Our earlier work on HIV-1 protease dynamics (Foulkes-Murzycki et. al., 2007) showed that the hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may be a mechanism by which the HIV-1 protease undergoes conformational changes that are required for natural substrate recognition and inhibitor binding. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease. Therefore these residues impact the dynamic balance between processing substrates and binding inhibitors, and thus contribute to drug resistance.
To further test the role of flexibility in this balance, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links.