Solution structure of a cyanovirin-N:Manα1-2Manα complex:: Structural basis for high-affinity carbohydrate-mediated binding to gp120

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) D1 D3 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 Man alpha1-2Man alpha, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man(8) D1 D3 and Man(9). The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Mans D1 D3 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 similar to 40 Angstrom spacing of the two binding sites provides a simple model for CVN:gp120 binding. Conclusions: The CVN:Man alpha1-2Man alpha. 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.