initiating event of basement membrane assembly that leads to alterations of the cell cytoskeleton accompanied by signaling (6Li S. Harrison D. Carbonetto S. Fassler R. Smyth N. Edgar D. Yurchenco P.D. J. Cell Biol. 2002; 157: 1279-1290Crossref PubMed Scopus (256) Google Scholar, 10Colognato H. Winkelmann D.A. Yurchenco P.D. J. Cell Biol. 1999; 145: 619-631Crossref PubMed Scopus (255) Google Scholar, 11Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar). Basic residues within the LG4-5 pair of several laminins mediate key interactions with three types of molecules: heparan sulfate chains that are attached to perlecan, agrin, collagen, and syndecan core proteins of basement membranes and cell surface; the glycoprotein α-DG that is a component of a larger transmembrane and submembrane complex associated with dystrophin and utrophin; and sulfated glycolipids, in particular the sulfatides, that can be present in the outer leaflet of the cell plasma membrane. Earlier mutagenesis analyses of recombinant laminin α1 and α2LG fragments revealed that there are binding similarities and differences between the two laminins. Heparin, representing the highly sulfated regions of glycosaminoglycan chains, α-DG, and galactosyl-3-sulfate ceramide (galactosyl sulfatide) all bind to an extensive basic surface region between the calcium sites of the laminin α2 LG4-5 domain pair (12Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar, 13Wizemann H. Garbe J.H. Friedrich M.V. Timpl R. Sasaki T. Hohenester E. J. Mol. Biol. 2003; 332: 635-642Crossref PubMed Scopus (65) Google Scholar). In contrast, a smaller topographical region confined to LG4 appears to bind the same cell surface components in the laminin α1 chain (12Talts J.F. Andac Z. Gohring W. Brancaccio A. Timpl R. EMBO J. 1999; 18: 863-870Crossref PubMed Scopus (398) Google Scholar, 14Andac Z. Sasaki T. Mann K. Brancaccio A. Deutzmann R. Timpl R. J. Mol. Biol. 1999; 287: 253-264Crossref PubMed Scopus (95) Google Scholar). The present study describes the crystal structure of laminin α1LG4-5 in conjunction with a site-directed mutagenesis and in vitro analysis of the binding sites for heparin, α-DG, and sulfatides. We report that the backbone of the α1LG4-5 domain pair is very similar to that of α2LG4-5, but there are significant differences in the distribution of charged residues on the protein surface. Binding of α1LG4-5 to heparin, α-DG, and sulfatides was found to be dependent upon partially overlapping basic amino acid residue clusters. Our results have led, we believe, to an improved understanding of the amino acid residues involved in each type of interaction and will aid in defining the biological roles of different laminin ligands. Laminin α1LG4-5 Vector Constructs—An expression construct containing the wild-type (WT) mouse laminin α1LG4-5 sequence (coding for residues 2666LHREH… PGPEP3060 of the mature laminin α1 chain (i.e. the numbering scheme used here omits the 24-residue signal peptide of SwissProt entry P19137)) was created by amplifying cDNA from laminin α1 pCIS (15Yurchenco P.D. Quan Y. Colognato H. Mathus T. Harrison D. Yamada Y. O'Rear J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10189-10194Crossref PubMed Scopus (123) Google Scholar) utilizing three successive PCRs. Three overlapping 5′ sense primers were used to place a 5′-terminal NheI site followed by the 5′-untranslated region and signal sequence of human BM-40 (cleaved by the signal peptidase) and a FLAG epitope tag (DYKDDDDK), whereas the 3′ primer placed a KpnI site downstream of the STOP codon at the 3′ terminus of the amplified product (oligonucleotide sequences are listed in supplemental Table 1). Pfx polymerase (Invitrogen) was used along with a PTC-100 thermal cycler (MI Research) to amplify the DNA, which was then purified after each reaction (UltraClean PCR DNA purification kit; MoBio). The NheI and KpnI sites were used to clone the PCR product into the analogous sites in the pcDNA3.1+/zeo vector (Invitrogen). The mutated α1LG4-5 DNAs were constructed by strand overlap extension PCR using the same three 5′ upstream sense primers and downstream 3′ antisense oligonucleotide in conjunction with internal primers introducing the desired mutations. Escherichia coli DH5α cells (Invitrogen) were transformed with the plasmids, and plasmid DNA was purified by alkaline lysis and spin columns (Ultra-Clean Standard