Exploring the Binding Mechanism of a Supramolecular Tweezer CLR01 to 14-3-3σ Protein Well-Tempered Metadynamics.

Using supramolecules for protein function regulation is an effective strategy in chemical biology and drug discovery. However, due to the presence of multiple binding sites on protein surfaces, protein function regulation selective binding of supramolecules is challenging. Recently, the functions of 14-3-3 proteins, which play an important role in regulating intracellular signaling pathways protein-protein interactions, have been modulated using a supramolecular tweezer, CLR01. However, the binding mechanisms of the tweezer molecule to 14-3-3 proteins are still unclear, which has hindered the development of novel supramolecules targeting the 14-3-3 proteins. Herein, the binding mechanisms of the tweezer to the lysine residues on 14-3-3σ (an isoform in 14-3-3 protein family) were explored by well-tempered metadynamics. The results indicated that the inclusion complex formed between the protein and supramolecule is affected by both kinetic and thermodynamic factors. In particular, simulations confirmed that K214 could form a strong binding complex with the tweezer; the binding free energy was calculated to be -10.5 kcal·mol with an association barrier height of 3.7 kcal·mol. In addition, several other lysine residues on 14-3-3σ were identified as being well-recognized by the tweezer, which agrees with experimental results, although only K214/tweezer was co-crystallized. Additionally, the binding mechanisms of the tweezer to all lysine residues were analyzed by exploring the representative conformations during the formation of the inclusion complex. This could be helpful for the development of new inhibitors based on tweezers with more functions against 14-3-3 proteins modifications of CLR01. We also believe that the proposed computational strategies can be extended to understand the binding mechanism of multi-binding sites proteins with supramolecules and will, thus, be useful toward drug design.