The fungal natural product azaphilone-9 binds to HuR and inhibits HuR-RNA interaction in vitro
Fig 4
NMR titrations of HuR RRM1/2 with AZA-9.
(A) Overlay of three 2D 1H-15N TROSY spectra of 15N HuR RRM1/2 titrated with increasing molar ratios of AZA-9. The peaks of some critical RNA-binding residues that undergo significant line broadening upon addition of AZA-9 are shown (expanded dashed box). (B) Representative residues showing peak broadening upon titration of AZA-9 are shown at similar contour level at individual titration points. (C) Relative peak intensity plot for non-overlapping amide resonances of HuR RRM1/2 in the ligand bound versus free state (I1:1 /I1:0). (D) Overlay of three 2D 1H-13C HSQC spectra of ILV-labeled HuR RRM1/2 titrated with increasing molar ratios of AZA-9. Analogous to 15N-titrations, ILV methyl groups of RNA-binding residues showed peak broadening with a few residues such as I103 and L138 also displaying chemical shift deviations (dashed box). (E) Relative peak intensity plot for assigned ILV methyl HuR RRM1/2 resonances in the ligand bound versus free state (I1:1 /I1:0). (F) Results of titrations mapped onto the co-crystal structure of HuR-AREc-fos complex (PDB 4ED5). Protein and RNA are shown as in Figs 2 and 3. RRM1/2 residues with peak intensity ratio (I1:1 /I1:0) lower than 1σ are colored red and the proposed AZA-9 binding site is shown by an arrow. Most of the AZA-9 affected residues are key RNA-binding residues. (C,E) Gray and red lines correspond to the mean and one standard deviation from the mean (1σ), respectively.