Mini Plasma Prep Kit, MoBio). All generated plasmids were completely sequenced. Recombinant α1LG4-5 Protein Production and Purification—Expression constructs were linearized with BglII (New England Biolabs) and transfected into the human kidney fibroblast cell line 293 (ATCC) using Lipofectamine 2000 (Invitrogen), and stable clones were selected under zeocin for secretion of α1LG4-5 protein. Cells were grown in Dulbecco's modified Eagle's medium with high glucose (Invitrogen) supplemented with 10% fetal bovine serum, 50 units/ml penicillin G, 50 μg/ml streptomycin, and 100 μg/ml zeocin (Invitrogen). Once cells had reached confluence, the growth medium was replaced with fresh medium minus zeocin and then collected 72 h later. The genomic DNA was isolated from the cells after medium harvesting (Exact-N-Amp kit; Sigma) and sequenced to verify the identity of the various α1LG4-5 proteins. The conditioned media were passed through a gravity column (Bio-Rad) packed with anti-FLAG M2-agarose resin (Sigma), and recombinant α1LG4-5 proteins were eluted with FLAG peptide (Sigma) in 90 mm NaCl, 1 mm CaCl2, 50 mm Tris-HCl, pH 7.4 (TBS50/Ca) at 4 °C. The eluted proteins were then loaded onto a heparin 5PW column (TosoHass) on anÄkta FPLC system (Amersham Biosciences), where the FLAG peptide (unbound component) was recovered for reuse, and the α1LG4-5 proteins were eluted using a 1 m NaCl gradient. The eluted α1LG4-5 proteins were concentrated, and the buffer was exchanged into TBS50/Ca at room temperature via centrifugal filtration with Amicon Ultra spin filters (Millipore). The recombinant proteins were further dialyzed against TBS50/Ca buffer with several buffer changes for 2 days using dialysis cassettes (Slide-A-Lyzer; Pierce). Deglycosylated WT α1LG4-5 protein was produced either by isolating WT α1LG4-5 from the medium of stably transfected cell lines grown in 2 μg/ml tunicamycin (Sigma) for 24 h or by treating 50 μg of purified α1LG4-5 with 1000 units of peptide N-glycosidase F (New England Biolabs) in TBS50/Ca for 1 h at 37 °C. Laminin α1LG4-5 Vector Construct for Crystallography—DNA coding for residues 2682QPELC… PGPEP3060 of the mature mouse laminin α1 chain was obtained by PCR amplification from laminin α1 pCIS (15Yurchenco P.D. Quan Y. Colognato H. Mathus T. Harrison D. Yamada Y. O'Rear J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10189-10194Crossref PubMed Scopus (123) Google Scholar). The PCR primers added NotI and NheI sites at the 5′ end and a STOP codon followed by XhoI and BamHI sites at the 3′ end. The PCR product was cloned into pBluescript II KS+ using NotI and BamHI, and four mutations (N2714Q, N2811K, N2900Q, and C3014S) were introduced by strand overlap extension PCR. The sequence-verified insert was cloned into the pCEP-Pu vector (16Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar) using NheI and XhoI. After cleavage of the BM-40 sequence signal, a vector-derived APLA sequence remains at the N terminus of the secreted recombinant α1LG4-5 protein. Protein Production for Crystallography—The laminin α1LG4-5 quadruple mutant (N2714Q/N2811K/N2900Q/C3014S) was purified from the conditioned medium of episomally transfected 293-EBNA cells. Cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum (Invitrogen), transfected using Fugene reagent (Roche Applied Science), and selected with 1 μg/ml puromycin (Sigma). Serum-free conditioned medium (1.5 liters) was dialyzed against 50 mm Na-HEPES, pH 7.5, and loaded onto a 2 × 5-ml heparin HiTrap column (GE Healthcare). Bound proteins were eluted using a linear NaCl gradient (0-1 m). The α1LG4-5 mutant protein eluted as a sharp single peak at ∼0.5 m NaCl and was further purified by size exclusion chromatography using a 24-ml Superdex 200 column (GE Healthcare) run at 0.5 ml/min in 20 mm Na-HEPES, pH 7.5, 150 mm NaCl. The final yield was 4 mg of pure protein. Crystal Structure Determination—The α1LG4-5 quadruple mutant protein was concentrated to 19 mg/ml in 10 mm Na-HEPES, pH 7.5. Crystals were obtained by hanging drop vapor diffusion using 20% polyethylene glycol 8000, 100 mm Tris-HCl, pH 8.5, 200 mm MgCl2 as precipitant. Crystals were frozen in liquid nitrogen in mother liquor supplemented with 20% glycerol. Diffraction data to 1.9 Å resolution were collected at 100 K on beamline 9.6 at the SRS Daresbury (λ = 0.87 Å). The crystals belong to space group P21, a = 