Orientation of Bound Ligands in Mannose-binding Proteins
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m200493200
ISSN1083-351X
AutoresKenneth Ng, Anand Kolatkar, S. Park-Snyder, H. Feinberg, Damon A. Clark, Kurt Drickamer, William I. Weis,
Tópico(s)Bacteriophages and microbial interactions
ResumoMannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca2+ coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or α1–6-linked mannose bind to MBP-A in one orientation, whereas α1–2- or α1–3-linked mannose binds in an orientation rotated 180° around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His189 to its MBP-C equivalent, valine, causes Manα1–3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands. Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca2+ coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or α1–6-linked mannose bind to MBP-A in one orientation, whereas α1–2- or α1–3-linked mannose binds in an orientation rotated 180° around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His189 to its MBP-C equivalent, valine, causes Manα1–3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands. mannose-binding protein carbohydrate-recognition domain noncrystallographic symmetry 2,4-methylpentanediol crystallography NMR system High mannose structures present on cell surfaces represent important recognition elements in the immune response. Serum mannose-binding proteins (MBPs)1 are C-type lectins that function in immune surveillance by recognizing high mannose structures present on pathogenic organisms such as bacteria and fungi (1Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (892) Google Scholar). MBPs trigger cell killing by activating the complement pathway, leading to opsonization or direct lysis of the target cell (2Ikeda K. Sannoh T. Kawasaki N. Kawasaki T. Yamashina I. J. Biol. Chem. 1987; 262: 7451-7454Abstract Full Text PDF PubMed Google Scholar, 3Kuhlman M. Joiner K. Ezekowitz R.A.B. J. Exp. Med. 1989; 169: 1733-1745Crossref PubMed Scopus (388) Google Scholar, 4Super M. Levinsky R.J. Turner M.W. Clin. Exp. Immunol. 1990; 79: 144-150Crossref PubMed Scopus (77) Google Scholar, 5Tenner A.J. Robinson S.L. Ezekowitz R.A. Immunity. 1995; 3: 485-493Abstract Full Text PDF PubMed Scopus (151) Google Scholar). The ability of these proteins to distinguish foreign from self through the recognition of unique carbohydrate structures thereby contributes to host defense independent of an antibody response. Rodents and some other mammals (6Mogues T. Ota T. Tauber A.I. Sastry K.N. Glycobiology. 1996; 6: 543-550Crossref PubMed Scopus (59) Google Scholar) express two related MBPs in the serum and the liver in which the rat are designated MBP-A and MBP-C, respectively (7Drickamer K. Dordal M.S. Reynolds L. J. Biol. Chem. 1986; 261: 6878-6886Abstract Full Text PDF PubMed Google Scholar). Both proteins can fix complement, although the biological role of the liver-associated MBP is unclear at present. Each MBP consists of a cysteine-rich N-terminal domain followed by a collagenous region, an α-helical "neck" and a C-terminal carbohydrate recognition domain (CRD) (7Drickamer K. Dordal M.S. Reynolds L. J. Biol. Chem. 1986; 261: 6878-6886Abstract Full Text PDF PubMed Google Scholar). The CRD mediates recognition of target cells, and the collagenous domain interacts with MBP-associated serine proteases that trigger the downstream complement response (8Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). MBP polypeptides are assembled into trimeric building blocks that contain collagenous triple helices. Trimer formation requires the presence of the neck region, which associates into an α-helical coiled-coil structure (9Sheriff S. Chang C.Y. Ezekowitz R.A.B. Nat. Struct. Biol. 1994; 1: 789-794Crossref PubMed Scopus (212) Google Scholar, 10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Disulfide bonds formed between cysteine-rich domains of MBP-A mediate higher order oligomerization of the trimeric building block, a property associated with more efficient complement fixation (8Wallis R. Drickamer K. J. Biol. Chem. 1999; 274: 3580-3589Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Wallis R. Drickamer K. Biochem. J. 1997; 325: 391-400Crossref PubMed Scopus (54) Google Scholar). MBPs have a broad carbohydrate specificity concordant with their need to recognize a variety of pathogenic cell surfaces. This specificity includes d-mannose,N-acetyl-d-glucosamine, and l-fucose (12Lee R.T. Ichikawa Y. Fay M. Drickamer K. Shao M.