Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

We analysed the tailspike from bacteriophage Sf6 in complex with the O-polysaccharide of the pathogen Shigella flexneri. The conformational space populated by the polyrhamnose backbone of the S. flexneri O-polysaccharide as studied by an octasaccharide in complex with Sf6TSP could be well described with 2D 1H,1H-trNOESY NMR, utilizing a combination of methine-methine and methine-methyl correlations. The results are in good agreement with the conformations obtained from molecular dynamics (MD) simulations. To examine the impact of amino acid exchanges in the glycan binding site of Sf6TSP, MD simulations were used to predict increased O-polysaccharide binding affinities. We used surface plasmon resonance on S. flexneri O-polysaccharide surfaces to measure affinity increases in the obtained mutants. <br /

Conformational Dynamics and Exchange Kinetics of <i>N</i>‑Formyl and <i>N</i>‑Acetyl Groups Substituting 3‑Amino-3,6-dideoxy-α‑d‑galactopyranose, a Sugar Found in Bacterial O‑Antigen Polysaccharides

Three dimensional shape and conformation of carbohydrates are important factors in molecular recognition events and the <i>N</i>-acetyl group of a monosaccharide residue can function as a conformational gatekeeper whereby it influences the overall shape of the oligosaccharide. NMR spectroscopy and quantum mechanics (QM) calculations are used herein to investigate both the conformational preferences and the dynamic behavior of <i>N</i>-acetyl and <i>N</i>-formyl substituents of 3-amino-3,6-dideoxy-α-d-galactopyranose, a sugar and substitution pattern found in bacterial O-antigen polysaccharides. QM calculations suggest that the amide oxygen can be involved in hydrogen bonding with the axial OH4 group primarily but also with the equatorial OH2 group. However, an NMR <i>J</i> coupling analysis indicates that the θ₁ torsion angle, adjacent to the sugar ring, prefers an <i>ap</i> conformation where conformations <180° also are accessible, but does not allow for intramolecular hydrogen bonding. In the formyl-substituted compound ⁴<i>J</i>_HH coupling constants to the <i>exo</i>-cyclic group were detected and analyzed. A van’t Hoff analysis revealed that the <i>trans</i> conformation at the amide bond is favored by Δ<i>G</i>° ≈ – 0.8 kcal·mol^–1 in the formyl-containing compound and with Δ<i>G</i>° ≈ – 2.5 kcal·mol^–1 when the <i>N</i>-acetyl group is the substituent. In both cases the enthalpic term dominates to the free energy, irrespective of water or DMSO as solvent, with only a small contribution from the entropic term. The <i>cis</i>–<i>trans</i> isomerization of the θ₂ torsion angle, centered at the amide bond, was also investigated by employing ¹H NMR line shape analysis and ¹³C NMR saturation transfer experiments. The extracted transition rate constants were utilized to calculate transition energy barriers that were found to be about 20 kcal·mol^–1 in both DMSO-<i>d</i>₆ and D₂O. Enthalpy had a higher contribution to the energy barriers in DMSO-<i>d</i>₆ compared to in D₂O, where entropy compensated for the loss of enthalpy

Molecular Dynamics Simulations of Membrane–Sugar Interactions

It is well documented that disaccharides in general and trehalose (TRH) in particular strongly affect physical properties and functionality of lipid bilayers. We investigate interactions between lipid membranes formed by 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) and TRH by means of molecular dynamics (MD) computer simulations. Ten different TRH concentrations were studied in the range <i>w</i>_TRH = 0–0.20 (w/w). The potential of mean force (PMF) for DMPC bilayer–TRH interactions was determined using two different force fields, and was subsequently used in a simple analytical model for description of sugar binding at the membrane interface. The MD results were in good agreement with the predictions of the model. The net affinities of TRH for the DMPC bilayer derived from the model and MD simulations were compared with experimental results. The area per lipid increases and the membrane becomes thinner with increased TRH concentration, which is interpreted as an intercalation effect of the TRH molecules into the polar part of the lipids, resulting in conformational changes in the chains. These results are consistent with recent experimental observations. The compressibility modulus related to the fluctuations of the membrane increases dramatically with increased TRH concentration, which indicates higher order and rigidity of the bilayer. This is also reflected in a decrease (by a factor of 15) of the lateral diffusion of the lipids. We interpret these observations as a formation of a glassy state at the interface of the membrane, which has been suggested in the literature as a hypothesis for the membrane–sugar interactions

Stochastic Modeling of Flexible Biomolecules Applied to NMR Relaxation. 2. Interpretation of Complex Dynamics in Linear Oligosaccharides

A computational stochastic approach is applied to the description of flexible molecules. By combining (i) molecular dynamics simulations, (ii) hydrodynamics approaches, and (iii) a multidimensional diffusive description for internal and global dynamics, it is possible to build an efficient integrated approach to the interpretation of relaxation processes in flexible systems. In particular, the model is applied to the interpretation of nuclear magnetic relaxation measurements of linear oligosaccharides, namely a mannose-containing trisaccharide and the pentasaccharide LNF-1. Experimental data are reproduced with sufficient accuracy without free model parameters

Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. ¹⁵N and ²H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy