The structure of the saccharide-binding site of concanavalin A.

Derewenda, Zygmunt S.; Yariv, J.; Helliwell, John R.; Kalb, A. Joseph; Dodson, E.J.; Papiz, Miroslav Z.; Wan, Tian; Campbell, J.A.

doi:10.1002/j.1460-2075.1989.tb08341.x

Cited by 295 publications

(200 citation statements)

References 25 publications

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“…Substitution of N156 with A or Mutation of AD120I 121 to VA Prevents Binding to a Mannose Column Structural analysis by high resolution x-ray crystallography of legume lectins co-crystallized with their appropriate ligands has shown that the two conserved residues corresponding to D121 and N156 in ERGIC-53 (see Figure 1) interact via hydrogen bonds with the calcium ion and the monosaccharide (Derewenda et al, 1989;Bourne et al, 1990;Shaanan et al, 1991). The role of the conserved asparagine in carbohydrate binding was previously tested by its substitution with aspartate in the bean lectin Phaseolus vulgaris leucoagglutinin (PHA-L), and in pea lectin.…”

Section: Ergic-53 Mutants Containing An N-terminalmentioning

confidence: 99%

“…Figure 6 shows that in the absence of divalent cations ERGIC-53 myc/6xHis failed to bind to the mannose column, while in the (Sharon and Lis, 1990;Sharon, 1993 (Derewenda et al, 1989;Bourne et al, 1990;Sharon and Lis, 1990;Shaanan et al, 1991). In the case of concanvalin A, removal of the cations by EDTA widens the binding site (Reeke et al, 1978), an effect that explains the loss of ligand binding in the absence of cations (Kalb and Levitzki, 1968).…”

Section: Elution Of Ergic-53 Is Selective For D-mannosementioning

confidence: 99%

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ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Itin

Roche

Monsigny

et al. 1996

MBoC

166

148

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Based on sequence homologies with leguminous lectins, the intermediate compartment marker proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independently, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannosecolumn binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glycoproteins in the early secretory pathway.

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Section: Ergic-53 Mutants Containing An N-terminalmentioning

confidence: 99%

Section: Elution Of Ergic-53 Is Selective For D-mannosementioning

confidence: 99%

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Itin

Roche

Monsigny

et al. 1996

MBoC

166

148

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“…These conditions are as previously described (Derewenda et al, 1989). These crystallization conditions are as identical as possible to those for the saccharide-free (I222) crystals.…”

Section: Crystallandation Data Collection and Processingmentioning

confidence: 99%

“…The model for the tetramer from Derewenda et al (1989), PDB coordinate file 4CNA, was treated as a molecular-replacement solution for the present study. The starting R factor was 45%.…”

Section: Model Refinementmentioning

confidence: 99%

“…The crystal structure of the methyl a-D-mannopyranoside concanavalin A complex was solved at 2.9/~ by Derewenda et al (1989) and the details of the binding site were reported. That study showed that the methyl a-D-mannopyranoside molecule is bound in the C1 chair conformation, 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site.…”

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confidence: 99%

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Refined structure of concanavalin A complexed with methyl α-D-mannopyranoside at 2.0 Å resolution and comparison with the saccharide-free structure

Naismith¹,

Emmerich²,

Habash³

et al. 1994

Acta Cryst D

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The three-dimensional structure of the complex between methyl a-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P21212~, a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0tr(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a f To whom all correspondence should be addressed.(" 1994 International Union or" Crystallography Printed in Great Britain -all rights reserved dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A 2 when it binds to the protein. This work builds upon the earlier studies of Derewenda et al.

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Variability in quaternary association of proteins with the same tertiary fold: A case study and rationalization involving legume lectins

1999

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Legume lectins constitute a family of proteins in which small alterations arising from sequence variations in essentially the same tertiary structure lead to large changes in quaternary association. All of them are dimers or tetramers made up of dimers. Dimerization involves side-by-side or back-to-back association of the flat six-membered beta-sheets in the protomers. Variations within these modes of dimerization can be satisfactorily described in terms of angles defining the mutual disposition of the two subunits. In all tetrameric lectins, except peanut lectin, oligomerization involves the back-to-back association of side-by-side dimers. An attempt has been made to rationalize the observed modes of oligomerization in terms of hydrophobic surface area buried on association, interaction energy and shape complementarity, by constructing energy minimised models in each of which the subunit of one legume lectin is fitted in the quaternary structure of another. The results indicate that all the three indices favor and, thus, provide a rationale for the observed arrangements. However, the discrimination provided by buried hydrophobic surface area is marginal in a few instances. The same is true, to a lesser extent, about that provided by shape complementarity. The relative values of interaction energy turns out to be a still better discriminator than the other two indices. Variability in the quaternary association of homologous proteins is a widely observed phenomenon and the present study is relevant to the general problem of protein folding.

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The structure of the saccharide-binding site of concanavalin A.

Cited by 295 publications

References 25 publications

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Refined structure of concanavalin A complexed with methyl α-D-mannopyranoside at 2.0 Å resolution and comparison with the saccharide-free structure

Variability in quaternary association of proteins with the same tertiary fold: A case study and rationalization involving legume lectins

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