-C. Lee Y.C. J. Biol. Chem. 1991; 266: 4810-4815Abstract Full Text PDF PubMed Google Scholar). The common feature of these sugars is the presence of vicinal equatorial hydroxyl groups in the stereochemistry of the 3- and 4-OH groups of d-mannose, and these sugars are referred to herein as "Man-type" ligands. The structural basis of MBP carbohydrate specificity has been investigated by high resolution x-ray crystallographic analysis of rat MBP-A (13Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (852) Google Scholar) and MBP-C (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The MBP CRD structure consists of a compactly folded domain that contains a series of loops stabilized by two Ca2+. Carbohydrate binding occurs through direct coordination of one of the Ca2+, which is designated the principal Ca2+, and hydrogen bond interactions with side chains of amino acids that also serve as ligands for this Ca2+, thereby forming an intimately linked ternary complex of protein Ca2+ and sugar (Fig. 1). Similar to most lectins, MBPs display only weak affinity for monovalent sugar ligands, with dissociation constants Kd in the millimolar range (12Lee R.T. Ichikawa Y. Fay M. Drickamer K. Shao M.-C. Lee Y.C. J. Biol. Chem. 1991; 266: 4810-4815Abstract Full Text PDF PubMed Google Scholar, 15Iobst S.T. Wormald M.R. Weis W.I. Dwek R.A. Drickamer K. J. Biol. Chem. 1994; 269: 15505-15511Abstract Full Text PDF PubMed Google Scholar), but bind avidly to target cell surfaces. This property allows discrimination between foreign cells and host cells, both of which display Man-type ligands on their surfaces. The trimeric structures of fragments of rat and human MBPs comprising the CRD plus α-helical neck regions reveal that the sugar binding sites on the trimer are spaced too far apart to bind to different branches of typical vertebrate high mannose oligosaccharides (9Sheriff S. Chang C.Y. Ezekowitz R.A.B. Nat. Struct. Biol. 1994; 1: 789-794Crossref PubMed Scopus (212) Google Scholar, 10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). However, they are presumably able to bind multivalently to the dense, repetitive arrays of Man-type ligands present on bacterial and fungal cell surfaces. The CRDs of MBP-A and MBP-C share 56% amino acid sequence identity, and x-ray analysis has shown that they have very similar structures (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The CRDs also show similar specificity for monosaccharide ligands, but differ in their affinities for oligosaccharides. MBP-C binds best to the trimannosyl core structures of N-linked carbohydrates, whereas MBP-A preferentially binds to terminal sugars of oligosaccharide chains (16Childs R.A. Feizi T. Yuen C.-T. Drickamer K. Quesenberry M.S. J. Biol. Chem. 1990; 265: 20770-20777Abstract Full Text PDF PubMed Google Scholar). Lee and co-workers (17Lee R.T. Lee Y.C. Glycoconj. J. 1997; 14: 357-363Crossref PubMed Scopus (18) Google Scholar, 18Quesenberry M.S. Lee R.T. Lee Y.C. Biochemistry. 1997; 36: 2724-2732Crossref PubMed Scopus (56) Google Scholar) have reported that the linear trisaccharide Manα1–2Manα1–6Man (17Lee R.T. Lee Y.C. Glycoconj. J. 1997; 14: 357-363Crossref PubMed Scopus (18) Google Scholar) and a bivalent mannose-terminated glycopeptide (18Quesenberry M.S. Lee R.T. Lee Y.C. Biochemistry. 1997; 36: 2724-2732Crossref PubMed Scopus (56) Google Scholar) bind with significantly higher affinity to MBP-C than either monosaccharides or other oligosaccharides, whereas MBP-A does not show such differences. The structural basis for these differences has not been apparent from the available crystal structures of MBP complexes, which are MBP-A bound to an asparaginyl oligosaccharide (13Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (852) Google Scholar) and MBP-C bound to a series of monosaccharides (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The major difference in the mode of sugar binding by MBP-A and MBP-C observed in these structures is that the orientation of the sugar ring is reversed in the two