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• W2090890529 abstract "The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewisx trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewisx and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewisx is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis. The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewisx trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewisx and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewisx is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis. The scavenger receptor C-type lectin (SRCL) 2The abbreviations used are: SRCL, scavenger receptor C-type lectin; CRD, carbohydrate recognition domain; DC-SIGN, dendritic cell-specific ICAM3 grabbing nonintegrin; DCSIGN-R, DC-SIGN-related protein; LNFP-III, lacto-N-fucopentaose III (Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc); r.m.s.d., root mean square deviation. 2The abbreviations used are: SRCL, scavenger receptor C-type lectin; CRD, carbohydrate recognition domain; DC-SIGN, dendritic cell-specific ICAM3 grabbing nonintegrin; DCSIGN-R, DC-SIGN-related protein; LNFP-III, lacto-N-fucopentaose III (Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc); r.m.s.d., root mean square deviation. is an unusual endothelial cell scavenger receptor. It contains a C-terminal Ca2+-dependent C-type carbohydrate recognition domain (CRD) that is projected from the cell surface by collagenous and coiled-coil domains that are characteristic of the class A scavenger receptors (1Ohtani K. Suzuki Y. Eda S. Kawai T. Kase T. Keshi H. Sakai Y. Fukuoh A. Sakamoto T. Itabe H. Suzutani T. Ogaswara M. Yoshia I. Wakamiya N. J. Biol. Chem. 2001; 276: 44222-44228Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 2Nakamura K. Funakoshi H. Miyamoto K. Tokunaga F. Nakamura T. Biochem. Biophys. Res. Comm. 2001; 280: 183-186Google Scholar). SRCL binds modified low density lipoproteins through these common domains, but the CRD additionally confers a glycan binding function not found in any other scavenger receptors. Recent studies have revealed that the CRD of human SRCL shows remarkably selective binding to glycans containing the Lewisx trisaccharide Galβ1-4(Fucα1-3)GlcNAc, along with weaker binding to the closely related Lewisa trisaccharide Galβ1-3(Fucα1-4)GlcNAc (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4Coombs P.J. Taylor M.E. Drickamer K. Glycobiology. 2006; 16: 1C-7CCrossref PubMed Scopus (58) Google Scholar). Among receptors containing C-type CRDs, only the selectins show specific binding to such a limited set of sugar structures, primarily sialylated and sulfated derivatives of Lewisx and Lewisa (5Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Crossref PubMed Scopus (821) Google Scholar). The endothelial localization of SRCL and its ability to interact selectively with a sugar epitope that is commonly displayed on adhesion molecules on the surface of various types of leukocytes and tumor cells suggest further parallels with the selectins. For example, recognition of Lewisx-containing glycoproteins on a breast cancer cell line by SRCL suggests that it might mediate interactions between tumor cells and endothelia during metastasis (6Elola M.T. Capurro M.I. Barrio M.M. Coombs P.J. Taylor M.E. Drickamer K. Mordoh J. Breast Cancer Res. Treat. 2007; 101: 161-171Crossref PubMed Scopus (44) Google Scholar). SRCL also shares several characteristics with the dendritic cell surface receptor DC-SIGN, which binds to Lewisx and Lewisa-containing glycans as well as to high mannose oligosaccharides (7Guo Y. Feinberg H. Conroy E. Mitchell D.A. Alvarez R. Taylor M.E. Weis W.I. Drickamer K. Nat. Struct. Mol. Biol. 2004; 11: 591-598Crossref PubMed Scopus (472) Google Scholar). Like DC-SIGN, SRCL has the ability to serve as a cell adhesion molecule as well as being an endocytic receptor. Despite these parallels, the structure of the CRD of SRCL suggests that it must bind Lewisx in a fundamentally different way from the way that the selectins and DC-SIGN bind such fucosylated ligands. In the CRDs of the latter receptors, the disposition of amino acid residues around the conserved Ca2+ generates a primary binding site that is configured to bind monosaccharides in which the 3 and 4 hydroxyl groups have the stereochemistry found in mannose or fucose (8Feinberg H. Mitchell D.A. Drickamer K. Weis W.I. Science. 