Exploring the Binding Mechanism of a Supramolecular Tweezer CLR01 to 14-3-3σ Protein via 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 via selective binding of supramolecules is challenging. Recently, the functions of 14-3-3 proteins, which play an important role in regulating intracellular signaling pathways via 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−1 with an association barrier height of 3.7 kcal·mol−1. 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 via 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.

These lysine residues are plotted using stick style and colored in green. The A chain of 14-3-3σ is colored in gray, and the B chain of 14-3-3σ is colored in purple. Figure S4. Initial structures of tweezer with 14-3-3σ.

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The tweezer was manually move to a position more than 12 Å from the selected lysine residue through VMD 1.9.1 software. S8 Figure S5. Collective variables selected for metadynamics in this work.
Black dotted lines represent the CV1. CV1 was the distance between the center of mass (COM) of the lysine side chain and the COM of the tweezer ring excluding the two phosphate groups. CV2 was the coordination number between the heavy atom in the side chain of lysine and the carbon atom in the tweezer ring. S9 S10 Figure S6. Two-dimensional free energy landscapes for the tweezer to these 13 lysine sites.
Free energies are in kcal/mol. The binding free energy value can be calculated from the free energy of the bound state minus the free energy of the unbound state. Herein, it can be found that K11, K27, K49, K109, K195 could not be recognized by tweezer with the binding free energies are greater than zero. S11 Figure  The first row mainly shows the changes of key residues in the binding process for each lysine site. The second row shows the changes of tweezer position in the binding process for each lysine site. Tweezer molecule and key residues are plotted using stick style. The unbound state, transition state and bound state are colored in magentas, blue, green, respectively. S16 Figure S10. Structure characteristics of K77 in apo-14-3-3σ.
Around K77, there are acidic residues E76 and E80, but K77 hardly interacts with them.
In addition, some short chain residues around K77, such as G73, S74, G78, and P79, further promote the complete exposure of K77 to the solvent environment. S17 Figure S11. Other representative snapshots for tweezer/K160 system on 14-3-3σ. The first row mainly shows the changes of key residues in the binding process for each site. The second row shows the changes of tweezer position in the binding process for each lysine site. The tweezer molecule and key residues are plotted using stick style.
The unbound state, transition state, semi-bound state and bound state are colored in magenta, blue, yellow, and green, respectively. S19 Figure S13. Semi-bound state for tweezer binding to (A) K87, (B)K159.
The key residues and tweezer are plotted using stick style. K87 and K159 are colored in green, the other residues and tweezer are colored in grey and blue, respectively. The lysine residue shows with stick style and yellow color. The tweezer is shown stick style and green color. The 14-3-3σ shows with cyan color. The additional residues with hydrogen bonds are shown with stick and blue. The tweezer/14-3-3σ was obtained from the metadynamics. S27 Figure S21. Sites of the lysine residues on 14-3-3σ with the active regions.
These lysine residues are plotted using stick style and colored in green. The A chain of 14-3-3σ is colored in blue, and the B chain of 14-3-3σ is colored in red. α-Helix of the 14-3-3σ monomer is labeled in red. Figure S22. Superimposed structures of MT-ExoS, CLR01, ExoS binding to 14-3-3.

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The structure of the MT-ExoS bound with 14-3-3σ (PDB ID: 6Y7T) is colored with green, and the tweezer molecule and K214 are plotted by stick style. The structure of tweezer/K214 on 14-3-3σ (PDB ID: 5OEG) is colored with blue, and the tweezer molecule and K214 are plotted by stick style. The structure of the ExoS peptide bound with 14−3−3ζ (PDB ID: 4N7G) is colored with magenta, and the ExoS peptide is plotted by stick style. S29 Table S1. Simulation time of tweezer binding to lysine residues on 14-3-3σ for metadynamics.

Lysine Site
Time ( Table S3. Distance of the upward phosphate group (P1) of tweezer and the ammonium group of lysine residue in the bound state.