The HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.
Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulphonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5-30 °C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a ΔC p of −80 ± 4 cal K −1 mol −1 indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.
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