©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Basis of Galactose Recognition by C-type Animal Lectins (*)

(Received for publication, November 27, 1995; and in revised form, January 16, 1996)

Anand R. Kolatkar William I. Weis (§)

From the Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The asialoglycoprotein receptors and many other C-type (Ca-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K.(1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta-methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild-type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca and form hydrogen bonds with amino acids that also serve as Ca ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K.(1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site.


INTRODUCTION

Ca-dependent (C-type) animal lectins are a family of proteins whose members contain one or more homologous carbohydrate-recognition domains (CRDs). (^1)The majority of C-type lectins bind to D-mannose, D-glucose, and related sugars (Man-type ligands), or to D-galactose and its derivatives (Gal-type ligands). The mammalian hepatocyte asialoglycoprotein receptors, which play a part in serum glycoprotein homeostasis(1, 2) , are the best known of the Gal-binding C-type lectins. C-type lectins with high affinity for glycoconjugates bearing terminal galactose residues have also been identified on the surfaces of peritoneal macrophages and Kupffer cells (3, 4) , and appear to mediate recognition of tumor cells(5, 6) . C-type CRDs with lower affinity for Gal-type ligands are found in proteoglycan core proteins of cartilage and other tissues and are presumed to contribute to the organization of the extracellular matrix(7) .

Previous crystallographic analyses of rat mannose-binding proteins (MBPs) A and C have shown that Man-binding C-type lectins recognize their sugar ligands by formation of direct coordination bonds between a Ca (designated site 2) and a lone pair of electrons from each of two vicinal hydroxyl groups possessing the same stereochemical arrangement as the equatorial 3- and 4-OH groups of D-mannose(8, 9) . The Ca is 8-coordinated in a pentagonal bipyramidal arrangement, with the two sugar hydroxyls bisecting one of the apical positions (8) (see Fig. 1a). In addition, the same OH groups form hydrogen bonds with amino acid side chains that are Ca site 2 ligands, producing an intimately linked ternary complex of protein, Ca, and sugar (see Fig. 1a). Only one other contact, an apolar van der Waals contact between a ring carbon and the C of residue 189 contributes significantly to binding(8, 10) .


Figure 1: Galactose binding to QPDWG. a, mannose binding to wild-type MBP-A as observed in a Man(6)GlcNAc(2)Asn-MBP-A complex(8) . Carbon, nitrogen, and oxygen atoms are shown as white, gray, and black spheres, respectively; Ca 2 is shown as a larger white sphere. Coordination and hydrogen bonds are represented by long and short dashed lines, respectively. Carbon atoms of the sugars are numbered. b, stereo pair of the final 2F(o) - F(c) electron density map in the binding site of the betaMeGal-QPDWG complex, contoured at 1.2 . c, galactose binding to QPDWG. Symbols are as described for a. Parts a and c were made with MOLSCRIPT(26) .



Studies with derivatized sugars have shown that free 3- and 4-OH groups are essential for binding to mammalian asialoglycoprotein receptors as well as Man-binding C-type lectins, whereas substitutions at other ring positions have little or no effect on binding(11) . However, the 3- and 4-OH groups of galactose have an equatorial/axial arrangement, so the mechanism of Gal- and Man-type ligand recognition must be different. Sequence analysis reveals that of the Ca 2 ligands, positions equivalent to Glu, Asn, and Asp of MBP-A are highly conserved among C-type lectins regardless of specificity. In contrast, positions 185 and 187 are found to be Glu and Asn in Man-binding family members, whereas Gal-binding C-type lectins have Gln and Asp at these positions. The Glu Gln/Asn Asp mutant of MBP-A, designated ``QPD'', binds to galactose in preference to mannose by a factor of 3 but with relatively low affinity for either sugar(12) . Position 189 of MBP-A (Fig. 1a) is not conserved among Man-binding C-type lectins but is always either Trp or Phe in Gal-binding family members. Replacement of His of MBP-A with Trp in the QPD mutant to make ``QPDW'' gives a protein with affinity for Gal comparable with natural Gal-binding C-type lectins but that still does not discriminate well between Gal and Man(13) . However, insertion of a glycine-rich loop found in the major form of the rat asialoglycoprotein receptor, rat hepatic lectin-1 (RHL-1), and other Gal-binding C-type lectins that display strong discrimination against mannose results in a mutant (``QPDWG'') with galactose affinity and selectivity comparable with RHL-1(13) . The affinity for galactose is comparable in QPDW and QPDWG, indicating that the determinants of affinity and selectivity are somewhat distinct.

