Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy

Abstract

NMR of a uniformly ¹³C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the ¹³C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the ¹³C spectral dispersion of ¹³C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

Figure 1

(a) Structural formula of Man₃ (Manα(1–2)Manα(1–2)ManαOMe). (b) Anomeric region and (c) ring proton region of the ¹H,¹³C-CT-HSQC spectrum of free Man₃ (red/magenta) superimposed on the ¹H,¹³C-CT-HSQC spectrum of CV-N-bound Man₃ (blue/cyan). An unidentified cross-peak is marked with an asterisk (*).

Figure 2

(a) ¹H NMR spectrum of Man₃ in D₂O buffer. (b) Selected region of the ¹H,¹³C-CT-HSQC spectrum of Man₃ bound to CV-N. (c) 1D ¹H,¹H-DPFGSE-T-ROESY spectrum of Man₃ with selective excitation on H1″ and a mixing time of 300 ms. (d) Selected region of a ¹H,¹³C-HSQC-NOESY spectrum of CV-N-bound Man₃ with a mixing time of 60 ms. (e-f) PANIC plots of I_j/I_i versus τ_mix, where I_j is the intensity of the NOE peak, -I_i is the intensity of the target peak (selective experiments) and I_i is the average intensity of the diagonal peaks (2D experiments). Plots are shown for H1″-H2 (red squares), H1′-H2″ (blue circles), and H1-H1″ (green diamonds) of (e) free and (f) bound Man₃. Cross-relaxation rates were obtained from the slopes of the lines.

Figure 3

(a) Superposition of the 2D version of the ¹³C-filtered NOESY-HSQC (red, 80 ms mixing time) and the ¹H,¹³C-CT-HSQC (grey) spectra of the CV-N/Man₃ complex. (b) Side-chain region of the ¹H,¹³C-HSQC-NOESY spectrum of the CV-N/Man₃ complex (60 ms mixing time). Intermolecular NOEs between sugar ring protons (correlated through ¹³C in f1) and protein side-chain protons are labeled.

Figure 4

(a) Amide proton region of the ¹H,¹³C-HSQC-NOESY spectrum (60 ms mixing time) of the CV-N/Man₃ complex. Intermolecular NOEs between sugar ring protons and protein amide protons are labeled. (b) ¹H,¹⁵N 2D version of the CNH-NOESY experiment (red, 20 ms mixing time) superimposed onto a ¹H,¹⁵N-HSQC spectrum (grey). Amide proton resonances exhibiting NOEs to ¹³C-attached sugar ring protons are connected by dashed lines.