The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to man(8) D1D3 and Man(9) with nanomolaraffinity: Implications for binding to the HIV envelope protein gp120

Cyanovirin-N (CVN) is a monomeric II kDa cyanobacterial protein that potent ly inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surf ace envelope glycoprotein gp120. Several lines of evidence have suggested t hat CVN-gp120 interactions are in part mediated by N-linked complex carbohy drates present on gp120, but experimental evidence has been lacking. To thi s end we screened a comprehensive panel of carbohydrates which represent st ructurally the N-linked carbohydrates found on gp120 for their ability to i nhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope -mediated cell fusion assay. Our results show that CVN specifically recogni zes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of ManpGlcN Ac(2). Nonlinear least squares best fitting of titration data generated usi ng the cell fusion assay show that CVN binds to gp120 with an equilibrium a ssociation constant (K-a) of 2.4 (+/- 0.1) x 10(7) M-1 and an apparent stoi chiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) DID3 acts as a dival ent ligand (2 CVN:I Man(8)) with a K-a of 5.4 (+/- 0.5) x 10(7) M-1, and Ma n(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K-a o f 1.3 (+/- 0.3) x 10(8) M-1 Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K-a = 1.5 (+/- 0.9) x 10(8) M-1), and the fitted data i ndicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1 :1 stoichiometry at micromolar concentrations suggested that CVN has not on ly a high affinity binding site-relevant to the studies at nM concentration s-but a lower affinity site as well that facilitates cross-linking of CVN-o ligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Mans over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Man alpha1 - -> 2Man alpha. By following the H-1-N-15 correlation spectrum of N-15-Iabel ed CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Man alpha1 --> 2Mana via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K, of 7.2 (+/- 4) x 10(6) M(- )1 and the low affinity site a kj of 6.8 (+/- 4) x 10(5) RI-l as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate b inding sites are presented, and implications for binding to gp120 are discu ssed.