NMR measurements reveal similar modes of galactose binding by QPDWG and RHL-1(13) , demonstrating that galactose specificity in C-type lectins is determined by a few residues and can be studied in the well characterized MBP-A background. Here we describe the structure of a trimeric fragment of QPDWG containing the neck and COOH-terminal CRD (14) , both alone and complexed with beta-methyl galactoside (betaMeGal) and N-acetylgalactosamine (GalNAc). The structures reveal the molecular basis of selective galactose recognition by C-type lectins. The structure of the QPDWG-GalNAc complex is consistent with results of site-directed mutagenesis experiments that have identified amino acid positions that contribute to the preferential binding of GalNAc over Gal by certain C-type lectins.


EXPERIMENTAL PROCEDURES

Materials

Unless otherwise specified, chemicals were obtained from J. T. Baker Inc. LB medium was obtained from Life Technologies, Inc. Guanidinium hydrochloride and isopropylthiogalactopyranoside were obtained from Boehringer Mannheim. Clostripain was obtained from Worthington Biochemical. Sepharose 6B, polyethylene glycol 8000, beta-methyl galactoside, and N-acetylgalactosamine were from Sigma Divinylsulfone was obtained from Fluka Chemical Co. 2-Methyl-2,4-pentanediol was obtained from Aldrich.

Purification of QPDWG

The QPDWG mutant of rat MBP-A was expressed in Escherichia coli as described previously(13) , except that the amount of isopropylthiogalactopyranoside used to induce expression was 1 mM. The clostripain-treated fragment of QPDWG, cl-QPDWG, was purified as described for wild-type cl-MBP-A(14) , except that galactose-Sepharose (prepared by the divinylsulfone method (15) ) was used in all affinity chromatography steps.

Crystallization and Data Collection

Crystals of cl-QPDWG were grown at 20 °C by hanging drop vapor diffusion by mixing equal volumes of 25 mg/ml cl-QPDWG in 10 mM CaCl(2)/10 mM NaCl and reservior solutions containing 12-15% polyethylene glycol 8000/100 mM Tris-HCl, pH 8.0/20 mM CaCl(2)/10 mM NaCl/0.02% NaN(3) (solution A). Crystals appeared within 3-4 days and grew to full size (typically 0.3 times 0.3 times 0.2 mm^3) in 7-10 days. Prior to data collection, the crystals were adapted in a stepwise fashion to solution A plus 0, 5, 7.5, 10, 15, and 20% 2-methyl-2,4-pentanediol. Complexes with monosaccharides were prepared by including 200 mM betaMeGal or GalNAc in the soaking solutions. Crystals were flash-cooled at 100 K, and diffraction data were measured on an R-AXIS II imaging plate detector mounted on a rotating copper anode operating at 4.5 kW. A total of 180 ° of data were collected in 1.2 ° oscillation scans from a single orientation and processed using DENZO and SCALEPACK(16) . A data set used in the initial stages of the structure determination was obtained to 2.0 Å resolution (R = 4.9%; 88.2% complete). A more complete data set was subsequently measured from another unliganded cl-QPDWG crystal and used in the final stages of refinement (see Table 1and Table 2).