cases (Fig. 1). To assess whether this difference is related to the fine specificity of these two proteins, we have determined high resolution crystal structures of MBP-A and MBP-C bound to various carbohydrate ligands. The results indicate that a single residue in the binding site can influence the mode of ligand binding, which is likely to impact on the ability of the protein to interact with oligosaccharides. We also report the structure of trimeric MBP-A cross-linked by an oligosaccharide and discuss the implications of this structure for multivalent cell surface recognition and complement activation. The O-methylglycosides of mannose,N-acetylglucosamine and fucose, and also Manα1–2Man, Manα1–3Man, and Manα1–6Man were obtained from Sigma. Man6GlcNAc2Asn, Manα1–3[Manα1–6]Man, and GlcNAc-β1–2Manα1–3[GlcNAc-β1–2Manα1–6]Man were purchased from V-Labs, Inc. Manα1–2Manα1–6Man and the bivalent glycopeptideN-Ac-Tyr-Asp-(Gly-Gly-NH-(CH2)6-O-Man)2were gifts from Dr. Reiko Lee (Johns Hopkins University, Baltimore, MD). A trimeric fragment of MBP-A, designated cl-MBP-A (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and a dimeric fragment of MBP-C (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) were expressed, purified, and crystallized as described previously. The His189 → Val mutant of MBP-A was generated by replacing appropriate restriction fragments with synthetic double-stranded oligonucleotides in the expression plasmid (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). The sequence of the MBP-A cDNA was altered to GTG at positions 658–660. The His189 → Val/Ile207 → Val double mutant of MBP-A was created by combining restriction fragments from the two singly mutated expression plasmids. The mutant proteins were expressed and purified as for the wild-type proteins. Crystals of the wild-type and mutant MBP-A trimer and MBP-C dimer were grown at 20–22 °C by hanging drop vapor diffusion. MBP-A crystals were prepared by mixing equal volumes of 12 mg ml−1 MBP-A in 10 mm NaCl and 10 mm CaCl2 with reservoir solution consisting of 8–13% polyethylene glycol 3350 or 8000, 100 mm Tris-Cl, pH 8.0, 10 mm NaCl, 20 mm CaCl2, and 2 mmNaN3. For data collection, crystals were adapted into 2,4-methylpentanediol (MPD) by serial transfer into synthetic mother liquor consisting of the reservoir solution and 0, 2.5, 5, 10, 15, 20, and 25% MPD at 10–15-min intervals. Sugar ligands were included in last two cryopreservation solutions at the concentrations indicated in Table I. MBP-C crystals were grown by mixing equal volumes of 12–20 mg ml−1 protein in 10 mm Tris-Cl, pH 7.5, and 12 mm CaCl2 with reservoir solution containing 11–14% polyethylene glycol 8000, 100 mmTris-Cl, pH 7.4, 100 mm NaCl, 12 mmCaCl2, and 2 mm NaN3. The crystals were adapted into MPD by serial transfer into synthetic mother liquor consisting of the reservoir solution and 0, 5, 10, 15, 20, and 25% MPD at 10-min intervals for data collection. Sugar ligands were included in the last two cryopreservation solutions at the concentrations indicated in Table II. The crystals were flash-frozen in a 100 K nitrogen gas stream for data collection.Table IData collection and refinement statistics for MBP-A trimer soaked with ligandsLigandResolution (last shell)Data collectionRefinementRsym1-aRsym = ∑h∑i(‖Ii(h)‖ − ‖〈I(h)〉‖)/∑h∑iIi(h), where Ii(h) = observed intensity and 〈I(h)〉 = mean intensity obtained from multiple measurements.% > 3σ(I)% completeAverage redundancyRfree1-bR and Rfree = ∑||Fo‖ − ‖Fc||/∑‖Fo‖, where ‖Fo‖ = observed structure factor amplitude and ‖Fc‖ = calculated structure factor amplitude for the working and test sets, respectively.R1-bR and Rfree = ∑||Fo‖ − ‖Fc||/∑‖Fo‖, where ‖Fo‖ = observed structure factor amplitude and ‖Fc‖ = calculated structure factor amplitude for the working and test sets, respectively.Bond length rmsdAngle rmsdRamachandran plot: most favored/ disallowed1-cAs defined in Procheck (26).