Atomic mapping of the sugar interactions in one-site and two-site mutants of cyanovirin-N by NMR spectroscopy.
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.
Sandström
Corine
C
Department of Chemistry, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden. corine.sandstrom@kemi.slu.se
Hakkarainen
Birgit
B
Matei
Elena
E
Glinchert
Anja
A
Lahmann
Martina
M
Oscarson
Stefan
S
Kenne
Lennart
L
Gronenborn
Angela M
AM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D018604:Epitope Mapping; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009844:Oligosaccharides; C028569:oligomannoside; C107733:cyanovirin N