Figure 8. The allosteric drug-induced Akt inhibited form is distinct from its native autoinhibited form.

(A) Overlay of ¹⁵N-¹H HSQC spectra of the PH domain in the context of full-length semisynthetic Akt with non-phosphorylated C-tail in the presence of the allosteric inhibitor MK2206 (red) or in its absence (blue). Select residue-specific assignments are shown. (B) Expanded ¹⁵N-¹H HSQC cross saturation transfer (CST) spectra around the peaks assigned to Phe27, Thr65 and Ala102. Reference (unsaturated) spectrum is shown in blue and its saturated counterpart in red. (C) Cartoon representation of secondary structure elements (rectangle for β-strands, zigzag for α-helices) in Akt PH domain. Color coding represents regions with distinct binding modes to the kinase domain. Star indicates that the α-helix is present only when Akt is bound to IP4. (D) Combined chemical shift perturbations corresponding to spectra in (A) plotted along the primary sequence for the PH domain in the context of semisynthetic full-length Akt with non-p C-tail with MK2206, referenced to the control without drug. Dashed red line corresponds to the standard deviation to the mean, excluding outliers (higher than 3xStDev). Grey bars indicate peaks that disappeared from the spectrum, also indicating strong interaction. Red area highlights the short hinge primarily studied here. Negative bars (−0.05) indicate non-assigned residues, negative bars (−0.025) indicate residues which assignment could not be easily transferred or recovered in the context of full-length Akt. (E) Saturation transfer efficiency derived from spectra in (B) plotted as the ratio of peak intensities of saturated over unsaturated spectra against the PH domain primary sequence. A ratio of 0 indicates maximum saturation transfer efficiency (very tight interaction), whereas a ratio of 1 indicate no saturation transfer (no interaction). An indicative dashed red line has been drawn at 0.5. Red area highlights the short hinge primarily studied here. Negative ratios (−0.05) indicate non-assigned residues, negative ratios (−0.025) indicate residues that were not present in 70% ²H₂O spectra. (F) Statistical bar and whisker plots of saturation transfer efficiencies from CST data (B,E), categorized and color coded according to secondary structure elements as in (C). (G) Structure of allosteric drug inhibited Akt (PDB: 3O96, [Wu et al., 2010]) with PH domain as ribbon in the front and kinase domain as surface in the back (N-lobe in light blue, C-lobe in dark blue). Significantly affected residues (chemical shift perturbations higher than the standard deviation) are colored in red. Non-affected residues are shown in light orange and non-assigned residues in grey. A representation rotated by 180° is shown.