Structure Solution and Refinement

Crystals of cl-QPDWG are nearly isomorphous with those of wild-type cl-MBP-A and permitted structure solution by rigid body refinement of the wild-type cl-MBP-A model (14) against the first cl-QPDWG data set. The side chains of residues 185 and 187, the entire loop from 189 to 198, Ca, and water molecules were omitted from the model. Temperature factors from the wild-type model were retained. All calculations were performed using X-PLOR(17) . The protomers were refined as individual rigid bodies against data from 10-4 Å and then 10-2.8 Å (R = 0.369). In order to remove model bias, this model was subjected to simulated annealing refinement (18) starting at 3000 K with 10% of the reflections omitted for calculation of R(19) , followed by positional and isotropic temperature factor refinement against data from 5.0-2.5 Å. Resolution-dependent weights were applied as 1/(1 - 5.5 (1/(2d) - 1/6))^2 where d is the Bragg spacing of the reflection. At this point, the glycine-rich loop and the omitted side chains were built using the program O(20) . Water molecules were added, and positional and temperature factor refinement was carried out against data from 5-2.0 Å. Reflections from 10-2.0 Å were then included and refinement continued. This model was then subject to rigid body refinement against the second data set from 10-2.8 Å, followed by several rounds of positional and isotropic temperature-factor refinement alternating with model adjustment (initially using data from 5-2.0 Å and then extending from 10 Å to the high resolution limit). Sugar complexes were refined by a similar strategy, starting from the same model used for refinement of the unliganded structure against its second data set. The methyl aglycon of betaMeGal was not modelled in the most poorly ordered copy. Only the beta anomer of GalNAc could be modelled reliably (the ratio of alpha:beta galactose is approximately 1:2 (21) but has not been reported for GalNAc). An overall anisotropic temperature factor (22) was applied to each structure, although it significantly reduced the R and R values only for the unliganded QPDWG model. Noncrystallographic symmetry restraints were not imposed at any point in the refinement.


RESULTS AND DISCUSSION

A trimeric fragment of QPDWG containing the neck and COOH-terminal CRD (14) was crystallized, and the structure was solved by molecular replacement, both alone and complexed with betaMeGal and GalNAc ( Table 1and Table 2). The structures were refined to resolutions of 2.0 Å or better ( Table 1and Table 2). Apart from the His Trp change and the glycine-rich insertion at the carbohydrate-binding site, the structures of wild-type MBP-A and the QPDWG mutant are identical to within the coordinate error. In particular, the Ca site 2 ligands of the two structures superimpose, with the side chain amide nitrogen of Gln and the carbonyl oxygen of Asp of QPDWG in the same positions as the carbonyl oxygen of Glu and the amide nitrogen of Asn in the wild-type protein.

Despite the different stereochemistry of the 3- and 4-OH groups, the mechanism of betaMeGal and GalNAc binding to QPDWG is similar to that of Man-type ligands to wild-type MBPs, with the full noncovalent bonding potential of 3- and 4-OH groups used for Ca coordination and hydrogen bond formation with Ca ligands (8, 9) (Fig. 1, a and c). However, maintenance of the pentagonal bipyramidal Ca coordination geometry forces the pyranose ring into a very different orientation from that observed in mannose binding to wild-type MBPs(8, 9) . The apolar patch formed by the 3, 4, 5, and 6 carbons of betaMeGal and GalNAc packs against the side chain of Trp, an interaction observed in all galactose-lectin interactions studied to date (23) (Fig. 1c). The angle between the least squares plane through the pyranose ring of galactose and the plane of the Trp side indole ring falls within the range found in other galactose-binding lectins (Table 3). This interaction is especially noteworthy given that no aromatic residues interact with the sugar ligand in Man-binding C-type lectins, which in fact make few nonpolar contacts with the sugar ligand(8, 9) .



Interaction of the Trp side chain with the Gly-rich loop is critical to the selectivity of QPDWG for galactose and discrimination against mannose. The loop is a rigid structure with a somewhat unusual conformation (Fig. 2, a and b). His tucks into the loop and stabilizes the structure by forming hydrogen bonds with a main chain amide and a carbonyl oxygen; Leu is on the outside of the loop and packs against Ala, thereby holding the loop down against the lower part of the protein (Fig. 2, a and b). The C of Gly packs against Trp and holds it in a slightly unfavorable (2) rotamer (+60 °). Modelling indicates that neither of the most favored (2) rotamers of Trp (±90 °) can be accommodated on the mutant protein, nor can other (1) rotamers. The Gly-rich loop thus serves as a ``doorstop'' that prevents Trp from adopting a more favorable conformation. Mutagenesis data show that changes in many of the loop residues are tolerated with only small effects on galactose selectivity (13) , consistent with the notion that the loop serves as a rigid unit that restricts the conformation of Trp rather than providing specific interactions with the sugar or other residues of the protein. Superposition of mannose bound as observed in a Man(6) oligosaccharide-MBP-A complex (8) (Fig. 1a) on QPDWG reveals that the exocyclic C6 clashes with Trp (Fig. 2c). Man-type ligands bind to the homologous MBP-C in an orientation reversed 180 ° from that shown in Fig. 1a, such that the positions 3- and 4-OH groups are exchanged(9) , and preliminary data indicate that MBP-A can also bind to monosaccharides in this manner. (^2)In this orientation, the anomeric oxygen in the alpha configuration sterically clashes with Trp. Thus, the position of Trp imposed by the Gly-rich loop excludes Man-type ligands from the site and explains the essential role of this loop in galactose selectivity.