2001; 294: 2163-2166Crossref PubMed Scopus (565) Google Scholar, 9Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Selective binding of Lewisx and related structures results from interaction of the terminal fucose with this primary binding site and additional interactions of the other terminal residues, such as galactose and sialic acid, with adjacent secondary binding sites on the surface of the CRD. In contrast, the amino acid sequence around the conserved Ca2+ in SRCL is characteristic of galactose-binding C-type CRDs, and it would not be expected to accommodate fucose. In the present studies, human and mouse SRCL are shown to have a similar narrow binding selectivity for LewisX-containing glycans. The structural basis for such selective binding in a galactose-type CRD has been elucidated by x-ray crystallography and site-directed mutagenesis. In addition, the molecular basis for ligand release at endosomal pH, required for endocytic function of the receptor, has been determined. Cloning and Expression of Mouse SRCL—The cDNA coding for mouse SRCL was amplified from a mouse lung cDNA library (Clontech). The portion of the DNA coding for the CRD, from residue 603 to the C terminus, was cloned into the pINII-IompA2 expression vector for expression in Escherichia coli as described for the CRD of human SRCL (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Mutations were introduced into the CRD using synthetic oligonucleotides. DNA coding for the extracellular domain of mouse SRCL, starting at residue 60, was fused to codons specifying the dog pre-proinsulin signal sequence and inserted into the vector pED for expression in DXB11 Chinese hamster ovary cells, as described for human SRCL (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Mouse SRCL extracellular domain and wild type and mutant CRDs were expressed and purified as described for human SRCL (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), except that in some cases, cell lysis was achieved by passing the washed cell suspension 2-3 times through an EmulsiFlex-C3 homogenizer (Avestin) at a pressure of 10-15,000 psi. For crystallization, the isolated protein was dialyzed against low salt buffer (25 mm NaCl, 10 mm Tris, pH 7.8, 10 mm CaCl2), applied to an anion exchange column (MonoQ; G.E. Healthcare), and eluted with a linear NaCl gradient from 25 to 1000 mm NaCl. Protein which eluted at ∼180 mm NaCl was exchanged back to the low salt buffer and concentrated to ∼15 mg/ml using a spin concentrator. Analysis of Ligand Binding—Fluorescein-labeled extracellular domain of mouse SRCL prepared as described for human SRCL (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) was used to probe the glycan array following the standard procedure of Core H of the Consortium for Functional Glycomics. The specificity of wild-type and mutant CRDs for Lewisx and galactose was determined using a solid-phase binding assay with CRDs immobilized to polystyrene wells (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Crystallization—Crystals of the CRD from human SRCL were grown at 21 °C, using the hanging drop method (1 μl of protein to 0.5 μl of reservoir in a drop). The protein solution contained 10 mg/ml protein, 8 mm CaCl2, 8 mm Tris, pH 7.8, 20 mm NaCl, and 10 mm Lewisx (V-labs, Inc. and Toronto Research Chemicals). The reservoir solution contained 8% polyethylene glycol 8000, 0.2 m Zn(CH3COO)2, and 0.1 m Tris-Cl, pH 7.0. Crystals were transferred to synthetic mother liquor consisting of all the salts and buffers that were present in the drop, as well as 10 mm Lewisx and 15% ethylene glycol, for 5 min and were then frozen in liquid nitrogen for data collection. Crystals used for the low resolution data set of this protein were grown at 21 °C (2 μl of protein to 1 μl of reservoir in a drop). The protein solution contained 13 mg/ml protein, 9 mm CaCl2, 9 mm Tris-Cl pH 7.8, 22.5 mm NaCl, 5 mm Lewisx. The reservoir solution contained 9% polyethylene glycol 8K, 0.1 m sodium cacodylate, pH 6.5, and 0.2 m Zn(CH3COO)2. Crystals were transferred to a fresh reservoir solution containing 5 mm Lewisx and 15% methyl pentane diol and then frozen in liquid nitrogen for data collection. Crystals of the CRD from mouse SRCL were grown at 21 °C (1 μl of protein to 1 μl of reservoir in a drop). The protein solution contained 6 mg/ml protein, 9 mm CaCl2, 9 mm Tris, pH 7.8, 22.5 mm NaCl, and 5 mm Lewisx. The reservoir solution contained 30% polyethylene glycol 8K, 0.2 m NaCl, and 0.1 m imidazole, pH 8.5. Crystals were transferred to a solution containing all the salts and buffers that are present in the drop, including 5 mm Lewisx, and then frozen in liquid nitrogen for data collection. Data Collection—Diffraction data were measured at 100 K on ADSC Q315 CCD detectors, at the Advanced Light Source beam line 8.2.1 (high and low resolution CRD from human SRCL) and the Stanford Synchrotron Radiation Laboratory beam line 11-1 (CRD from mouse SRCL). Data were processed with MOSFLM and SCALA (10Collaborative Computational Project N. