New carbohydrate specificity and HIV-1 fusion blocking activity of the cyanobacterial protein MVL: NMR, ITC and sedimentation equilibrium studies.
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 Man(6)GlcNAc(2) 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 approximately 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 Manbeta(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 IC(50) value of approximately 30 nM.
Bewley
Carole A
CA
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. carole_bewley@nih.gov
Cai
Mengli
M
Ray
Satyajit
S
Ghirlando
Rodolfo
R
Yamaguchi
Masato
M
Muramoto
Koji
K
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002151:Calorimetry; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D000458:Cyanobacteria / Q000737:chemistry*; D015497:HIV-1 / Q000187:drug effects* / Q000502:physiology; D008561:Membrane Fusion / Q000187:drug effects*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding
D001426:Bacterial Proteins; D002241:Carbohydrates