Extended Data Fig. 5: Linoleic acid activates AMPK through directly binding to AMPKγ.
From: Remodelling of the translatome controls diet and its impact on tumorigenesis
a. Relative AMPKα1β1γ1 activity on its substrate SAMStide in the presence of vehicle (Veh), BSA, 100 μM linoleic acid (LA), 10 μM linoleoyl-CoA (LA-CoA) or 100 μM AMP (n = 5 biological independent samples) b. Relative AMPKα1β1γ1 activity on its substrate peptide SAMStide in the presence of vehicle (Veh), or the indicated linoleic acid and AMP combinations (n = 3 biological independent samples). c. Silver staining of blue native gel with recombinant AMPK complex with bodipy or C12-bodipy. d. Immunoblot of indicated proteins in whole cell lysates from fed and 24 h fasted livers, and linoleic acid conjugated or butyric acid conjugated beads pull-downed proteins; the total proteins of each sample on membrane were shown using ponceau S staining. e. Immunoblot of residual AMPKγ in the purified AMPKα1β1γ1 incubated linoleic acid conjugated beads or butyric acid beads after competing with different concentrations of free linoleic acid or butyric acid. f. Relative AMPKα1β2γ1 activity on its substrate SAMStide, in the presence of vehicle (Veh), BSA, 100 μM linoleic acid (LA), 10 μM linoleoyl-CoA (LA-CoA), 10 μM palmitoyl-CoA (PA-CoA) or 100 μM AMP (n = 3 biological independent samples). g. Relative AMPKα1β2γ1 activity on its substrate SAMStide, with different concentrations of linoleic acid (n = 5 biological independent samples). h. The isolated AMPKγ1-subunit remains stable. Backbone RMSD values of the γ-subunit are report without (blue line) and with (orange line) AMP bound at CBS sites −1, −3, and −4. The relatively low and stable RMSDs over these simulations confirm that simulations of γ can be run in the absence of the rest of the AMPK complex. i. A heat map of linoleic acid contacts with AMPKγ1 reveals primary sites of interaction. Structure of the γ subunit (PDB 4RER) with residues colored by contact time with linoleic acid aggregated across all lipid binding simulations, highlighting identified lipid binding Sites 1 and 2. The color spectrum runs from white to red, with white being no/minimal fatty acid contact and red being high contact. Site 1 is a hydrophobic pocket buried among the main and side chains of L173, K177, L285, I289, V293, L315, I318, and Y29; Site 2 is buried within F91, I94, L95, A250, Y255, K58, F62, V65, and L109. A third site, Site 3, at the bottom of the figure also sampled frequent contact with linoleic acid, but was not tested as a candidate for mutation given the site’s proximity to the binding interface with the β-subunit, reduced contact times compared to Sites 1 and 2, and the diffuse nature of the interaction surface. j. Proposed mutations to Sites 1 (left column) and 2 (right column) for disrupting linoleic acid binding. The images highlight the wild type residues (top row) and proposed mutations (bottom row). Mutations were selected based on fatty acid binding simulations, aiming to crowd out the hydrophobic pockets that linoleic acid was seen to enter in order to prevent binding. k. Scheme of expression of different AMPKγ1 mutants in Hek293T cells, and purification of AMPKα1β1γ1 complex with different AMPKγ mutants using anti-flag beads; AMPKα1 and AMPKγ1 levels in the pulled-down samples were shown using immunoblot; relative AMPK activity of AMPK complex with different AMPKγ1 mutants when used SAMStide as substrate and reaction with AMPK inhibitor compound C was used as a negative control (n = 3 biological independent samples). l. Immunoblot of different AMPKγ1 mutants in the input and bound to the beads. m. Silver staining of purified human wild type or mutated AMPKα1β1γ1 complex. All values represent the mean ± SEM; One-way ANOVA for a, b, f, linear regression analysis was used for g, and two-sided Student’s t test for g, i, j, k. Schematics in d, e, k, m is created using BioRender (BioRender.com).
