Abstract
The seed lectin from Dioclea grandiflora (DGL) has recently been shown to possess high affinity for 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranose, the core trimannoside of asparagine-linked carbohydrates, but lower affinity for biantennary complex carbohydrates. In the previous paper, the thermodynamics of DGL binding to deoxy analogs of the core trimannoside and to a biantennary complex carbohydrate were determined by isothermal titration microcalorimetry. The data suggest that DGL recognizes specific hydroxyl groups of the trimannoside similar to that of the jack bean lectin concanavalin A (ConA) (Gupta, D. Dam, T. K., Oscarson, S., and Brewer, C. F. (1997) J. Biol. Chem. 272, 6388-6392). However, the thermodynamics of DGL binding to certain deoxy analogs and to the complex carbohydrate are different from that of ConA. In the present paper, the x-ray crystal structure of DGL complexed to the core trimannoside was determined to a resolution of 2.6 A. The overall structure of the DGL complex is similar to the structure of the ConA-trimannoside complex (Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976). The location and conformation of the bound trimannoside as well as its hydrogen-bonding interactions in both complexes are nearly identical. However, differences exist in the location of two loops outside of the respective binding sites containing residues 114-125 and 222-227. The latter residues affect the location of a network of hydrogen-bonded water molecules that interact with the trisaccharide. Differences in the arrangement of ordered water molecules in the binding site and/or protein conformational differences outside of the binding site may account for the differences in the thermodynamics of binding of the two lectins to deoxy analogs of the trimannoside. Molecular modeling studies suggest how DGL discriminates against binding the biantennary complex carbohydrate relative to ConA.
Highlights
EXPERIMENTAL PROCEDURESDGL was isolated from D. grandiflora seeds obtained from northeastern Brazil (Albano Ferreira Martin Ltd., Sao Paulo, Brazil) as described previously (11), and the amino acid sequence was verified. Crystals of DGL were produced at 4 °C using the hanging drop vapor diffusion technique (20)
Differences in the binding specificities of DGL and ConA for complex carbohydrate 2 appear to be due to relatively unfavorable contact interactions of the (1,2) GlcNAc residue on the ␣(1– 6) arm of 2 in DGL
Summary
DGL was isolated from D. grandiflora seeds obtained from northeastern Brazil (Albano Ferreira Martin Ltd., Sao Paulo, Brazil) as described previously (11), and the amino acid sequence was verified. Crystals of DGL were produced at 4 °C using the hanging drop vapor diffusion technique (20). The solvent content of the crystals is 75%, and the asymmetric unit contains two subunits of the tetramer. The atomic coordinates of a single subunit of ConA (Protein Data Bank, entry code 5CNA) were used as the search model (with all sugar atoms and water molecules removed). The appropriate amino acid changes were made to convert the molecular model replacement of ConA to DGL, and simulated annealing refinement was performed (R value ϭ 26.7%; Rfree ϭ 34.3%) using XPLOR. Two molecules of 1 (one for each subunit of the dimer) and 190 ordered water molecules were placed in the model over the course of cycling between manual model building using the program CHAIN (24) and XPLOR positional refinement followed by individual temperature factor refinement (final R value ϭ 18.9%, final Rfree ϭ 25.2%).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