Figure 2: Galactose selectivity imposed by the glycine-rich loop. a, ribbon diagram of wild-type and QPDWG mutant of MBP-A in the vicinity of the binding site. The Ca 2 ligands, as well as the residue at position 189, are indicated along with the positions of Ca 1 and 2 (spheres). The loop following residue 189, which differs between wild-type and QPDWG, is highlighted in black. The van der Waals contact between Leu of the Gly-rich loop and Ala is shown as a dashed line. b, stereo pair showing the detailed structure of the glycine-rich loop and the packing of Trp against Gly. Symbols are as described in the legend to Fig. 1. c, superposition of mannose observed in the wild-type MBP-A binding site on the mutant binding site. The steric clash between mannose and Trp is emphasized. The figure was made with MOLSCRIPT(26) .



No significant differences in the protein are observed between unliganded and sugar complex structures. The sugar complexes were prepared using sugar concentrations in approximately 100-fold excess over the K(d)(13) , and the average temperature factors of the sugar and its liganding residues are quite similar. Thus, the sugars appear to be fully occupied in the binding sites, although the correlation of temperature factor and occupancy at the resolutions used in this study precludes refinement of the sugar occupancy. In two of the three crystallographically independent copies (protomers 1 and 3; Table 2), the glycine-rich loops in both the unliganded and complexed structures have similar temperature factors. The glycine-rich loop of protomer 2 of each structure has consistently higher temperature factors and is most likely a consequence of participating in relatively few lattice contacts. The average temperature factors of the loop in protomer 2 differ by approximately 25 Å^2 between the unliganded and complexed structures. It is possible that lattice contacts immobilize the loop so that any effect of sugar binding on loop mobility would be detectable only in the copy with no lattice contacts. However, the entire protomer 2 of the unliganded structure has significantly higher temperature factors than the equivalent protomer in the complexed structures (Table 2), so it cannot be concluded that sugar binding significantly affects the mobility of the binding site region.

The QPDWG structures explain binding, mutagenesis, and spectroscopic data obtained from several galactose-binding mutants of MBP-A. Proton NMR spectra of betaMeGal in the presence of QPDWG show upfield shifts of the H5, H6, and H6` protons of Gal consistent with their interaction with the delocalized electron system of the Trp ring observed in the crystal structure(13) . The line widths of the aromatic protons of Trp are broadened upon Gal binding to QPDW, whereas they are broad in the absence or the presence of Gal in QPDWG, consistent with the notion that the Gly-rich loop immobilizes Trp in a position optimal for interaction with Gal(13) . The proteoglycan core protein CRDs have Phe instead of Trp at position 189 and exhibit relatively poor selectivity against Man-type ligands. The corresponding MBP-A mutant QPDFG, which includes Phe, binds to Gal-type ligands only 6-fold more strongly than Man-type ligands, as opposed to the 40-fold selectivity for Gal-type ligands shown by QPDWG (13) . These properties are explained by exclusion of Man by the 6-membered portion of the Trp ring (Fig. 2c), which extends farther out than the side chain of Phe.

Several Gal-binding C-type lectins, including RHL-1, display strong preference for GalNAc over Gal, whereas others do not discriminate between these two sugars. An example of the latter is the macrophage galactose receptor (MGR), and the QPDWG mutant of MBP-A mimics MGR in this respect. Site-directed mutagenesis of MGR based on sequence comparisons with the asialoglycoprotein receptors has identified residues in four regions of the sequence that provide selectivity for GalNAc over Gal(24) . Of these regions, the residue equivalent to Ser of MBP-A provides 20-fold of the observed 60-fold selectivity for GalNAc over Gal by RHL-1(24) . Moreover, a histidine equivalent to Thr of QPDWG is found in both RHL-1 and MGR and must be present in order to observe the enhancement provided by the residue at 154. The structure of QPDWG complexed with GalNAc shows that the 2-acetamido substituent is in the vicinity of Thr, which in turn lies near Ser (Fig. 3), and is thus consistent with the formation of a GalNAc-specific binding site by residues in these positions in RHL-1.