ÅÅ°%Native (MPD)1.950.04582.195.42.80.2340.1980.0071.489.5/0.3(2.01–1.95)(0.296)(55.3)(83.9)(2.5)αMe-O-Man1.95 .03979.394.11.90.2410.2070.0071.389.5/0.0(2.01–1.95)(0.257)(49.0)(88.9)(1.8)αMe-O-GlcNAc2.000.05278.798.62.80.2490.2120.0071.389.2/0.0(2.07–2.00)(0.270)(45.8)(97.3)(2.6)αMe-O-Fuc1.900.05089.086.02.30.2240.1890.0071.390.2/0.3(1.96–1.90)(0.186)(65.9)(65.2)(1.5)βMe-O-Fuc2.000.04485.498.23.00.2290.2000.0081.490.5/0.3(2.07–2.00)(0.218)(60.5)(94.5)(2.8)Manα1–3Man2.000.04787.198.82.90.2310.1980.0091.489.7/0.3(2.07–2.00)(0.169)(59.6)(95.0)(2.4)Manα1–3Man1.900.06385.794.62.90.2520.2230.0081.490.0/0.5 (H189V mutant)(1.94–1.90)(0.307)(49.1)(83.0)(2.4)Manα1–3Man2.000.04682.993.22.50.2320.2010.0081.389.0/0.0 (H189V/I207V mutant)(2.05–2.00)(0.244)(51.2)(92.0)(2.4)The ligand concentration in the soak solution was 200 mm. The space group is C2 with approximate unit cell constantsa = 79.0 Å, b = 85.3 Å,c = 98.3 Å, β = 106° with one trimer in the asymmetric unit. Numbers in parentheses are for last shell. rmsd, root-mean-square deviation.1-a Rsym = ∑h∑i(‖Ii(h)‖ − ‖〈I(h)〉‖)/∑h∑iIi(h), where Ii(h) = observed intensity and 〈I(h)〉 = mean intensity obtained from multiple measurements.1-b R and Rfree = ∑||Fo‖ − ‖Fc||/∑‖Fo‖, where ‖Fo‖ = observed structure factor amplitude and ‖Fc‖ = calculated structure factor amplitude for the working and test sets, respectively.1-c As defined in Procheck (26Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Table IIData collection and refinement statistics for MBP-C trimer soaked with ligandsLigandResolution (last shell)Data collectionRefinementRsym2-aSee Table I.% > 3σ(I)% completeAverage redundancyRfree2-bSee Table I.R2-bSee Table I.Bond length rmsdAngle rmsdRamachandran plot: most favored/ disallowed2-bSee Table I.ÅÅ°%Manα1–3Man1.740.06583.699.99.40.2350.2130.0071.493.0/0.0 (200 mm)(1.77–1.74)(0.271)(54.8)(99.1)(7.0)Trimannosyl core2-cManα1–3[Manα1–6]Man.1.800.06875.294.83.50.2590.2120.0061.391.8/0.0 (100 mm)(1.86–1.80)(0.111)(36.2)(81.8)(1.8)High affinity linear trimannose2-dManα1–2Manα1–6Man (17).1.850.06875.399.93.70.2420.2100.0061.392.9/0.0 (100 mm)(1.92–1.85)(0.252)(48.0)(99.9)(3.7)GlcNAc-terminated core2-eGlcNAcβ1–2Manα1–3[GlcNAcβ1–2Manα1–6]Man.1.900.06475.197.43.70.2390.1980.0061.393.9/0.0 (70 mm)(1.97–1.90)(0.183)(51.4)(91.2)(2.5)Bivalent Man-terminated glycopeptide2-fN-Ac-Tyr-Asp-[(Gly-Gly-NH-(CH2)6-O-Man)]2(18).1.800.04879.497.13.10.2430.2110.0061.393.4/0.0 (50 mm)(1.86–1.80)(0.156)(50.8)(89.5)(2.4)The ligand concentration in the soak solution is indicated in the first column. The space group is P212121 with approximate unit cell constants a = 60.6 Å,b = 75.3 Å, c = 57.6 Å with one dimer in the asymmetric unit. Numbers in parentheses are for last shell. rmsd, root-mean-square deviation.2-a See Table I.2-b See Table I.2-c Manα1–3[Manα1–6]Man.2-d Manα1–2Manα1–6Man (17Lee R.T. Lee Y.C. Glycoconj. J. 1997; 14: 357-363Crossref PubMed Scopus (18) Google Scholar).2-e GlcNAcβ1–2Manα1–3[GlcNAcβ1–2Manα1–6]Man.2-f N-Ac-Tyr-Asp-[(Gly-Gly-NH-(CH2)6-O-Man)]2(18Quesenberry M.S. Lee R.T. Lee Y.C. Biochemistry. 1997; 36: 2724-2732Crossref PubMed Scopus (56) Google Scholar). Open table in a new tab The ligand concentration in the soak solution was 200 mm. The space group is C2 with approximate unit cell constantsa = 79.0 Å, b = 85.3 Å,c = 98.3 Å, β = 106° with one trimer in the asymmetric unit. Numbers in parentheses are for last shell. rmsd, root-mean-square deviation. The ligand concentration in the soak solution is indicated in the first column. The space group is P212121 with approximate unit cell constants a = 60.6 Å,b = 75.3 Å, c = 57.6 Å with one dimer in the asymmetric unit. Numbers in parentheses are for last shell. rmsd, root-mean-square deviation. Data were measured on an RAXIS-IIc-imaging plate detector mounted on a rotating Cu anode x-ray generator operating at 4.5 kilowatts. For MBP-A, between 90 and 180° of data were measured with typical exposures of 60–75 min/1.2° image. For MBP-C, 100–120° of data were measured with exposures of 15–20 min/1–1.5° image. Data were integrated and scaled with Denzo and Scalepack (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). Data collection statistics are presented in Tables I and II. In all cases, the crystals were sufficiently isomorphous to the unliganded MBP-A (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) or MBP-C (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) structures to permit direct solution by rigid body refinement. All water and other solvent molecules were removed as were the alternate side chain conformations. Ca2+ and the refined individual temperature factors were retained in the starting models. All refinement calculations were performed in crystallography NMR system (20Brünger 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. