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J. Biol. Chem., Vol. 282, Issue 15, 11573-11581, April 13, 2007

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Crystal Structure and Cell Surface Anchorage Sites of Laminin 1LG4-5^*

David Harrison¹, Sadaf-Ahmahni Hussain¹, Ariana C. Combs^¶, James M. Ervasti^¶, Peter D. Yurchenco, and Erhard Hohenester²

From the Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, the ^¶Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, and the Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, United Kingdom

Received for publication, November 16, 2006 , and in revised form, January 11, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 1LG4-5 domains and provide a mutational analysis of heparin, -dystroglycan, and galactosylsulfatide binding. The two domains of 1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin 2 LG4-5 but with a substantially different interdomain angle. Recombinant 1LG4-5 binding to heparin, -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 -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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Laminin-111, recently renamed from laminin-1 to better reflect its 1 11 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 (1, 2). Later in development, laminin-111 is strongly expressed in placenta, liver, kidney, and testis, where it is thought to play a role in organogenesis (3). In the adult, the laminin 1 chain has very limited expression and is largely supplanted by the laminin 5 chain. Targeted inactivation of the LAMA1 gene coding for the 1 chain was found to result in a failure of Reichert's membrane with developmental arrest by embryonic day 6.5 in the mouse (4). In-frame deletion of the mouse laminin 1 exons corresponding to laminin G-like (LG)³ domains 4 and 5 was found to result in similar stage lethality accompanied by defective epiblast differentiation without loss of basement membrane (5), the last possibly a result of the partially redundant expression of laminin-511 (4).

The 1 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 1 and 1 chains. The C-terminal LG domains, LG1-3, bind to the ₆₁ integrin, whereas LG4-5 bind to heparin, sulfated glycolipids, and -dystroglycan (-DG). The polymerization and cell-anchoring activities are thought to act in concert to assemble a functional basement membrane on a cell surface (6).

Our earlier understanding of the laminin 1 LG1-5 structure was based on crystal structures of LG4 and LG4-5 from the related 2 chain (40% sequence identity). The LG domain fold was revealed as a multistranded -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 (7, 8). 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 (9).

Analysis of contributions of 1LG4-5 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 (6, 10, 11). 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 -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 1 and 2LG fragments revealed that there are binding similarities and differences between the two laminins. Heparin, representing the highly sulfated regions of glycosaminoglycan chains, -DG, and galactosyl-3-sulfate ceramide (galactosyl sulfatide) all bind to an extensive basic surface region between the calcium sites of the laminin 2 LG4-5 domain pair (12, 13). In contrast, a smaller topographical region confined to LG4 appears to bind the same cell surface components in the laminin 1 chain (12, 14).

The present study describes the crystal structure of laminin 1LG4-5 in conjunction with a site-directed mutagenesis and in vitro analysis of the binding sites for heparin, -DG, and sulfatides. We report that the backbone of the 1LG4-5 domain pair is very similar to that of 2LG4-5, but there are significant differences in the distribution of charged residues on the protein surface. Binding of 1LG4-5 to heparin, -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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Laminin 1LG4-5 Vector Constructs—An expression construct containing the wild-type (WT) mouse laminin 1LG4-5 sequence (coding for residues ²⁶⁶⁶LHREH... PGPEP³⁰⁶⁰ of the mature laminin 1 chain (i.e. the numbering scheme used here omits the 24-residue signal peptide of SwissProt entry P19137 [GenBank] )) was created by amplifying cDNA from laminin 1 pCIS (15) 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 1LG4-5 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 DH5 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 1LG4-5 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 1LG4-5 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 1LG4-5 proteins. The conditioned media were passed through a gravity column (Bio-Rad) packed with anti-FLAG M2-agarose resin (Sigma), and recombinant 1LG4-5 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 anÄ_kta FPLC system (Amersham Biosciences), where the FLAG peptide (unbound component) was recovered for reuse, and the 1LG4-5 proteins were eluted using a 1 M NaCl gradient. The eluted 1LG4-5 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 1LG4-5 protein was produced either by isolating WT 1LG4-5 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 1LG4-5 with 1000 units of peptide N-glycosidase F (New England Biolabs) in TBS50/Ca for 1 h at 37 °C.

