Solution structure of a cyanovirin-N : Man alpha 1-2Man alpha complex: Structural basis for high-affinity carbohydrate-mediated binding to gp120

Background: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein th at potently inhibits viral entry by diverse strains of HIV through high-aff inity carbohydrate-mediated interactions with the viral envelope glycoprote in gp120. CVN contains two symmetry-related carbohydrate binding sites of d iffering affinities that selectively bind to Man(8) D1 D3 and Man(9) with n anomolar 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 t his unprecedented specificity. Results: We have determined by multidimensional heteronuclear NMR spectrosc opy the three-dimensional solution structure of CVN in complex with two equ ivalents 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 an d Man(9) over other oligomannose structures, and presents two novel carbohy drate binding sites that account for the differing affinities of the two si tes. The high-affinity site comprises a deep pocket that nearly envelops th e disaccharide, while the lower-affinity site comprises a semicircular clef t that partially surrounds the disaccharide. The similar to 40 Angstrom spa cing 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 wi th nanomolar affinity and presents a new carbohydrate binding motif, as wel l as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.