Studies of the Binding Specificity of Concanavalin A. Nature of the Extended Binding Site for Asparagine-Linked Carbohydrates

Dipak K. Mandal, Lokesh Bhattacharyya, Seymour H. Koenig, Rodney D. Brown, Stefan Oscarson, C. Fred Brewer

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

In the preceding paper [Mandal, D. K., Kishore, N., & Brewer, C. F. (1994) Biochemistry (preceding paper in this issue)] the trisaccharide 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was shown by titration microcalorimetry to bind to the lectin concanavalin A (Con A) with nearly -6 kcal mol-1 greater enthalpy change (ΔH) than methyl α-D-mannopyranoside (MeαMan). These results indicate that Con A possesses an extended binding site for the trisaccharide. In the present paper, we have investigated the binding of a series of synthetic analogs of the methyl α-anomer of the trisaccharide using hemagglutination inhibition, solvent proton magnetic relaxation dispersion (NMRD), near ultraviolet circular dichroism, and titration microcalorimetry measurements. Four of the analogs tested possess an α-glucosyl or α-galactosyl residue substituted at either the α(1-6) or α(1-3) position. Analysis of the data indicates that the α(1-6) residue of the parent trimannoside binds to the so-called monosaccharide site and the α(1-3) residue to a weaker secondary site. Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the α(1-3)Glc derivative since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser ΔH than the trimannoside, whereas the α(1-3)-2-deoxyGlc analog possesses essentially the same affinity and ΔH as the trimannoside. NMRD data show that the α(1-3) 2-, 3-, 4-, and 6-deoxy derivatives of the trimannoside induces essentially the similar conformational changes in the protein as that of the parent trimannoside. However, the calorimetry data show that only the 3-deoxy analog binds with ∼ 10-fold lower affinity and -3.4 kcal mol-1 lesser enthalpy change. This indicates that the 3-hydroxyl of the α(1-3)Man makes a specific hydrogen bond with the protein at a secondary binding site. The ΔH of -11 kcal mol-1 for the 3-deoxy analog is still, however, greater than that of MeαMan (-8.4 kcal mol-1) which indicates another site of contact between the trimannoside and Con A, most likely with the “core” Man residue. Thus, Con A has an extended binding site which includes a high-affinity site that recognizes the 3-, 4-, and 6-hydroxyl groups of the α(1-6)Man residue of the trimannoside (the monosaccharide site), a lower affinity site that binds the 3-hydroxyl of the α(1-3)Man residue, and a third site which appears to involve the “core” Man residue.

Original languageEnglish (US)
Pages (from-to)1157-1162
Number of pages6
JournalBiochemistry
Volume33
Issue number5
DOIs
StatePublished - Feb 1 1994

ASJC Scopus subject areas

  • Biochemistry

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