Insights from NMR Spectroscopy into the Conformational Properties of Man-9 and Its Recognition by Two HIV Binding Proteins

Abstract

Man₉ GlcNAc₂ (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D ¹³ C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of ¹³ C,¹⁵ N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.

Keywords: NMR spectroscopy; carbohydrate recognition; glycan conformation; isotopic labeling; molecular dynamics.

Figure 1

NMR data for Man₉GlcNAc₂ (Man-9). a) Symbol representation of Man-9 showing nomenclature for individual rings, glycosidic linkages and the D1, D2 and D3 arms; b) ¹H-¹³C HSQC-TOCSY spectrum of unlabeled Man-9 versus c) HCCH-TOCSY spectrum of ¹³C-labeled Man-9 showing TOCSY correlations from H1 through H6/6′ for each pyranose ring. (See Figure S3, Supporting Information, for an expansion.) d) Glycosidic torsion angle ω (O5′-C5′-C6′-O) of Man3 and Man4′ rings (shown in blue) were determined from NOEs and scalar couplings: in e) overlay of HSQC-NOESY (blue) and HSQC (red) spectra of Man-9 showing NOEs of equal intensities from H5 of Man4′ to H6 and H6′ indicating an anti conformation for H5-O6, and respective dihedral angles of +60 and −60 for H5–H6 and H5–H6′. f) High-resolution (1.7 Hz/point, Figures S4–5, Supporting Information) HSQC spectrum showing two large H-H couplings for H4, which is anti to H3 and H5, compared to the large geminal and small gauche couplings for H6/6′ of Man4 and Man4′ consistent with dihedral angles determined by NOEs.

Figure 2

NMR structure and MD simulations of Man-9. a) Key inter-ring NOEs used for structure calculations. Strong, medium and weak NOEs are designated by bold, regular and dashed lines, respectively. b) NMR ensemble of 30 structures calculated by Xplor-NIH using NOE derived distance restraints, and NOE- and J coupling-derived dihedral angle restraints. c) Temporal analysis comparing distances observed in 1.6 μs simulations of Man-9 in explicit solvent versus NOE-derived distances. NOE-assigned distances appear along the x-axis and simulation time in nsec along the y-axis. |Δ_NOE| is defined as the absolute difference between the distance measured during the free simulation and the NOE-computed distance (used as restraints in solving the free Man-9 NMR structure). d) Distributions of the glycosidic ω torsion angles for the unrestrained MD simulation. ω, φ, ψ torsion angles were defined as O-C6′-C5′-O5′, H1-C1-O- C6′ and C1-O- C6′-C5′, respectively.

Figure 3

Binding site, stoichiometry and modes of binding of Man-9 and terminal trisaccharides to MVN. a) Overlay of ¹H-¹⁵N HSQC spectra of free MVN (black) and MVN in the presence of stoichiometric amounts of Man-9 (red). b–e) Expansion of Asn55 resonance showing different effects on MVN in the presence of (d) D2 and (e) D3 arm trisaccharides compared to (b) Man-9 and (c) the D1 arm trisaccharide. f) Surface representation of MVN showing the Man-9 D1 arm binding site (blue) is more extensive than Manα1–2Man (pink surface). Full description is shown in Figure S8, Supporting Information.

Figure 4

NMR-determined conformation of MVN-bound Man-9 and principal component analysis of receptor-bound Man-9 structures. a) NOESY spectra of free (left) and MVN-bound (right) Man-9 showing minor differences in NOE patterns. b) NMR ensemble of Man-9 in the presence of MVN. c) Superposition of restrained minimized, mean NMR structures of Man-9 (blue) and MVN-bound Man-9 (green). d) PCA of MD-observed and NMR-derived glycosidic torsion angles.

Figure 5

Man-9 binding to DC-SIGN. a) Anomeric region of HSQC spectrum of ¹³C-Man-9 free (blue) and in a 1:1 complex with DC-SIGN (red). Resonances that disappear upon DC-SIGN binding are labeled. b) Region of Man-9 in contact with DC-SIGN in solution.