Investigation Of TEAD Protein Structure And Function In Hippo Pathway

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities.Based on irreversible inhibitors of PATs, we developed activity-based chemical probes 2-bromopalmitate (2-BP) and cerulenin, which inhibit palmitoylating activities by alkylating the active site cysteines of the enzymes or autopalmitoylated proteins. We have synthesized analogs of 2-BP and cerulenin with an alkyne tail, which serve as bioorthogonal chemical reporters for covalently labeling and profiling PATs and autopalmitoylated proteins. Through proteomic and biochemical studies, we determined that the TEAD transcription factors are palmitoylated at evolutionarily conserved cysteine residues. We found that TEADs undergo PAT-independent autopalmitoylation at physiological concentrations of palmitoyl-CoA.We constructed, expressed and purified the hTEAD2 YBD protein. We obtained crystals. carried out X-ray diffraction, data collection and analysis, and determined the crystal structure is indeed a palmitate bound TEAD2 complex. We solved the structure of hTEAD2-PLM complex (PDB:5HGU) and found that TEADs and revealed a new ligand-binding site in TEADs. Furthermore, we found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket, pointing to C380. This indicates a new drug binding target of TEADs. Autopalmitoylation plays critical roles in regulating TEAD-YAP association and their physiological functions in vitro and in vivo. Therefore, palmitoylation of TEADs plays important roles in regulating Hippo pathway transcriptional complexes. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.Through drug screening, we found a chemical molecule inhibitor of TEADS - flufenamic acid (FA), which occupies the central hydrophobic pocket and binds with TEAD YBD. We successfully soaked FA molecule into the crystal of hTEAD2-PLM complex, carried out X-ray crystal diffraction and solved the structure of hTEAD2-FA complex (PDB:5DQ8). We found that FA localized at the central hydrophobic pocket of TEAD YBD and binds with C380. To confirm that the omit electronic density in the structure is donated by FA molecule, we designed the bromo-homolog of FA, Bromofenamic acid (BFA).We also soaked BFA molecule into the crystal of hTEAD2-PLM, carried out X-ray crystal diffraction and solved the structure of BFA-hTEAD2217-447 complex (PDB:5DQE). And the anomalous difference map proved that the omit map comes from BFA. We found that FA/BFA also binds with C348. The binding sites of FA/BFA in TEAD2 YBD are just the palmitoylation sites, indicating that FA/BFA inhibits the palmitoylation of TEAD YBD.We also carried out the mutation of TEAD2 YBD’s palmitoylation site (C348, C380) from C to S, and found that the affinity between TEAD and YAP becomes weaker. We also mutated A235, A304 sites to V and detected the decrease of the binding level of TEAD and YAP. In sum, inhibition of palmitoylaiton of TEADs weakens the binding of TEAD and YAP.