70.53 Å, b = 55.81 Å, c = 100.99 Å, β = 98.48°. There are two α1LG4-5 molecules in the asymmetric unit, resulting in a solvent content of ∼45%. The diffraction data were processed with MOSFLM (available on the World Wide Web at www.mrc-lmb.cam.ac.uk/harry/mosflm) and programs of the CCP4 suite (17Collaborative Computing Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The structure was solved by molecular replacement with PHASER (18Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1103) Google Scholar), using the laminin α2LG4-5 structure (8Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar) as a search model; the LG domains had to be placed individually to obtain a solution. The structure was rebuilt with O (19Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refined with CNS (20Brunger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) without noncrystallographic symmetry restraints. Data collection and refinement statistics are summarized in Table 1. The figures were made with PYMOL (available on the World Wide Web at www.pymol.org).TABLE 1Crystallographic statisticsParameterValueData collection and reductionSpace groupP21Unit cell dimensionsa = 70.53 Å, b = 55.81 Å, c = 100.99 Å, β = 98.48°Resolution range (Å)20.0 (2.00) to 1.90Unique reflections59,682Multiplicity2.6 (2.1)Completeness (%)97.3 (91.5)Mean I/σ(I)10.2 (2.2)Rmerge0.070 (0.333)RefinementReflections (working set/test set)56,655/3015Atoms (protein/solvent)5781/334Rcryst/Rfree0.230/0.262Root mean square deviationsBond lengths (Å)0.006Bond angles (degrees)1.4B-factors (Å2)aDifference in B-factors of covalently bonded atoms.2.8Ramachandran plot (%)bResidues in most favored, additionally allowed, generously allowed, and disallowed regions (33). In both crystallographically independent molecules, two residues assume unfavorable main chain conformations: Lys2791, which is part of the heparin binding site, and Arg2896, whose peptide carbonyl oxygen receives a hydrogen bond from a buried lysine.86.9/11.9/0.8/0.5a Difference in B-factors of covalently bonded atoms.b Residues in most favored, additionally allowed, generously allowed, and disallowed regions (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). In both crystallographically independent molecules, two residues assume unfavorable main chain conformations: Lys2791, which is part of the heparin binding site, and Arg2896, whose peptide carbonyl oxygen receives a hydrogen bond from a buried lysine. Open table in a new tab Heparin Binding of Laminin α1 E3 Fragment and Recombinant α1LG4-5—The laminin-111 E3 fragment was prepared from laminin-111 purified from Engelbreth-Holm-Swarm sarcoma tumor (21Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). E3 fragment, recombinant α1LG4-5, α1LG4-5 treated with enterokinase to remove the FLAG tag, and mutant α1LG4-5 proteins were loaded onto a TosoHass Heparin 5PW column in TBS50/Ca at 4 °C on anÄkta FPLC system and eluted with a 0-1 m NaCl salt gradient, and the NaCl concentration required for elution was determined. Analysis of α1LG4-5 Binding to α-Dystroglycan—α-DG was purified from rabbit muscle as described (22Combs A.C. Ervasti J.M. Biochem. J. 2005; 390: 303-309Crossref PubMed Scopus (69) Google Scholar). Equal aliquots of α-DG (1 μg) were loaded into the slots of SDS-polyacrylamide gels and electrophoresed under reducing conditions. The protein bands were then electroeluted onto nitrocellulose membranes, blocked in phosphate-buffered saline containing 5% nonfat dry milk for 1 h at room temperature, and assessed for binding to each α1LG4-5 protein (1 μg/ml) using a previously described overlay assay (23Ervasti J.M. Burwell A.L. Geissler A.L. J. Biol. Chem. 1997; 272: 22315-22321Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Binding of the α1LG4-5 proteins was detected with 1 ml of 1.1 μg/ml horseradish peroxidasecoupled anti-FLAG antibody M2 (Sigma) per 3.5 cm2 of blot membrane. The solid phase assay was performed in 96-well microtiter plates with α-DG bound to the plate (100 μl of 1 μg/ml per well) and incubated with various concentrations of α1LG4-5 proteins as previously described (24Smirnov S.P. McDearmon E.L. Li S. Ervasti J.M. Tryggvason K. Yurchenco P.D. J. Biol. Chem. 2002; 277: 18928-18937Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), except that horseradish peroxidase-linked monoclonal FLAG antibody M2 (100 μl of 1.1 μg/ml per