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). Before any refinement commenced, a subset of the data was removed as the test set for the calculation of the free R-value (21Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3864) Google Scholar). The oligomer structures (MBP-A trimer or MBP-C dimer) were first refined as a single rigid body, and then the individual protomers were used as rigid bodies at 4 Å resolution. The resolution was then moved to 2.8 Å where another round of rigid body refinement of the protomers was run before switching to standard positional refinement. Refinement proceeded in several rounds of increasing resolution and consisted of alternate positional and individual temperature factor refinement. An overall anisotropic temperature factor (22Sheriff S. Hendrickson W.A. Acta Crystallogr. Sec. A. 1987; 43: 118-121Crossref Scopus (63) Google Scholar) and bulk solvent correction (23Jiang J.-S. Brünger A.T. J. Mol. Biol. 1994; 243: 100-115Crossref PubMed Scopus (340) Google Scholar) were applied throughout. For the first four structures of each protein, water molecules were independently identified inFo − Fcmaps and refined. The water molecules common to the four structures were then identified to generate a common set that could be added to subsequent structures without repicking them individually (275 for MBP-A, 192 for MBP-C). Use of these sets allowed the remaining waters and other solvent molecules to be found rapidly and the structures to be completed. Refinement statistics are presented in Tables I and II. Hanging drop vapor diffusion at 20 °C was used to prepare crystals of the MBP-A trimer-Man6GlcNAc2Asn complex. A 5 mg ml−1 solution of cl-MBP-A in 10 mmTris-Cl, pH 8.0, 10 mm NaCl, 20 mmCaCl2 and 1.1 mmMan6GlcNAc2Asn was mixed with an equal volume of reservoir solution containing 10% (w/v) polyethylene glycol 8000, 100 mm Tris-Cl pH 8.0, 10 mm NaCl, 20 mm CaCl2 and 2 mm NaN3. The oligosaccharide was required for the growth of the crystal, and crystallization was sensitive to the precise ratio of protein to sugar. grow in two habits: hexagonal plates and hexagonal rods with missing corners. The latter form was used for structure determination. A single crystal of dimensions 0.50 × 0.35 × 0.15 mm3 was transferred directly to a containing the reservoir plus 20% MPD for 3σ(I) (60.7% last shell). Data were measured an average of 1.9 times (1.4 × last shell). The data are 94.4% complete overall (last shell 75.9%). Based on the typical protein partial specific volume of 1.21 Å3/Da, the asymmetric unit could have between 1 and 4 trimers. A self-rotation function revealed a 3-fold noncrystallographic symmetry (NCS) axis oriented 18° from the b axis and 60° from the a axis, and three 2-fold NCS axes perpendicular to the 3-fold axis. The structure was solved by molecular replacement using the MBP-A trimer (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) with Ca2+, solvent molecules, and alternate conformations removed as a search model. All molecular replacement calculations were performed using X-PLOR (24Brünger A.T. X-PLOR manual, Version 3.1. Yale University, New Haven, CT1992Google Scholar). A cross-rotation function calculated using data in the resolution range of 10–4 Å and a maximum Patterson integration radius of 45 Å produced six solutions of approximately the same height that were significantly higher than any other solution (a range of the six solutions 6.2–6.7 standard deviations (σ) over the mean, next solution 4.9 σ over the mean). Patterson-correlation refinement (25Brünger A.T. Acta Crystallogr. Sec. A. 1990; 46: 46-57Crossref Scopus (361) Google Scholar) of the top 20 cross-rotation function peaks was carried out in two steps for each solution, first using the trimer as a rigid body and then using the three protomers as individual rigid bodies. The six most significant solutions from the cross-rotation function remained as such after this procedure. The six peaks fell into two groups of three. Within each group, the three peaks are related by the 3-fold NCS, and the two groups