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19667) Google Scholar), and are summarized in Table 1.TABLE 1Crystallographic data and refinement statisticsHuman SRCL-CRDMouse SRCL-CRDData collectionSpace groupP32P1Unit cell parameters (Å)a = b = 80.42, c = 67.16a = 48.0, b = 53.76, c = 59.08(°)α = 67.75, β = 76.70, γ = 85.37Resolution Å(last shell)2.5 (2.57)1.95 (2.06)Number of unique reflections (F > 0)1652535627Number of reflections marked for Rfree8401785Rsym (last shell)aRsym = ∑h∑i (|Ii(h)| - |<I(h)>|)/∑h∑i Ii(h) where Ii(h) is the observed intensity, and <I(h)> is the mean intensity obtained from multiple measurements.6.9 (24.0)10.9 (11.3)% completeness (last shell)98.4 (99.5)92.2 (91.7)Average multiplicity1.9 (1.9)2.8 (1.9)RefinementRfreebR and Rfree = ∑||Fo| - |Fc||/∑|Fo|, where |Fo| is the observed structure factor amplitude and |Fc| is the calculated structure factor amplitude for the working and test sets, respectively.30.8 (35.6)27.3 (32.0)RbR and Rfree = ∑||Fo| - |Fc||/∑|Fo|, where |Fo| is the observed structure factor amplitude and |Fc| is the calculated structure factor amplitude for the working and test sets, respectively.23.3 (26.4)22.4 (26.0)Average B (Å2)31.724.5Bond length r.m.s.d. (Å)0.0070.006Angle r.m.s.d. (°)1.321.33Ramachandran plot: (% in most favored/allowed/generous/disallowed regions)74.1/21.9/3.1/0.984.6/13.6/1.8/0a Rsym = ∑h∑i (|Ii(h)| - |<I(h)>|)/∑h∑i Ii(h) where Ii(h) is the observed intensity, and <I(h)> is the mean intensity obtained from multiple measurements.b R and Rfree = ∑||Fo| - |Fc||/∑|Fo|, where |Fo| is the observed structure factor amplitude and |Fc| is the calculated structure factor amplitude for the working and test sets, respectively. Open table in a new tab Structure Determination—A lower resolution (2.8 Å) data set was measured for the human CRD. These data scaled with P6 symmetry and gave a molecular replacement solution in space group P65 using the program Amore (11Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar), with the CRD of DC-SIGNR, Protein Data Bank (PDB) ID 1k9j, as a search model. The best solution gave a correlation coefficient of 42% and an R value of 49% (resolution range 15-3 Å). A partial model for SRCL CRD was built into the electron density map, and although the electron density maps were unambiguous, refinement did not lower the Rfree below 34%. The original data were incomplete along the 00l axis. However, the high resolution data set showed systematic absences along this axis, with significant intensities only for 00l = 3n. This observation is incompatible with space group P65, implying a lower symmetry trigonal space group (P62 and P64, the only hexagonal space groups consistent with these absences, did not give translation function solutions). Molecular replacement for the higher resolution data set was performed with the program COMO (12Tong L. Acta Crystallogr. Sect. A. 1996; 52: 782-784Crossref Scopus (39) Google Scholar) using the partially refined model from the lower resolution data set as a search model. The best solution had two monomers in space group P32, with a correlation coefficient of 42% and R value of 41% in the resolution range 12-3.5 Å. Maximum likelihood amplitude refinement was performed using the program CNS (13Brünger A.T. Adams P.D. Clore G.M. 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. 1998; 54: 905-921Crossref PubMed Scopus (16918) Google Scholar), with bulk solvent and anisotropic temperature factor corrections applied at all stages. Missing loops were built in gradually, and the resolution was increased to 2.5 Å. After several rounds of positional and isotropic temperature factor refinement alternating with manual model adjustment, most of the residues in the two monomers, designated A and B, could be added to the model. Given the presence of 200 mm ZnCl2 in the crystallization medium, several strong difference electron density peaks were modeled as Zn2+, based on the geometry of surrounding ligands and the fact that their refined temperature factors were comparable in magnitude to the surrounding ligands. The human CRDs showed binding to 5 Zn2+ per monomer, but did not show density for Ca2+ or the Lewisx trisaccharide in the expected binding site. Each monomer is cross-linked to its crystallographic