The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to man8 D1D3 and Man9 with nanomolar affinity:: Implications for binding to the HIV envelope protein gp120

Cyanovirin-N (CVN) is a monomeric II kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of ManpGlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K-a) of 2.4 (+/- 0.1) x 10(7) M-1 and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) DID3 acts as a divalent ligand (2 CVN:I Man(8)) with a K-a of 5.4 (+/- 0.5) x 10(7) M-1, and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K-a of 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 indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose 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-Iabeled 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 binding sites are presented, and implications for binding to gp120 are discussed.