Molecular Conformations in the Pentasaccharide LNF-1 Derived from NMR Spectroscopy and Molecular Dynamics Simulations

Abstract

The conformational dynamics of the human milk oligosaccharide lacto-N-fucopentaose (LNF-1), α-L-Fucp-(1 → 2)-β-D-Galp-(1 → 3)-β-D-GlcpNAc-(1 → 3)-β-D-Galp-(1 → 4)-D-Glcp, has been analyzed using NMR spectroscopy and molecular dynamics (MD) computer simulations. Employing the Hadamard (13)C-excitation technique and the J-HMBC experiment, (1)H,(13)C trans-glycosidic J coupling constants were obtained, and from one- and two-dimensional (1)H,(1)H T-ROESY experiments, proton-proton cross-relaxation rates were determined in isotropic D(2)O solution. In the lyotropic liquid-crystalline medium consisting of ditetradecylphosphatidylcholine, dihexylphosphatidylcholine, N-cetyl-N,N,N-trimethylammonium bromide, and D(2)O, (1)H, (1)H and one-bond (1)H, (13)C residual dipolar couplings (RDCs), as well as relative sign information on homonuclear RDCs, were determined for the pentasaccharide. Molecular dynamics simulations with explicit water were carried out from which the internal isomerization relaxation time constant, τ(N), was calculated for transitions at the ψ torsion angle of the β-(1 → 3) linkage to the lactosyl group in LNF-1. Compared to the global reorientation time, τ(M), of ∼0.6 ns determined experimentally in D(2)O solution, the time constant for the isomerization relaxation process, τ(N(scaled)), is about one-third as large. The NMR parameters derived from the isotropic solution show very good agreement with those calculated from the MD simulations. The only notable difference occurs at the reducing end, which should be more flexible than observed by the molecular simulation, a conclusion in complete agreement with previous (13)C NMR relaxation data. A hydrogen-bond analysis of the MD simulation revealed that inter-residue hydrogen bonds on the order of ∼30% were present across the glycosidic linkages to sugar ring oxygens. This finding highlights that intramolecular hydrogen bonds might be important in preserving well-defined structures in otherwise flexible molecules. An analysis including generalized order parameters obtained from nuclear spin relaxation experiments was performed and successfully shown to limit the conformational space accessible to the molecule when the number of experimental data are too scarce for a complete conformational analysis.

Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition

“…However, in contrast to proteins, glycans display large internal motions that prevents their accurate description by any single 3D shape 20,21 . Fortunately, MD simulations allow accurate prediction of the 3D shapes and motions of glycans, as confirmed by comparison to solution NMR data [22][23][24] , and such simulations have been widely applied to glycoproteins [25][26][27][28][29] .…”

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

“…Therefore, knowledge of the structure and dynamics of N-glycans is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. An oligosaccharide chain is flexible in solution and has an ensemble of diverse conformations rather than a single well-defined structure [18]–[20]. The inherent flexibility of oligosaccharides often hinders crystallographic structure determination, and there are only a few crystal structures of oligosaccharides longer than 2–3 residues in the Cambridge Structure Database [21].…”

Recognition of Dimeric Lewis X by Anti-Dimeric Lex Antibody SH2

“…Extended epitope presentation from the non-reducing end Le x trisaccharide in DimLe x to the mAb SH2 binding site should also be considered in the context of the β- d -Glc N Ac-(1→3)- d -Gal glycosidic bond conformation. Indeed, this glycosidic bond, that links the non-reducing end Le x trisaccharide to the reducing end Le x moiety in DimLe x ( 6 ) or to galactose in Lex[1,3]Gal (9), has been shown to be highly flexible in various oligosaccharides [ 60 , 61 , 62 , 63 , 64 , 65 , 66 ]. Conformations around glycosidic bonds are defined by two dihedral angles: Φ (O5-C1-O1-Cx) and Ψ (C1-O1-Cx + 1).…”