symmetry equivalent copy by a Zn2+ (Fig. 1, A and B): His610 from one monomer A and His641 from a symmetry-related monomer A bind to the same Zn2+, and the same holds for monomer B and its symmetry equivalent. Monomers A and B are related to each other by a -60° rotation and a translation of 1/6 along the z-axis. These two monomers are cross-linked to each other by another Zn2+, with His700 from one monomer and Asp616 and Asp733 from another providing the coordination ligands. The six monomers (three A and three B) in the unit cell are related to each other by a 65 screw axis, to form a hexameric “barrel” that surrounds a large central space (Fig. 1, A and C). Electron density outside of monomers A and B was seen in the center of the hexameric barrel, with four large peaks (>6σ) present in an Fo - Fc map of the asymmetric unit. After fixing monomers A and B, a search for an additional CRD was performed in COMO, using a CRD model with the Zn2+ and some loops removed. The search yielded two equivalent solutions related by a 65 screw axis, but which overlap each other (monomers C and D, Fig. 1A). Surprisingly, the four large peaks seen in the Fo - Fc map calculated only with monomers A and B fit two Zn2+ positions for both monomers C and D (Zn2+ number 1 and 2, Fig. 2A). One of the Zn2+ cross-links each monomer with its symmetry-related copy in the same manner observed for monomers A and B (Fig. 1B), supporting the validity of the solution for monomers C and D. A 2-fold rotation axis relates monomers A or B to monomers C or D, causing the filament formed by cross-linking C or D to run in the opposite direction from monomers A and B along the z-axis. Note that although monomers C and D are related by a 65 screw axis, their packing is not compatible with space group P65, as application of a -60° rotation and a 1/6 translation along z to either C or D results in overlap with its symmetry mate. Instead, the crystal has the lower symmetry of P32, with the unit cell containing monomers A, B, and either C or D. Presumably, C and D are randomly distributed through crystal to give a statistical mixture that effectively makes them present at 50% occupancy (Fig. 1). To refine the arrangement of molecules in P32, monomers C and D were treated as alternative conformations each with 50% occupancy, i.e. there are three independent copies in the asymmetric unit. Because C and D overlap, the maps around them are not as clear as for monomers A and B, but it appears that they have loop conformations and Ca2+ in similar positions as in the mouse SRCL CRD (see below). Water molecules were added to peaks >3σ in Fo - Fc maps and were within hydrogen bond distance to monomers A and B or to other water molecules. Because the quality of the maps around monomers C and D is not as high as for monomers A and B, the only water molecules that were added in the vicinity of monomers C and D are ligands bound to the Zn2+ or Ca2+. Temperature factor refinement suggested that in some cases Cl- serves asaZn2+ ligand instead of water. The final human CRD model contains residues 606-734 for all protein monomers, 16 Zn2+, 6 Ca2+, 12 Cl-, and 33 water molecules. Molecular replacement for the mouse SRCL CRD data set, using the program COMO and the partially refined model of the human SRCL CRD as a search model, gave a solution for four monomers in the P1 unit cell. The best solution had a correlation coefficient of 31% and an R value of 43% for the resolution range 12-3.5 Å. The rotation between monomers A and C, and between B and D, is almost 180°, whereas the rotation between the A-C pair and the B-D pair is about 80°. The structure was refined in CNS using a maximum likelihood amplitude target, and bulk solvent and anisotropic temperature factor corrections were applied throughout. Test set reflections for calculating Rfree were chosen in thin shells. Strict non-crystallographic symmetry was initially applied, but was released later in the refinement as some side chains showed different conformations among the four independent copies. These loops were built in gradually and the resolution was increased to 1.95 Å. In each of the four monomers, four Ca2+ and one Lewisx molecule were visible. The final model contains residues 606-735 for monomers A and D, 607-698 and 704-738 for monomer B, 607-737 for monomer C, 16 Ca2+, 4 Lewisx trisaccharides, and 357 water molecules. Glycan Ligands for SRCL—To facilitate structural and functional analysis of SRCL, both the human and mouse proteins were investigated. The sequences of human SRCL and mouse SRCL are 