Laminin 1LG4-5 Vector Construct for Crystallography—DNA coding for residues ²⁶⁸²QPELC... PGPEP³⁰⁶⁰ of the mature mouse laminin 1 chain was obtained by PCR amplification from laminin 1 pCIS (15). 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 (16) 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 1LG4-5 protein.

Protein Production for Crystallography—The laminin 1LG4-5 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 x 5-ml heparin HiTrap column (GE Healthcare). Bound proteins were eluted using a linear NaCl gradient (0-1 M). The 1LG4-5 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 1LG4-5 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 ( = 0.87 Å). The crystals belong to space group P2₁, a = 70.53 Å, b = 55.81 Å, c = 100.99 Å, = 98.48°. There are two 1LG4-5 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 (17). The structure was solved by molecular replacement with PHASER (18), using the laminin 2LG4-5 structure (8) as a search model; the LG domains had to be placed individually to obtain a solution. The structure was rebuilt with O (19) and refined with CNS (20) 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).

Analysis of 1LG4-5 Binding to -Dystroglycan—-DG was purified from rabbit muscle as described (22). Equal aliquots of -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 1LG4-5 protein (1 µg/ml) using a previously described overlay assay (23). Binding of the 1LG4-5 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 -DG bound to the plate (100 µl of 1 µg/ml per well) and incubated with various concentrations of 1LG4-5 proteins as previously described (24), 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 (25). We verified that the signal readout was linear to OD > 3 by collecting kinetic data on color development. Estimates of half-maximal binding (K_D) and binding capacity (B_max) were determined by curve fitting of the binding data of WT LG4-5 using a single-site model (fitted values = B_max x L/(K_D + L), where L is the molar ligand concentration), with the calculated B_max 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 1LG4-5 Binding to Galactosyl Sulfatide and Other Lipids—The ammonium salt of HSO₄-3Gal1-1'ceramide (brain sulfatides; Avanti%20Polar%20Lipids">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. 1LG4-5 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 1LG4-5 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%20Polar%20Lipids">Avanti Polar Lipids and Sigma), sphingomyelin (Avanti%20Polar%20Lipids">Avanti Polar Lipids), phosphatidic acid (Avanti%20Polar%20Lipids">Avanti Polar Lipids), cholesterol 3-sulfate (Sigma), and GM1 ganglioside (Avanti%20Polar%20Lipids">Avanti Polar Lipids). Half-maximal binding was estimated in the same manner as described for -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 (21). Images are shown with reversed contrast.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Crystal Structure of Laminin 1LG4-5—The C-terminal LG domain pair of the laminin 1 chain contains an unpaired cysteine (Cys³⁰¹⁴) and three predicted N-linked glycosylation sites (Asn²⁷¹⁴, Asn²⁸¹¹, and Asn²⁹⁰⁰). Because we found that WT 1LG4-5 preparations always contained a small fraction of disulfide-linked dimers (data not shown), we mutated Cys³⁰¹⁴ to serine. An 1LG4-5 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 1LG4-5 quadruple mutant (N2714Q/N2811K/N2900Q/C3014S) devoid of any N-linked carbohydrate. The crystal structure of this mutant, hereafter termed simply 1LG4-5, was refined at 1.9 Å resolution to R_free = 0.261 (Table 1).

The asymmetric unit of the crystals contains two crystallographically independent 1LG4-5 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 C 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.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Structure of laminin 1LG4-5. A, superposition of the two 1LG4-5 molecules in the asymmetric unit. Molecules A (mol A; light brown) and B (mol B; blue) were superimposed on their LG4 domains. The position of Tyr2871 (see"Results") is indicated. B, schematic diagram of molecule B (cyan, LG4; green, LG5). The N and C termini are labeled. Disulfide bonds are shown as yellow ball-and-stick models. Metal ions are shown as purple spheres. The positions of Asn2714 and Asn2811 (N-linked glycosylation sites) and Cys3014 (unpaired cysteine) are indicated. The third glycosylation site at Asn2900 is located at the back of LG5.