well) was used for detection followed by color development with 3,3′,5,5′-tetramethylbenzidine (Bio-Rad). Color development was quantitated at 655 nm using a Molecular Dynamics Spectramax 340 UV-visible microplate reader (25McDearmon E.L. Combs A.C. Ervasti J.M. J. Biol. Chem. 2003; 278: 44868-44873Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We verified that the signal readout was linear to OD > 3 by collecting kinetic data on color development. Estimates of half-maximal binding (KD) and binding capacity (Bmax) were determined by curve fitting of the binding data of WT LG4-5 using a single-site model (fitted values = Bmax × L/(KD + L), where L is the molar ligand concentration), with the calculated Bmax value used for subsequent determinations of all other half-maximal binding, an approach employed to minimize the errors inherent in estimating binding from plots that are low and nearly linear over the concentration range evaluated. Analysis of α1LG4-5 Binding to Galactosyl Sulfatide and Other Lipids—The ammonium salt of HSO4-3Galβ1-1′ceramide (brain sulfatides; Avanti Polar Lipids) was dissolved in methanol, and 10 μg was added per immulon-1B microtiter plate well (ThermoLabsystems). The plate was dried at 37 °C for 2 h, and the wells were washed four times with 200 μl of enzyme-linked immunosorbent assay wash buffer (EWB; 1% bovine serum albumin in TBS50/Ca) at room temperature. The wells were then blocked for 1 h at room temperature with 200 μl of EWB, followed by three 200-μl washes of EWB. α1LG4-5 proteins in varying concentrations in EWB were added to each well and incubated for 1.5 h at room temperature. The wells were then washed four times with 200 μl of EWB, and horseradish peroxidase-linked monoclonal FLAG antibody (Sigma) in EWB was added. After 1 h at room temperature, the wells were washed four times with EWB, and 150 μl of substrate solution (4 mm o-phenylenediamine (Sigma), 50 mm citric acid, 100 mm Na2HPO4, 0.012% H2O2) was added. The developing color reaction was stopped after 2-10 min by the addition of 60 μl of 2 m H2SO4, followed by 50 μl of ethanol, and the plates were read in a TECAN SpectraFluor microtiter spectrophotometer at 492 nm. Samples with OD > 2 were diluted and remeasured to correct for any deviation from linearity. A molecular mass of 44.3 kDa was used to calculate molar α1LG4-5 concentrations. Inhibition studies were performed in the presence of either 10 μg/ml low molecular weight heparin (Sigma) or 5 mm EDTA. The assay was also performed with several other lipids: galactosyl ceramide (Avanti Polar Lipids and Sigma), sphingomyelin (Avanti Polar Lipids), phosphatidic acid (Avanti Polar Lipids), cholesterol 3-sulfate (Sigma), and GM1 ganglioside (Avanti Polar Lipids). Half-maximal binding was estimated in the same manner as described for α-DG. Electron Microscopy—Pt/C rotary shadowing of proteins was performed by deposition of 0.9-nm metal at an 8° angle as previously described (21Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Images are shown with reversed contrast. Crystal Structure of Laminin α1LG4-5—The C-terminal LG domain pair of the laminin α1 chain contains an unpaired cysteine (Cys3014) and three predicted N-linked glycosylation sites (Asn2714, Asn2811, and Asn2900). Because we found that WT α1LG4-5 preparations always contained a small fraction of disulfide-linked dimers (data not shown), we mutated Cys3014 to serine. An α1LG4-5 C3014S construct with an N-terminal His tag failed to crystallize, as did several other constructs with additional mutations of asparagine residues modified by glycosylation. Eventually, crystals could be obtained of an untagged α1LG4-5 quadruple mutant (N2714Q/N2811K/N2900Q/C3014S) devoid of any N-linked carbohydrate. The crystal structure of this mutant, hereafter termed simply α1LG4-5, was refined at 1.9 Å resolution to Rfree = 0.261 (Table 1). The asymmetric unit of the crystals contains two crystallographically independent α1LG4-5 molecules, A and B. We observed clear electron density for both molecules, with the exception of residues 2987-2990, 3032-3034, and 3060 of molecule A and residues 2682-2684 and 3060 of molecule B. Molecules A and B are very similar in their LG4 and LG5 domain structures (root mean square deviation 0.36 and 0.58 Å, respectively, for all Cα atoms) but differ substantially in their respective domain arrangements. When