91% identical overall, with no insertions or deletions, indicating that this protein is highly conserved between the two species. Soluble fragments of human SRCL consisting of just the CRD or the whole extracellular domain containing the coiled-coil region, the collagen-like region and the CRD have been characterized previously (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For this study, the equivalent fragments of mouse SRCL were produced. The binding specificity of the human receptor was previously characterized by probing a glycan array consisting of biotinylated oligosaccharides immobilized on streptavidin in polystyrene wells (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For comparison, the trimeric extracellular domain of the mouse receptor expressed in Chinese hamster ovary cells was tested against a second generation glycan array, in which oligosaccharides are covalently immobilized on a glass surface (14Blixt O. Head S. Mondala T. Scanlan C. Huflejt M.E. Alvarez R. Bryan M.C. Fazio F. Calarese D. Stevens J. Razi N. Stevens D.J. Skehel J.J. van Die I. Burton D.R. Wilson I.A. Cummings R. Bovin N. Wong C.H. Paulson J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17033-17038Crossref PubMed Scopus (950) Google Scholar). Despite the difference in the assay format, the results reveal that, like human SRCL, mouse SRCL is highly specific for Lewisx- and Lewisa-containing oligosaccharides and shows some preference for Lewisx compared with Lewisa (Fig. 3A). The mouse receptor also binds to forms of these ligands in which the 6 position of GlcNAc or glucose is sulfated (glycans 274 and 275), but as expected it does not bind forms in which the 3 position of galactose bears sulfate (glycans 28 and 259-262). Thus, this receptor shows partial similarity in specificity to the selectins, which can also bind sulfated ligands (5Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Crossref PubMed Scopus (821) Google Scholar). The fact that the mouse and human receptors show the same restricted specificity for Lewisx and Lewisa is not surprising given that the amino acid sequences of the CRDs of the two proteins are very similar. The CRD sequences are 86% identical overall and in the region shown to form the sugar binding site in other C-type CRDs there is only one amino acid difference between the mouse and human proteins (Fig. 3B). Recognition of Lewisx by SRCL by a Novel Mechanism—With the goal of elucidating the mechanism of SRCL binding to Lewisx, attempts were made to crystallize the carbohydrate recognition domain of human SRCL with bound ligand. These efforts proved unsuccessful, but parallel studies on the mouse CRD resulted in determination of the structure in the presence of Ca2+ and Lewisx trisaccharide. The crystals contain four independent copies, each of which reveals four Ca2+ and a Lewisx molecule. The CRD adopts the typical long form C-type lectin fold (Fig. 2B), including a third β-strand at the bottom of the domain (β0) and a disulfide bond that connects the loops preceding β0 and β1. As predicted from the amino acid sequence of SRCL, the galactose residue in the Lewisx oligosaccharide interacts with the conserved Ca2+ site in the CRD: the equatorial 3- and axial 4-hydroxyl groups form coordination and hydrogen bonds similar to those seen in other galactose-binding C-type CRDs (Figs. 4 and 5). Carbonyl oxygen atoms from the side chains of Gln694 and Asn718 act as Ca2+ ligands, and the amide groups of these side chains serve as hydrogen bond donors to the 3 and 4 hydroxyl groups of galactose. The side chains of Asp696 and Glu706 also serve as Ca2+ ligands and act as hydrogen bond acceptors from the same sugar hydroxyl groups. Interactions of the apolar face of galactose with an aromatic side chain are a hallmark of galactose-binding lectins (15Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (994) Google Scholar). In this case, both C4 and the exocyclic C6 pack against Trp698.FIGURE 5Comparison of galactose-binding sites in C-type CRDs. Carbon, nitrogen, oxygen, and calcium are represented as white, blue, red, and green spheres, respectively. Hydrogen bonds are shown as dashed gray lines, Ca2+ coordination bonds are dashed black lines and hydrophobic interactions are in dashed blue lines. A, mouse SRCL. For simplicity only the galactose residue of Lewisx is shown. B, Gal/GalNAc-binding mutant of mannose-binding protein complexed with GalNAc (1FIH, copy A) (16Feinberg H. Torgerson D. Drickamer K. Weis W.I. J. Biol. Chem. 2000; 275: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). C, rattlesnake venom lectin complexed with lactose (1JZN) (17Walker J.R. Nagar B. Young N.M. Hirama T. Rini J.M. Biochemistry. 