Both LG4 and LG5 contain one bound metal ion, located on the rim of the -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 (7). 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 1LG4-5, 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 1LG4-5 and 2LG4-5—Mouse laminin 1LG4-5 is 41% identical to the homologous region of the mouse 2 chain (2LG4-5) (Fig. 2A), whereas the sequence identity to the 3-5 chains is substantially lower (<30%). A structural comparison of 1LG4-5 and 2LG4-5 (8) reveals only a few notable differences at the level of individual LG domains. LG4 of laminin 1 and 2 can be superimposed with a root mean square deviation of 0.91 Å for 148 C atoms; the major differences are concentrated in the spatially adjacent B-C and L-M loops and in the edge -strand J, which is irregular in 1LG4-5 (Fig. 2B). The LG5 domains can be superimposed with a root mean square deviation of 0.59 Å for 153 C 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 1LG4-5 and 2LG4-5 is also similar, with 2LG4-5 more closely resembling molecule B than molecule A of 1LG4-5 (not shown). In terms of their interdomain angles, the two crystallographically independent 1LG4-5 molecules are, in fact, more different than 2LG4-5 is from molecule B of 1LG4-5. 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 1LG4-5) stacks against a proline (Pro³⁰⁵⁶ in 1LG4-5). Further away from the hinge, a conserved leucine in LG4 (Leu²⁷⁰³ in 1LG4-5) makes a van der Waals contact with a proline in LG5 (Pro³⁰⁵² in 1LG4-5). Finally, a conserved glutamine (Gln²⁷⁰⁰ in 1LG4-5) points its side chain into the water-filled cavity within the interdomain interface (not shown).

View larger version (66K): [in this window] [in a new window]   FIGURE 2. Comparison of 1LG4-5 and 2LG4-5. A, sequence alignment of 1LG4-5 and 2LG4-5. Identical residues are shaded yellow, cysteines are shaded black, and metal ion ligands are shaded purple. The sequence numbering and -strands of mouse 1LG4-5 are indicated above the alignment, and the sequence numbering of mouse 2LG4-5 is indicated below the alignment. Residues implicated in receptor binding to 1LG4-5 (this work) and 2LG4-5 (13) are indicated in red. B, superposition of 1LG4 (this work) and 2LG4 (8). A total of 148 C atoms were superimposed with a root mean square deviation of 0.91 Å. C, superposition of 1LG5 (this work) and 2LG5 (8). A total of 153 C atoms were superimposed with a root mean square deviation of 0.59 Å.

View larger version (95K): [in this window] [in a new window]   FIGURE 3. Characterization of recombinant 1LG4-5 proteins. A, reducing 12% SDS-polyacrylamide gel stained with Coomassie Blue of recombinant 1LG4-5 proteins (WT, wild type; A-I, mutants as defined in Table 2). The E3 fragment purified from Engelbreth-Holm-Swarm sarcoma tumor laminin-111 is included for comparison. Recombinant 1LG4-5 proteins were purified by anti-FLAG affinity chromatography from the conditioned medium of 293 cell lines. The molecular masses of marker proteins are indicated on the left. B, Coomassie Blue-stained SDS-polyacrylamide gel of WT 1LG4-5 before (right lane) and after incubation (left lane) with enterokinase to remove the FLAG tag. C, Western blot with anti-FLAG-horseradish peroxidase of gel in B. D, Western blot with mouse 1LG4-5 specific polyclonal antibody after stripping of blot in C. E, rotary shadow electron micrographs, contrast reversed, of E3 and selected recombinant LG4-5 proteins. The insets show selected molecules enlarged 2.5-fold.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Heparin affinity elution of 1LG4-5 proteins. The indicated proteins were loaded on a heparin-5PW column in 50 mM Tris-HCl, 1 mM CaCl2, pH 7.4, at 4 °C and eluted with a 0-1 M NaCl gradient. The peak values were used to estimate relative affinities.

