Based on computational predictions and subsequent experimental validation, we identified the spatially clustered hot spot residues I688, Y689, I692, and L696 in the Hsp90 CTD interface, which are located on α-helix H5, form a functional epitope, and account for most of the protein dimerization energy. Furthermore, conformational analysis by 2D NMR and MD simulations revealed for the recently introduced tripyrimidonamide scaffold that it can act as a potential α-helix mimetic, mimicking side chains at positions i, i+4 (dimeric compound) or i, i+4, i+8 (trimeric compound). This side chain pattern is concordant with the succession of the hot spot residues in the Hsp90 CTD interface.