Cryptococcus neoformans is a fungal pathogen that is the causative agent of cryptococcosis and cryptococcal meningitis. It is encapsulated by a polysaccharide (PS) which functions to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of the capsular PS is being explored as a conjugate vaccine to protect against C. neoformans infection. In this combined NMR and Molecular Dynamics (MD) study, we delineate the structure of a synthetic decasaccharide derived from a C. neoformans capsular PS. The synthetic O-acetylated decasaccharide alleviates the NMR complications of the capsular PS which arise from signal degeneracy and sample heterogeneity. Thus, we used NMR techniques that enhance spectral resolution to fully assign an O-acetylated C. neoformans synthetic decasaccharide mimetic. The synthetic decasaccharide is composed of a five residue α-1,3 linked mannose (Man) backbone, substituted with a β-1,2- linked glucuronic acid (GlcA) on the third Man and β-1,2 linked xylose (Xyl) on all other Man. Man I, V, and VII are O-acetylated at C6. We used 1H-1H NOE data and transglycosidic J-values to determine the O-acetylated decasaccharide structure. Our experimental data were complemented by three distinct 5 µs MD simulations on an α-1,3-linked pentamannose, a de-O-acetylated decasaccharide, and a decasaccharide. NMR and MD data agree that the mannose backbone is in a single (semi-rigid) conformation and the xylose branches are found on alternating sides of the Man backbone. The Xyl and GlcA branches are in two main confirmations “face-on,” where the plane of the ring is aligned with the Man backbone, and “edge-on,” in which the plane of the ring is oriented perpendicular to the Man backbone. Determining the decasaccharide structure provides a foundation for future structural studies aimed at elucidating the conformation of the decasaccharide recognized by C. neoformans protective antibodies.