Dystroglycan Binding—The ability of the 1LG4-5 proteins to bind -DG was evaluated using two different assay formats. In a blot overlay assay (Fig. 5A), all 1LG4-5 mutants showed reduced binding to immobilized -DG compared with wild type protein, with mutants A and J showing no detectable binding. A very similar result was obtained using an enzyme-linked immunosorbent assay-based solid phase assay with immobilized -DG (Fig. 5B). All mutations decreased -DG binding, in the following order: WT > A2 G > D > I > A J; mutants A and J had very low -DG binding activity. The finding that A2 bound -DG with higher affinity than A was surprising, given the greater loss of charge of the former compared with the latter. The sequences of A and A2 were reconfirmed by PCR of genomic DNA isolated from cells secreting the two laminin fragments (data not shown). We cannot offer a mechanistic explanation for the unexpected behavior of mutants A and A2 in the -DG binding assays.

View larger version (50K): [in this window] [in a new window]   FIGURE 5. Binding of 1LG4-5 proteins to -dystroglycan. A, blot-immobilized -DG overlaid with recombinant 1LG4-5 proteins. B, microtiter plate wells coated with -DG were incubated with increasing concentrations of the indicated 1LG4-5 proteins. In each assay (A and B), bound proteins were detected with horseradish peroxidase-linked anti-FLAG monoclonal antibody. Values are presented as mean ± S.D. (n = 3) and were fitted for single-site binding.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Lipid binding of 1LG4-5 proteins. A, binding of WT 1LG4-5 (0.4 mg/ml) to the indicated lipids (10 µg/well) immobilized in microtiter plates. B, binding of WT and mutant 1LG4-5 proteins to immobilized galactosyl (gal)-sulfatide. Values are presented as mean ± S.D. (n = 4) and were fitted for single-site binding.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Interactions of the distal LG4-5 portion of the laminin 1 chain with cellular receptors (heparan sulfate proteoglycans and -DG) and plasma membrane components (sulfatides) are responsible for laminin anchorage during basement membrane assembly as well as for cell signaling (6, 10, 11). Previous mutagenesis studies have identified basic amino acid residues within LG4 contributing to ligand binding (14, 28), but, in the absence of a 1LG4-5 structure, the precise structure of binding sites remained unknown. We now have determined the crystal structure of 1LG4-5 and analyzed the binding properties of this laminin 1 portion.

An electrostatic surface representation of the 1LG4-5 structure reveals a large, contiguous surface area of positive potential extending over both LG domains (Fig. 7A). Basic residues previously implicated in receptor binding (14, 28) are clustered around the metal ion binding site in LG4 (Fig. 7B). A particularly striking feature is the spatial proximity of two basic sequences, ²⁷¹⁹RKR and ²⁷⁹¹KRK, which are located, respectively, at the start of -strand C and in the H-I turn (Fig. 7C). Five of the six basic side chains are fully surface-exposed and available for receptor binding. The only exception is Arg²⁷⁹², which donates two hydrogen bonds to main chain carbonyl groups in the long J-K loop, which in turn makes rather loose contacts with the body of the domain. Two other basic motifs, ²⁷⁶⁶KGRTK and ²⁸³¹RAR, are located closer to the cleft between the LG4 and LG5 domains.

Regarding heparin binding, we found similar elution behavior to the earlier study of Andac et al. (14) for those mutants evaluated in common (supplemental Table 2). Hozumi et al. (28) used an energy-minimized homology model of 1LG4-5 to gain insight into the interactions of LG4 with heparin/heparan sulfate proteoglycans and integrin ₂₁, the latter a cryptic activity in intact laminin. These authors found heparin binding to LG4 to depend upon contributions from ²⁷¹⁹RKR and ²⁷⁹¹KRK and to a lesser extent from ²⁷⁶⁶KGRTK, in full agreement with our heparin binding data.

View larger version (53K): [in this window] [in a new window]   FIGURE 7. Functionally important sites in 1LG4-5. A, electrostatic surface representation of the 1LG4-5 structure. Positive and negative potential are indicated by blue and red coloring, respectively. B, mapping of functionally important residues (compare Fig. 2A and Table 2) onto a surface representation of 1LG4-5. The view direction is the same as in A. The locations of the metal ion binding site and two asparagines modified by glycosylation are also shown (DG, dystroglycan; hep, heparin; S, sulfatide). C, atomic details of the major basic region in LG4. A magnesium ion and selected side chains are shown as ball-and-stick models. Hydrogen bonds are indicated by dashed lines. Selected -strands are labeled. Asn2811 carries an N-linked glycan in WT 1LG4-5.

