TY - JOUR
T1 - New carbohydrate specificity and HIV-1 fusion blocking activity of the cyanobacterial protein MVL
T2 - NMR, ITC and sedimentation equilibrium studies
AU - Bewley, Carole A.
AU - Cai, Mengli
AU - Ray, Satyajit
AU - Ghirlando, Rodolfo
AU - Yamaguchi, Masato
AU - Muramoto, Koji
N1 - Funding Information:
We thank C. Broder for the CCR5-encoding recombinant vaccinia virus, I. Hamachi for Manα(1-2)Manα-containing trisaccharides, M. Clore & D. Williams for helpful discussions, and the Intramural AIDS Targeted Antiviral Program of the Office of the Director, NIH (C.A.B.) for financial support.
PY - 2004/6/11
Y1 - 2004/6/11
N2 - Carbohydrate-binding proteins that bind their carbohydrate ligands with high affinity are rare and therefore of interest because they expand our understanding of carbohydrate specificity and the structural requirements that lead to high-affinity interactions. Here, we use NMR and isothermal titration calorimetry techniques to determine carbohydrate specificity and affinities for a novel cyanobacterial protein, MVL, and show that MVL binds oligomannosides such as Man6GlcNAc2 with sub-micromolar affinities. The amino acid sequence of MVL contains two homologous repeats, each comprising 54 amino acid residues. Using multi-dimensional NMR techniques, we show that MVL contains two novel carbohydrate recognition domains composed of four non-contiguous regions comprising ∼15 amino acid residues each, and that these residues make numerous intermolecular contacts with their carbohydrate ligands. NMR screening of a comprehensive panel of di-, tri-, and high-mannose oligosaccharides establish that high-affinity binding requires at least the presence of a discrete conformation presented by Manβ(1→4)GlcNAc in the context of larger oligomannosides. As shown by sedimentation equilibrium and gel-filtration experiments, MVL is a monodisperse dimer in solution, and NMR data establish that the three-dimensional structure must be symmetric. MVL inhibits HIV-1 Envelope-mediated cell fusion with an IC50 value of ∼30nM.
AB - Carbohydrate-binding proteins that bind their carbohydrate ligands with high affinity are rare and therefore of interest because they expand our understanding of carbohydrate specificity and the structural requirements that lead to high-affinity interactions. Here, we use NMR and isothermal titration calorimetry techniques to determine carbohydrate specificity and affinities for a novel cyanobacterial protein, MVL, and show that MVL binds oligomannosides such as Man6GlcNAc2 with sub-micromolar affinities. The amino acid sequence of MVL contains two homologous repeats, each comprising 54 amino acid residues. Using multi-dimensional NMR techniques, we show that MVL contains two novel carbohydrate recognition domains composed of four non-contiguous regions comprising ∼15 amino acid residues each, and that these residues make numerous intermolecular contacts with their carbohydrate ligands. NMR screening of a comprehensive panel of di-, tri-, and high-mannose oligosaccharides establish that high-affinity binding requires at least the presence of a discrete conformation presented by Manβ(1→4)GlcNAc in the context of larger oligomannosides. As shown by sedimentation equilibrium and gel-filtration experiments, MVL is a monodisperse dimer in solution, and NMR data establish that the three-dimensional structure must be symmetric. MVL inhibits HIV-1 Envelope-mediated cell fusion with an IC50 value of ∼30nM.
KW - HIV-1 envelope-mediated fusion
KW - HIV-1, human immunodeficiency virus type 1
KW - ITC, isothermal titration calorimetry
KW - NMR chemical shift mapping
KW - carbohydrate binding proteins
KW - gp120
KW - high-mannose oligosaccharides
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U2 - 10.1016/j.jmb.2004.04.019
DO - 10.1016/j.jmb.2004.04.019
M3 - Article
C2 - 15165858
AN - SCOPUS:2542433994
VL - 339
SP - 901
EP - 914
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -