Computational Insights Into the Mechanism of EGCG's Binding and Inhibition of the TDP‐43 Aggregation

ABSTRACT Misfolding and aggregation of TAR DNA‐binding protein, TDP‐43, is linked to devastating proteinopathies such as ALS. Therefore, targeting TDP‐43's aggregation is significant for therapeutics. Recently, green tea polyphenol, EGCG, was observed to promote non‐toxic TDP‐43 oligomer formation disallowing TDP‐43 aggregation. Here, we investigated if the anti‐aggregation effect of EGCG is mediated via EGCG's binding to TDP‐43. In silico molecular docking and molecular dynamics (MD) simulation suggest a strong binding of EGCG with TDP‐43's aggregation‐prone C‐terminal domain (CTD). Three replicas, each having 800 ns MD simulation of the EGCG‐TDP‐43‐CTD complex, yielded a high negative binding free energy (ΔG) inferring a stable complex formation. Simulation snapshots show that EGCG forms close and long‐lasting contacts with TDP‐43's Phe‐313 and Ala‐341 residues, which were previously identified for monomer recruitment in CTD's aggregation. Notably, stable physical interactions between TDP‐43 and EGCG were also detected in vitro using TTC staining and isothermal titration calorimetry which revealed a high‐affinity binding site of EGCG on TDP‐43 (Kd, 7.8 μM; ΔG, −6.9 kcal/mol). Additionally, TDP‐43 co‐incubated with EGCG was non‐cytotoxic when added to HEK293 cells. In summary, EGCG's binding to TDP‐43 and blocking of residues important for aggregation can be a possible mechanism of its anti‐aggregation effects on TDP‐43. TTC staining and isothermal titration calorimetry (ITC) show direct in vitro binding of EGCG with TDP‐43. In silico MD simulation predicts stable binding of EGCG to the C‐terminal domain of TDP‐43. TDP‐43 aggregates are cytotoxic, whereas TDP‐43 oligomers formed upon EGCG co‐incubation are not.