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.

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

“…3A [see SI Appendix for NMR assignment (SI Appendix, Table S1)]. To precisely map ligand epitopes in close contact with the protein, we acquired STD build up curves by collecting spectra at different saturation times (25)(26)(27)(28) and fitting experimental data with the monoexponential equation: STD = STD max [1 -exp (−k sat t)] (see Materials and Methods, NMR Spectroscopic Analysis for complete details). This method avoids artifacts in the epitope definition, which are consequences of differences in ability to accumulate saturation in the free state, allowing to obtain the real STD effect of each proton, independently of T1 bias, and to prevent any misinterpretation of STD enhancements.…”

Protein-Carbohydrate Interactions Studied by NMR: From Molecular Recognition to Drug Design

“…Using phage display, a weakly immunogenic carbohydrate-mimetic peptide with the amino acid sequence MDWNMHAA, of the S. flexneri Y O-linked polysaccharide, was identified to be cross-reactive with the SYA/J6 monoclonal antibody [78] STD-NMR combined with CORCEMA-ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) analysis were used to probe the bioactive solution conformation of the carbohydrate mimic MDWNMHAA when bound to mAb SYA/J6. [79] …”

Specificity of a UDP‐GalNAc Pyranose–Furanose Mutase: A Potential Therapeutic Target for Campylobacter jejuni Infections

“…[16][17][18][19][20] All of the conserved residues noted as important for enzyme activity [19,20] are located in the same positions in cjUNGM as in the other structures, with the noted exception of the residues in the mobile loop. Given that we have been unsuccessful in obtaining a cocrystal of the enzyme bound to a substrate, we used STD-NMR spectroscopy in conjunction with molecular dynamics and CORCEMA-ST calculations [21][22][23][24][25][26] to provide a model of the complex structures.…”

“…Yet, STD intensities alone cannot directly afford substrate binding modes, and therefore we resorted to MD simulations in conjunction with CORCEMA-ST calculations to develop a semiquantitative model of substrate recognition. [21][22][23][24][25][26] Molecular dynamics studies of binding modes…”