Figure 3: N-Acetylgalactosamine binding to QPDWG. a, stereo pair of the final 2F(o) - F(c) electron density map in the binding site of the GalNAc-QPDWG complex, contoured at 1.2 . b, stereo ribbon diagram (MOLSCRIPT(26) ) showing location of GalNAc with respect to the CRD.



The present structures leave unclear how the Glu Gln/Asn Asp differences lead to specificity for Man- or Gal-type ligands. The residues in the binding sites of wild-type and mutant MBP-A superimpose closely, so it is not obvious why galactosides do not bind to wild-type MBPs in the orientation observed in the present structures. Indeed, free galactose binds to MBP-C through the 1- and 2-OH groups, emphasizing the selectivity of the wild-type site for equatorial OH groups having the same stereochemical arrangement as the 3- and 4-OH of mannose(9) . These OH groups are related by a 2-fold rotation axis that bisects the pyranose ring and form hydrogen bonds with side chain carbonyl oxygen and amide nitrogen atoms that conform approximately to this symmetry in the wild-type site but not in the QPD site (Fig. 4). Although the mechanism is not obvious, this difference in symmetry may be related to the weaker affinity of QPD for either Gal- or Man-type ligands(12) . The absolute affinity of wild-type MBP-A for Man is similar to that of QPDW or QPDWG for Gal, which implies that the binding energy of Man to the wild-type Ca site is greater than that of Gal to the QPD mutant site. Thus the favorable interaction with the aromatic residue at position 189 can be viewed as compensating for the loss of symmetry in the mutant site to provide affinity for Gal comparable with that of wild-type MBP-A for Man. In the absence of the glycine-rich loop, mannose is not excluded from the site but interacts with lower affinity due to the asymmetric arrangement of its hydrogen-bonding partners.


Figure 4: Symmetry of hydrogen bonding partners in wild-type and mutant MBP-As. Arrangement of the four side chain groups that form hydrogen bonds with the 3- and 4-OH groups of the sugar ligand in wild-type MBP with Man and QPDWG mutant with Gal. The symbols as described in the legend to Fig. 1. The figure was made with MOLSCRIPT(26) .



Another potential source of the different specificities of wild-type and QPD sites is the displacement of ordered water molecules upon sugar binding. High resolution structures of MBP-C show that the 3- and 4-OH of Man-type ligands replace two water molecules that form the same set of hydrogen and Ca coordination bonds(9) . Unfortunately, the amount of visible, ordered water structure in the uncomplexed QPDWG site varies among the three crystallographically independent copies, making it difficult to draw firm conclusions. In the best ordered site, two water molecules that form hydrogen bonds with the Ca 2 ligands at 185, 187, 198, and 210 equivalent to those formed by Gal can be discerned. These water molecules are in approximately the same position as the 3- and 4-OH groups of Gal but only one of them appears to be close enough to Ca 2 to form a coordination bond. Only one water molecule is observed in another copy, and no water molecules can be placed with confidence in the third site. Higher resolution structures of the uncomplexed QPDWG site will be required to assess whether or not there is a change in Ca coordination number upon ligand binding.

The different locations of the bound pyranose ring seen in the present structures and the structures of wild-type MBPs complexed with Man-type ligands are a consequence of Ca coordination geometry. This observation and the fact that few other contacts are made with the protein demonstrate the dominant role that Ca coordination plays in sugar recognition by C-type lectins. The different pyranose ring locations dictated by Ca coordination geometry forms the basis of selective recognition of galactose by steric exclusion of Man-type ligands provided by Trp and the glycine-rich loop.


FOOTNOTES

*
This work was supported by Grant GM50565 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1AFA (betaMeGal complex), 1AFB (GalNAc complex), and 1AFD (unliganded QPDWG)) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed: Dept. of Structural Biology, Fairchild Bldg., Stanford University School of Medicine, Stanford, CA 94305-5400. Tel.: 415-725-4623; Fax: 415-723-8464; weis{at}fucose.stanford.edu.

