Atomic Mapping of the Sugar Interactions in One-Site and Two-Site Mutants of Cyanovirin-N by NMR Spectroscopy

Abstract

The details of the interaction between two mutants of Cyanovirin-N (CV-N), an HIV inactivating protein, and di- and trimannosides, substructures of Man-9, were investigated by STD NMR spectroscopy. One mutant, CV-N (mutDB), contains only one carbohydrate-binding site on domain A, whereas in CV-N (mutDA), the specificity of domain A for trimannose was changed while the site in domain B was kept intact, allowing for a dissection of the overall binding. Results of the STD NMR experiments revealed close contact between the protein binding site on domain A and H2, H3, and H4 of the nonreducing terminal mannose unit for Manalpha(1-2)Manalpha OMe, Manalpha(1-2)Manalpha(1-3)Manalpha OMe, and Manalpha(1-2)Manalpha(1-6)Manalpha OMe. The Manalpha(1-2)Manalpha(1-2)Manalpha OMe trisaccharide interacted with CV-N with the highest affinity. Further dissection of the interaction was achieved by NMR experiments with synthetic 2'-, 3'-, 4'-, and 6'-deoxy analogues of the disaccharide Manalpha(1-2)Manalpha OMe. STD and (1)H- (15)N HSQC NMR spectroscopy revealed that the 2'- and 6'-deoxy dimannosides were recognized by CV-N, whereas no binding was detected for the 3'- and 4'-deoxy sugars. These results demonstrate that the 3'- and 4'-hydroxyl groups on the terminal residue are engaged in key polar interactions with the protein and are required for high-affinity binding.

Longer simulations sample larger subspaces of conformations while maintaining robust mechanisms of motion

“…42–45 We compared the dynamic information retrieved from 1 ns to 400 ns MD runs. As depicted in the Fig.…”

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

“…18,19 Earlier STD NMR studies with Man 3 resulted in low STD signals, 20 and STD NMR with Man 3 for single binding site mutants did not yield STD signals because the off-rate was too small. 21 NMR studies on a 19 F-labeled Man 3 revealed a single binding mode on domain A, but two binding modes in the binding site of domain B. 22 Previous work on CV-N mannose binding primarily used the disaccharide Manα(1–2)Manα.…”

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

“…[34] These results prompted us to proceed to studying the interaction with the lectin by 19 F-NMR spectroscopy.…”

“…[28,31,32] The availability of detailed STD data for these ligands as well as results with specific deoxy sugars allowed us to identify the pivotal hydroxyl groups responsible for binding. [33,34] In particular, we determined that the 3′- and 4′-hydroxyl groups are essential for the interaction, whereas the 2′- and 6′-hydroxyl groups of the Manα(1–2)Manα (Man2) terminal unit are not directly involved in CV-N binding. [34] We therefore prepared methyl 2-deoxy-2-fluoro-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside ( 19 F-Manα(1–2)Manα: 19 F-Man2) and methyl 2-deoxy-2-fluoro-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside ( 19 F-Manα(1–2)Manα(1–2)Manα: 19 F-Man3) (Supplementary Scheme S1) for use in our current work.…”