title: Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations.
abstract: Saturation transfer difference (STD)-NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O-polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD™ Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA-ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD-NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor-ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R(eff), was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody-bound solution conformation of the peptide which also differed from the bound conformation determined by X-ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD-NMR intensity-restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O-polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build-up, which, in turn, hindered a rigorous analysis via full STD build-up curves.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.
authors.forename: Monica G
authors.initials: MG
authors.lastname: Szczepina
authors.forename: Dustin W
authors.initials: DW
authors.lastname: Bleile
authors.forename: B Mario
authors.initials: BM
authors.lastname: Pinto
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012763:Shigella flexneri / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000906:Antibodies; D008024:Ligands; D010455:Peptides