Exploring the druggability of the binding site of aurovertin, an exogenous allosteric inhibitor of FOF1-ATP synthase

In addition to playing a central role in the mitochondria as the main producer of ATP, FOF1-ATP synthase performs diverse key regulatory functions in the cell membrane. Its malfunction has been linked to a growing number of human diseases, including hypertension, atherosclerosis, cancer, and some neurodegenerative, autoimmune, and aging diseases. Furthermore, inhibition of this enzyme jeopardizes the survival of several bacterial pathogens of public health concern. Therefore, FOF1-ATP synthase has emerged as a novel drug target both to treat human diseases and to combat antibiotic resistance. In this work, we carried out a computational characterization of the binding sites of the fungal antibiotic aurovertin in the bovine F1 subcomplex, which shares a large identity with the human enzyme. Molecular dynamics simulations showed that although the binding sites can be described as preformed, the inhibitor hinders inter-subunit communications and exerts long-range effects on the dynamics of the catalytic site residues. End-point binding free energy calculations revealed hot spot residues for aurovertin recognition. These residues were also relevant to stabilize solvent sites determined from mixed-solvent molecular dynamics, which mimic the interaction between aurovertin and the enzyme, and could be used as pharmacophore constraints in virtual screening campaigns. To explore the possibility of finding species-specific inhibitors targeting the aurovertin binding site, we performed free energy calculations for two bacterial enzymes with experimentally solved 3D structures. Finally, an analysis of bacterial sequences was carried out to determine conservation of the aurovertin binding site. Taken together, our results constitute a first step in paving the way for structure-based development of new allosteric drugs targeting FOF1-ATP synthase sites of exogenous inhibitors.

Atom types and partial charges used for AUR B

Figure S1
Conformational relaxation of the AUR binding site sites with and without inhibitor.

Figure S2
Contact analysis at the AUR binding site in βE

Figure S3
Protein-inhibitor hydrogen bonding at the AUR binding site in βE  Dihedral angle free energy landscapes (FEL) for αTP residues Cumulative per-residue squared covariance ( 2 ) for the βE site from a side chain dPCA

Figure S7
Conformational variability in the βTP AUR binding site of experimental structures of BtF1

Figure S8
Side chain dihedral angle free energy landscapes (FEL) for the nucleotide binding residues in βE

Figure S9
Residue-wise free energy decomposition and solvent site identification in βE     Backbone dPCA. One and two metastable conformational states were observed for AUR + (S1) and AUR ─ (S1,S2), respectively. The percentage of cumulative frequencies are shown. The main difference between S1 and S2 was the  angle value of I 344 . C,D) Side chain dPCA. Black lines delimit the macrostates identified through a Markov-state model analysis. E,F) Network transition pathway of the Markov-state model. The thickness of the connecting arrows is proportional to the transition probability. G,H) Superimposition of representative conformations for each attraction basin in E,F). Macrostates were labeled S1, S2 and so on from lowest to highest occupancy.

Figure S9. Per-residue free energy decomposition and solvent site identification in βE.
Per-residue decomposition of the binding free energy (GPB) was calculated with the MMPBSA method. Residues that favor interaction with the inhibitor are shown in green. The identified hydrophobic solvent sites (SSHP) are shown as spheres.