Abstract
By using near-UV circular dichroism (CD) and solvent proton nuclear magnetic relaxation dispersion measurements, three different conformational states have been detected in Ca(2+)-Mn(2+)-concanavalin A upon binding a variety of asparagine-linked carbohydrates. Two of these transitions have been described previously, one for the binding of monosaccharides such as methyl alpha-D-mannopyranoside and oligosaccharides with terminal alpha-Glc or alpha-Man residues, and the second for the binding of oligomannose and complex type carbohydrates (Brewer, C. F., and Bhattacharyya, L. (1986) J. Biol. Chem. 261, 7306-7310). The third transition occurs upon binding a bisected biantennary complex type carbohydrate with terminal GlcNAc residues. Temperature-dependent nuclear magnetic relaxation dispersion and CD measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes. The results support our previous conclusions that the rotamer conformation of the (alpha 1,6) arm of bisected complex type oligosaccharides binds to concanavalin A with dihedral angle omega = -60 degrees whereas nonbisected complex type oligosaccharides bind with omega = 180 degrees (Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299). The present findings also explain the effects of increasing chain length of bisected complex type carbohydrates on their interactions with the lectin.
Highlights
From the $Departmentsof Molecular Pharmacology, Microbiology, and Immunology, $Albert EinsteinCollege of Medicine, Bronx, New York10461 and SZBM Thomas J
Measurements, three different conformational states ConA has been shown to be specific for a-Glc[2] and a-Man have been detected in Ca2+-Mnz+-concanavaliAn upon residues with free 3, 4, and 6-hydroxyl groups .The binding a variety of asparagine-linkedcarbohydrates. lectin is a tetramer above pH 7 and a dimer below pH 6
Temperature-dependent nuclear magnetic relaxation dispersion and circular dichroism (CD) measurements have identified regions of the protein near the two metal ion binding sites that are associated with the conformation changes, and Tyr-12, which is part of the monosaccharide binding site, as responsible for the CD changes
Summary
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 266, No 15, Issue of May 25, pp. 9835-9U0,1991 Printed in U.S.A. The paramagnetic relaxivity values, in turn, are sensitive to conformational changes in the protein induced by saccharidebinding (11, 13,15-18) Binding of monosaccharides such a-MM and simple linear oligosaccharides with nonreducing a-Glc or a-Man residues uniformily reduces the relaxivity values of the protein at 25 “C by approximately 20% (11, 13). Similar studies showed that the presence of a "bisecting" GlcNAc in certain complex type oligosaccharides such as[2] and 3 reduced their affinitiesfor ConA by an order of magnitude relative t o 1, and that 2 induced pendence of the longitudinal (spin-lattice) relaxation rat(els/Tl),i.e. nuclear magnetic relaxation dispersion (NMRD) profile, of solvent water protons corresponding to proton Larmforrequencies from 0.01 to 50 MHz, were madeusing a field cycling relaxometer (12, 25). For these 2 residues to bind and cross-link the protein they must face in opposite direc-
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