2004; 43: 3783-3792Crossref PubMed Scopus (47) Google Scholar). D, tunicate lectin complexed with galactose (1TLG) (18Poget S.F. Freund S.M.V. Howard M.J. Bycroft M. Biochemistry. 2001; 40: 10966-10972Crossref PubMed Scopus (16) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interactions of galactose at the principal Ca2+ site orient Lewisx so that the central GlcNAc residue points away from the protein, while the terminal fucose residue contacts the protein in a secondary binding site, providing specificity for Lewisx over other galactose-containing ligands. In the secondary site, Lys691 forms hydrogen bonds with the 4-hydroxyl group and the ring oxygen of fucose, and there are van der Waals contacts between the exocyclic methyl group of fucose and Cδ1 of Ile712. Changing Ile712 to valine results in a 3-fold loss in selectivity for Lewisx compared with galactose, confirming the importance of this interaction (Table 2). Mutation of Ile712 to Ala results in a reduction in sugar-binding activity. Although this mutant still bound weakly to galactose-Sepharose so that some protein could be purified, binding to the LNFPIII-BSA reporter ligand was too weak to allow quantification of binding in solid phase assays. In addition to contacting the fucose residue, Ile712 also makes contact with Asn718, so reducing the size of the side chain at position 712 probably allows Asn718 to move out of position, disrupting the primary binding site. In previous studies, a mutant CRD in which Lys691 was changed to alanine still showed preferential binding to Lewisx (3Coombs P.J. Graham S.A. Drickamer K. Taylor M.E. J. Biol. Chem. 2005; 280: 22993-22999Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus, in the absence of Lys691, hydrogen bonds between the fucose oxygens and water are energetically equivalent to the bonds with the amino group of the lysine residue in the wild-type CRD, probably because of the high solvent accessibility of these hydrogen bonds. Finally, Phe720 appears to play a critical role in organizing both the primary and secondary binding sites as it packs against Ile712 as well as main chain and side chain atoms of residues that form the conserved Ca2+-binding site. Mutation of this residue to alanine results in complete loss of sugar binding activity, as shown by the inability of the mutant to bind to galactose-Sepharose.TABLE 2Mouse SRCL binding to saccharide ligandsMutant CRDK1, Lewisx/K1, galactoseaRelative inhibition constants for galactose and Lewisx were determined in binding competition assays in which the reporter ligand 125I-labeled LNFPIII-BSA was bound to CRDs immobilized in polystyrene wells.KD for LNFPIII-BSAbKD values for LNFPIII-BSA were determined in binding assays in which increasing concentrations of 125I-labeled LNFPIII-BSA and unlabeled LNFPIII-BSA bound to CRDs immobilized in polystyrene wells.μg/mlWild type0.0063 ± 0.00021.24 ± 0.0011712V0.03 ± 0.0013.60 ± 0.30a Relative inhibition constants for galactose and Lewisx were determined in binding competition assays in which the reporter ligand 125I-labeled LNFPIII-BSA was bound to CRDs immobilized in polystyrene wells.b KD values for LNFPIII-BSA were determined in binding assays in which increasing concentrations of 125I-labeled LNFPIII-BSA and unlabeled LNFPIII-BSA bound to CRDs immobilized in polystyrene wells. Open table in a new tab The interaction of SRCL with Lewisx is fundamentally different from the way that DC-SIGN and the selectins bind to related glycans, although the conformation of the Lewisx trisaccharide is similar in the DC-SIGN and SRCL complexes (Fig. 4, A-D). When bound to SRCL, the trisaccharide is oriented with the central GlcNAc residue tipped away from the protein so that the terminal fucose residue contacts the protein in the secondary binding site. In contrast, with the fucose residue in the primary binding site of DC-SIGN, the internal GlcNAc residue points away from the protein in the opposite direction and galactose mak" @default.
• W2090890529 created "2016-06-24" @default.
• W2090890529 creator A5043309599 @default.
• W2090890529 creator A5063106344 @default.
• W2090890529 creator A5086277029 @default.
• W2090890529 date "2007-06-01" @default.
• W2090890529 modified "2023-09-30" @default.
• W2090890529 title "Scavenger Receptor C-type Lectin Binds to the Leukocyte Cell Surface Glycan Lewisx by a Novel Mechanism" @default.
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