Solution Structure of a Cyanovirin-N:Manα1-2Manα Complex: Structural Basis for High-Affinity Carbohydrate-Mediated Binding to gp120

Abstract

Background: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein that potently inhibits viral entry by diverse strains of HIV through high-affinity carbohydrate-mediated interactions with the viral envelope glycoprotein gp120. CVN contains two symmetry-related carbohydrate binding sites of differing affinities that selectively bind to Man 8 D1D3 and Man 9 with nanomolar affinities, the carbohydrates that also mediate CVN:gp120 binding. High-resolution structural studies of CVN in complex with a representative oligosaccharide are desirable for understanding the structural basis for this unprecedented specificity. Results: We have determined by multidimensional heteronuclear NMR spectroscopy the three-dimensional solution structure of CVN in complex with two equivalents of the disaccharide Manα1-2Manα, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man 8 D1D3 and Man 9. The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Man 8 D1D3 and Man 9 over other oligomannose structures, and presents two novel carbohydrate binding sites that account for the differing affinities of the two sites. The high-affinity site comprises a deep pocket that nearly envelops the disaccharide, while the lower-affinity site comprises a semicircular cleft that partially surrounds the disaccharide. The ∼40 Å spacing of the two binding sites provides a simple model for CVN:gp120 binding. Conclusions: The CVN:Manα1-2Manα complex provides the first high-resolution structure of a mannose-specific protein-carbohydrate complex with nanomolar affinity and presents a new carbohydrate binding motif, as well as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.