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Solution NMR analyses of the C-type carbohydrate recognition domain of DC-SIGNR protein reveal different binding modes for HIV-derived oligosaccharides and smaller glycan fragments.

ABSTRACT: The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)2Man3; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man9GlcNAc2, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man3, Man5, and (GlcNAc)2Man3 were investigated alongside Man9GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man9GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). (15)N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces.

SUBMITTER: Probert F

PROVIDER: S-EPMC3829359 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:'In dopaminergic neurons, alpha-synuclein (alphaS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of alphaS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which alphaS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of alphaS. Exchange between the free state and the lipid-bound alphaS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s(-1). Partitioning of the binding modes is dependent on lipid/alphaS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of alphaS adopts an alpha-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound alphaS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large alpha-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this "dark state." Titration of alphaS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where alphaS lies in an alpha-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by alphaS.

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Solution NMR analyses of the C-type carbohydrate recognition domain of DC-SIGNR protein reveal different binding modes for HIV-derived oligosaccharides and smaller glycan fragments.

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