the molecules are superimposed on their LG4 domains, a rotation by 14.5° is required to bring their LG5 domains into superposition; the pivot point of this rotation is in the interdomain linker, near Tyr2871 (Fig. 1A). The following description of the structure is based upon the more complete molecule B. The α1LG4-5 structure consists of two canonical LG domains (9Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (258) Google Scholar), LG4 and LG5, connected by a short linker and interacting through a small interface near the domain termini (Fig. 1B). Each LG domain folds into a curved β-sandwich built from two antiparallel sheets and contains a single disulfide bond near the C terminus. A third disulfide bond tethers the segment preceding LG4 to an α-helical turn in LG5. The interface between LG4 and LG5 is water-filled and predominantly polar, and the different conformations of molecules A and B are likely to be due to the paucity of specific interactions in the LG4-LG5 interface. Both LG4 and LG5 contain one bound metal ion, located on the rim of the β-sandwich opposite the interdomain linker. These ions have been modeled as magnesium, given their coordination geometry and the high magnesium concentration in the crystals, but we assume that the binding sites are occupied by calcium under physiological conditions (7Hohenester E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Magnesium ion 1 is coordinated octahedrally by the side chains of Asp2747 and Asp2816, the main chain carbonyl oxygens of residues 2764 and 2814, and two water molecules; the average metal-ligand distance is 2.17 Å. Magnesium ion 2 is coordinated octahedrally by the side chains of Asp2923 and Asp2996, the main chain carbonyl oxygens of residues 2940 and 2994, and two water molecules; the average metal-ligand distance is 2.15 Å. The unpaired cysteine of α1LG4-5, Cys3014, is located in the convoluted loop that occupies most of the concave face of LG5. Two predicted N-linked glycosylation sites are located in LG4 (Asn2714 and Asn2811) and one in LG5 (Asn2900). Asn2811 is close to the metal ion binding site in LG4 (Fig. 1B). Structural Comparison of α1LG4-5 and α2LG4-5—Mouse laminin α1LG4-5 is 41% identical to the homologous region of the mouse α2 chain (α2LG4-5) (Fig. 2A), whereas the sequence identity to the α3-5 chains is substantially lower (<30%). A structural comparison of α1LG4-5 and α2LG4-5 (8Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar) reveals only a few notable differences at the level of individual LG domains. LG4 of laminin α1 and α2 can be superimposed with a root mean square deviation of 0.91 Å for 148 Cα atoms; the major differences are concentrated in the spatially adjacent B-C and L-M loops and in the edge β-strand J, which is irregular in α1LG4-5 (Fig. 2B). The LG5 domains can be superimposed with a root mean square deviation of 0.59 Å for 153 Cα atoms; the major differences are again concentrated in the B-C and L-M loops (Fig. 2C). The relative arrangement of LG4 and LG5 in α1LG4-5 and α2LG4-5 is also similar, with α2LG4-5 more closely resembling molecule B than molecule A of α1LG4-5 (not shown). In terms of their interdomain angles, the two crystallographically independent α1LG4-5 molecules are, in fact, more different than α2LG4-5 is from molecule B of α1LG4-5. Notably, only a few contacts in the LG4-LG5 interface are conserved in the two laminin isoforms. Near the pivot point of interdomain flexibility, an aromatic side chain (Tyr2871 in α1LG4-5) stacks against a proline (Pro3056 in α1LG4-5). Further away from the hinge, a conserved leucine in LG4 (Leu2703 in α1LG4-5) makes a van der Waals contact with a proline in LG5 (Pro3052 in α1LG4-5). Finally, a conserved glutamine (Gln2700 in α1LG4-5) points its side chain into the water-filled cavity within the interdomain interface (not shown). The coordination of the two magnesium ions in α1LG4-5 is identical to that of the two calcium ions in α2LG4-5, with equivalent protein residues acting as metal ligands (Fig. 2A) and water molecules observed in equivalent positions in both structures. Thus, we expect that calcium would readily occupy the metal sites of α1LG4-5 under more physiological conditions than those used for crystallization. Characterization of Recombinant Laminin α1LG4-5 Proteins—To evaluate the contributions of basic residues to ligand binding by laminin α1LG4-5