(^1)
The abbreviations used are: CRD, carbohydrate-recognition domain; MBP, mannose-binding protein; betaMeGal, beta-methyl-D-galactoside; GalNAc, N-acetyl-D-galactosamine; MGR, macrophage galactose receptor; RHL-1, rat hepatic lectin-1; cl-, clostripain-treated fragment.

(^2)
S. Park-Snyder and W. I. Weis, unpublished results.


ACKNOWLEDGEMENTS

We thank Shaun Park-Snyder for technical assistance and Kurt Drickamer for providing expression constructs and comments on the manuscript.


REFERENCES

  1. Spiess, M. (1990) Biochemistry 29, 10009-10018 [Medline] [Order article via Infotrieve]
  2. Drickamer, K. (1991) Cell 67, 1029-1032 [Medline] [Order article via Infotrieve]
  3. Ii, M., Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298 [Abstract/Free Full Text]
  4. Hoyle, G. W., and Hill, R. L. (1988) J. Biol. Chem. 263, 7487-7492 [Abstract/Free Full Text]
  5. Vavasseur, F., Berrada, A., Heuze, F., Jotereau, F., and Meflah, K. (1990) Int. J. Cancer 248, 744-751
  6. Sato, M., Kawakami, K., Osawa, T., and Toyoshima, S. (1992) J. Biochem. 111, 331-336 [Abstract]
  7. Drickamer, K., and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9, 237-264 [CrossRef]
  8. Weis, W. I., Drickamer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134 [CrossRef][Medline] [Order article via Infotrieve]
  9. Ng, K. K.-S., Drickamer, K., and Weis, W. I. (1996) J. Biol. Chem. 271, 663-674 [Abstract/Free Full Text]
  10. Iobst, S. T., Wormald, M. R., Weis, W. I., Dwek, R. A., and Drickamer, K. (1994) J. Biol. Chem. 269, 15505-15511 [Abstract/Free Full Text]
  11. Lee, R. T., Ichikawa, Y., Fay, M., Drickamer, K., Shao, M.-C., and Lee, Y. C. (1991) J. Biol. Chem. 266, 4810-4815 [Abstract/Free Full Text]
  12. Drickamer, K. (1992) Nature 360, 183-186 [CrossRef][Medline] [Order article via Infotrieve]
  13. Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519 [Abstract/Free Full Text]
  14. Weis, W. I., and Drickamer, K. (1994) Structure 2, 1227-1240 [Medline] [Order article via Infotrieve]
  15. Fornstedt, N., and Porath, J. (1975) FEBS Lett. 57, 187-191 [CrossRef][Medline] [Order article via Infotrieve]
  16. Otwinowski, Z. (1993) in Proceedings of the CCP4 Study Weekend: Data Collection and Processing, 29-30 January 1993 (Sawyer, L., Isaacs, N., and Bailey, S., and Daresbury, U. K., eds) pp. 56-62, Science and Engineering Research Council Daresbury Laboratory, Warrington, UK
  17. Br ü nger, A. T. (1992) X-PLOR Manual, Version 3.1 , Yale University, New Haven, CT
  18. Brünger, A. T., Krukowski, A., and Erickson, J. W. (1990) Acta Crystallogr. Sect. A 46, 585-593 [CrossRef][Medline] [Order article via Infotrieve]
  19. Brünger, A. T. (1992) Nature 355, 472-475 [CrossRef]
  20. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119 [CrossRef][Medline] [Order article via Infotrieve]
  21. Angyal, S. J., and Pickles, V. A. (1972) Aust. J. Chem. 25, 1695-1710
  22. Sheriff, S., and Hendrickson, W. A. (1987) Acta Crystallogr. Sect. A 43, 118-121 [CrossRef]
  23. Weis, W. I., and Drickamer, K. (1996) Annu. Rev. Biochem. 65, in press
  24. Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693 [Abstract/Free Full Text]
  25. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. T., Brice, M. D., Rogas, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. J. (1977) J. Mol. Biol. 112, 535-542 [Medline] [Order article via Infotrieve]
  26. Kraulis, P. J. (1991) J. Appl. Cryst. 24, 946-950

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