Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G′ loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B′-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9

Recent advances in the application of NMR methodologies to analyze the conformation, dynamics, and interactions of saccharides

25 p.-7 fig. Carbohydrate Chemistry: Chemical and Biological Approaches.Volume 44. Editors: Amélia Pilar Rauter, Thisbe K Lindhorst, Yves QueneauThis chapter is dedicated to the presentation of different examples of the application of solution NMR to the study of conformation, dynamics of sugar molecules (oligo and polysaccharides, glycopeptides and glycomimetics) including the investigation of glycanrelated molecular recognition events. It is not our intention to be exhaustive, rather to highlight diverse examples since 2016. They are presented depending on the chemical nature of the saccharide molecule, and on the different methodologies that can be employed.We thank MINECO (Spain, Projects) and the European Union (Project RECGLYCANMR and BACTIVAX) for financial support. AF also thanksMINECO for Juan de la Cierva contract.Peer reviewe