are related by the 2-fold NCS, implying that the asymmetric unit contains two trimers (64% solvent content). Translation searches (10–4 Å) run separately on the two trimers gave one significant peak (10.3 σ, next peak 2.9 σ over the mean). After placing one trimer, the second trimer was placed with respect to the first in a one-dimensional search along the y axis calculated for the four possible choices of origin. This procedure gave a single solution 8.9 σ (next peak 2.6 σ). The two placed trimers were initially refined as separate rigid bodies, and then as six individual protomers, first using data from 10–4 Å and then using data from 10–2.9 Å. The R-value at the end of this procedure was 0.385. The trimers are packed to give a hexagonal unit cell geometry, but because the trimer axes are tipped 18° off of the b axis, the space group is P21 rather than its supergroup P63. Refinement of the structure was performed in CNS using positional and individual temperature factor refinement. The trimers do not obey perfect NCS, and free R value tests indicated that there was no advantage in imposing NCS restraints. After the first round of refinement, most of the Man6GlcNAc2Asn could be built into the model. The final model comprises 894 amino acids, 14 sugar residues (only the terminal sugars are visible in one of the three copies; in the others, the six mannose residues are visible), and 18 Ca2+. The R and Rfreevalues are 24.4 and 28.9%, respectively. The bond and angle root-mean-square deviation from ideality were 0.007 Å and 1.3°, respectively. A total of 82.2% of the amino acids are in the most favored regions of the Ramachandran plot (26Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and 0.6% are in disallowed regions. The previously determined structure of the MBP-A CRD complexed with Man6GlcNAc2Asn (13Weis W.I. Drickamer K. Hendrickson W.A. Nature. 1992; 360: 127-134Crossref PubMed Scopus (852) Google Scholar) defined the binding of terminal mannose groups to the CRD through the 3- and 4-OH groups (Fig.1, left). Each of these OH groups forms a coordination bond with the principal Ca2+and two hydrogen bonds with amino acids that are also Ca2+ligands. The CRDs in these crystals are cross-linked by the oligosaccharide, precluding experiments in which other ligands are soaked into the crystal. The crystals of the homologous MBP-C CRD grow in the absence of sugar and were used to examine the structures of a series of cognate monosaccharides (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Surprisingly, the orientation of monosaccharides bound to MBP-C is reversed relative to MBP-A (14Ng K.K.-S. Drickamer K. Weis W.I. J. Biol. Chem. 1996; 271: 663-674Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) (Fig. 1A, right). Although the monosaccharides are asymmetric structures, the two orientations can be generated by rotation around a local 2-fold symmetry axis that relates the equatorial 3- and 4-OH groups of mannose or their stereochemical equivalents. In this paper, we define the orientation observed in the MBP-A-Man6GlcNAc2Asn (i.e. with the 3-OH of mannose or its equivalent bound to Glu185 and Asn187) as orientation I (Fig. 1, left) and the reverse as orientation II in which the 4-OH occupies this position (Fig. 1, right). Unlike the MBP-A CRD bound to Man6GlcNAc2Asn, the trimeric fragment containing the neck and CRD crystallizes in the absence of sugar (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Therefore, the crystals of the trimeric fragment were used to visualize the binding of Man-type ligands to MBP-A. The original structure determination of this fragment employed 25% glycerol as a cryoprotectant (10Weis W.I. Drickamer K. Structure. 1994; 2: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). In that structure, a molecule of glycerol was bound at the Ca2+ site through vicinal OH groups in a manner identical to that observed in sugars bound to MBPs. Therefore, high concentrations glycerol or any other compound containing vicinal OH groups cannot be used as cryoprotectants, because they would compete with sugar ligands for binding. The compound MPD, which does not have vicinal OH groups, was found to be an effective cryoprotectant. A "
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