In spite of the abundance of glycoproteins in biological processes, relatively little three-dimensional structural data is available for glycan structures. Here, we study the structure and flexibility of the vast majority of mammalian oligosaccharides appearing in N- and O-glycosylated proteins using a bottom up approach. We report the conformational free-energy landscapes of all relevant glycosidic linkages as obtained from local elevation simulations and subsequent umbrella sampling. To the best of our knowledge, this represents the first complete conformational library for the construction of N- and O-glycan structures. Next, we systematically study the effect of neighboring residues, by extensively simulating all relevant trisaccharides and one tetrasaccharide. This allows for an unprecedented comparison of disaccharide linkages in large oligosaccharides. With a small number of exceptions, the conformational preferences in the larger structures are very similar as in the disaccharides. This, finally, allows us to suggest several efficient approaches to construct complete N- and O-glycans on glycoproteins, as exemplified on two relevant examples.

Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N-glycan modifications of IgA1 and IgA2 on binding to the Fcα receptor (FcαRI), which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1), and IgA2m(2) variants in Nicotiana benthamiana ΔXT/FT plants lacking the enzymes responsible for generating nonhuman N-glycan structures. By coinfiltrating IgA with the respective glycan-modifying enzymes, we generated IgA carrying distinct homogenous N-glycans. We demonstrate that distinctly different N-glycan profiles did not influence antigen binding or the overall structure and integrity of the IgA antibodies but did affect their thermal stability. Using size-exclusion chromatography, differential scanning and isothermal titration calorimetry, surface plasmon resonance spectroscopy, and molecular modeling, we probed distinct IgA1 and IgA2 glycoforms for binding to four different FcαRI glycoforms and investigated the thermodynamics and kinetics of complex formation. Our results suggest that different N-glycans on the receptor significantly contribute to binding affinities for its cognate ligand. We also noted that full-length IgA and FcαRI form a mixture of 1:1 and 1:2 complexes tending toward a 1:1 stoichiometry due to different IgA tailpiece conformations that make it less likely that both binding sites are simultaneously occupied. In conclusion, N-glycans of human IgA do not affect its structure and integrity but its thermal stability, and FcαRI N-glycans significantly modulate binding affinity to IgA.

Targeted glycosylation of recombinant proteins may provide an advantage in therapeutic applications. Despite increasing biotechnological interest in IgE antibodies, knowledge and impact of glycosylation on this antibody class are scarce. With the ability to glyco-engineer recombinant IgE, we provide an important step towards the generation of IgE with other targeted N-glycans. This will facilitate detailed structure-function studies and may lead to the production of IgE with optimized activities.

The binding energies for cation-π complexation are underestimated by traditional fixed-charge force fields owing to their lack of explicit treatment of ion-induced dipole interactions. To address this deficiency, explicit treatment of cation-π interactions has been introduced into the OPLS-AA force field. Following prior work with atomic cations, it is found that cation-π interactions can be handled efficiently by augmenting the usual 12-6 Lennard-Jones potentials with 1/r 4 terms. Results are provided for prototypical complexes as well as protein-ligand systems of relevance for drug design. Alkali cation, ammonium, guanidinium, and tetramethylammonium were chosen for the representative cations, while benzene and six heteroaromatic molecules were used as the π systems. The required non-bonded parameters were fit to reproduce structures and interactions energies for gas-phase complexes from DFT calculations at the ωB97X-D/6-311++G(d,p) level. The impact of solvent was then examined by computing potentials of mean force (pmfs) in both aqueous and THF solutions using free-energy perturbation (FEP) theory. Further testing was carried out for two cases of strong and one case of weak cation-π interactions between drug-like molecules and their protein hosts, namely, the JH2 domain of JAK2 kinase and macrophage migration inhibitory factor. FEP results reveal greater binding by 1.5 -4.4 kcal/mol from addition of the explicit cation-π contributions. Thus, in the absence of such treatment of cation-π interactions, errors for computed binding or inhibition constants of 10 1 −10 3 are expected.

By using molecular dynamics simulations with an efficient enhanced sampling technique and in combination with nuclear magnetic resonance (NMR) spectroscopy quantitative structural information on α -2,8-linked sialic acids is presented. We used a bottom-up approach to obtain a set of larger ensembles for tetra- and deca-sialic acid from model dimer and trimer systems that are in agreement with the available J-coupling constants and nuclear Overhauser effects. The molecular dynamic (MD) simulations with enhanced sampling are used to validate the force field used in this study for its further use. This empowered us to couple NMR observables in the MD framework via J-coupling and distance restraining simulations to obtain conformations that are supported by experimental data. We used these conformations in thermodynamic integration and one-step perturbation simulations to calculate the free-energy of suggested helical conformations. This study brings most of the available NMR experiments together and supplies information to resolve the conflict on the structures of poly- α -2,8-linked sialic acid.