Summarizing all of the binding data in the light of our new crystal structure, we can safely assign the heparin/heparan sulfate binding site of laminin 1LG4 to the basic patch made up of ²⁷¹⁹RKR and ²⁷⁹¹KRK; of the ²⁷⁶⁶KGRTK sequence, only the side chain of Lys²⁷⁶⁶ is positioned to contribute to heparin binding (Fig. 7, B and C). The -DG binding site appears to be formed by a larger, semicircular arrangement of basic side chains, with our new results assigning a key role to residues located away from the heparin binding site (i.e. ²⁸³¹RAR and ²⁸⁵⁸KDR). The metal ion bound to LG4 (probably calcium under physiological conditions) is likely to be essential for -DG, given that mutation of calcium ligands in LG4 of the related laminin 2 chain abolished -DG binding (13) and that equivalent calcium sites in the LG domains of neurexin (29) and agrin (30) are critical for biological function. Finally, sulfatide binding in our hands is most strongly affected by mutations of basic residues on the upper face (view of Fig. 7B) of LG4. Two asparigine residues in laminin 1LG4, Asn²⁷¹⁴ and Asn²⁸¹¹, carry bulky glycan modifications, and the glycosylation status of these residues might influence ligand binding. However, whether and how glycosylation of laminin affects its interaction with cell surfaces has not been studied.

Laminin 2LG4-5 binds to the same cell surface molecules as 1LG4-5. Using a similar approach as in the present study, Wizemann et al. (13) found that heparin and sulfatide binding to 2LG4-5 were most strongly affected by mutation of ²⁸⁷⁰KK in LG4 and ³⁰⁸⁸KLTKGTGK in LG5, whereas -DG binding was particularly dependent upon Arg²⁸⁰³ and ²⁸⁷⁰KK as well as upon the calcium ion in LG4. Remarkably, neither of these critical sequences correspond to the binding sites identified in 1LG4-5 (Fig. 2A and Table 2). The lack of conservation of functionally important residues in two closely related proteins is unusual. We speculate that the predominance of electrostatic interactions in ligand binding by the laminin LG4-5 portion accounts for the poor conservation of binding sites. Evidently, it is sufficient to maintain the general basic character of the binding surfaces.

Our earlier findings do not support a role of heparin/heparan sulfates in basement membrane anchorage but instead argue for a prominent role of sulfated glycolipids with a signaling rather than an anchorage contribution arising from -DG (11). Analysis of general and tissue-specific DG knockouts has revealed an essential role of DG for Reichert's membrane, but not muscle, peripheral nerve and other basement membranes, where it may function primarily as a signaling receptor (31). A recent analysis of cultured breast epithelial cells (32) revealed an anchoring activity for DG, raising the possibility that some cells employ DG as an anchor in a manner similar to sulfated glycolipids. In this regard, it will be informative to analyze cell surface assembly of heterotrimeric laminins bearing mutations that distinguish between -DG and sulfated glycolipid binding (i.e. A(²⁷¹⁹RKR to AKA) and I (²⁸³¹RAR to AAA)). Based on the results presented here, we would predict reduced anchorage only of the latter mutant, whereas both mutants could be compromised in their ability to signal through -DG.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2JD4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a Wellcome Senior Fellowship (to E. H.) and National Institutes of Health Grant R37-DK36425 (to P. D. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2.

¹ These two authors contributed equally to this work.

² To whom correspondence should be addressed: Division of Cell and Molecular Biology, Biophysics Section, Blackett Laboratory, Imperial College London SW7 2AZ, London. Tel.: 44-20-7594-7701; Fax: 44-20-7589-0191; E-mail: e.hohenester{at}imperial.ac.uk.

³ The abbreviations used are: LG, laminin G-like domain; DG, dystroglycan; WT, wild type (native sequence); EWB, enzyme-linked immunosorbent assay wash buffer; GM1, Gal3GalNAc4(NeuAc3)Gal4Glc1Cer.

	ACKNOWLEDGMENTS

 
We thank Jason Howitt and the staff at beamline 9.6 at the SRS Daresbury for help with data collection.

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