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Structure of autoinhibited Akt1 reveals mechanism of PIP 3 -mediated activation

ABSTRACT: To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

INSTRUMENT(S): impact HD

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Burke

LAB HEAD: Dr. John E Burke

PROVIDER: PXD022061 | Pride | 2021-09-01

REPOSITORIES: Pride

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Project description:Previous studies from our laboratory have shown that the resistance increase observed after biological systemic acquired resistance (SAR) induction in plants can be mimicked by exogenous plant treatment with N-hydroxypipecolic acid (NHP, Hartmann et al., 2018, Schnake et al., 2020). Moreover, exogenous application of the NHP biosynthetic precursor pipecolic acid (Pip) induced a transcriptional response that was overlapping with the SAR transcriptional response and fully depended on the NHP synthase FMO1 (Hartmann et al., 2018; E-MTAB-6243). In order to investigate whether elevations of NHP lead to a SAR-like transcriptional reprogramming, we supplied individual Arabidopsis wildtype Col-0 plants, as well as sid2-1 and npr1-3 mutant plants, with doses of 10 µmol NHP and determined the transcriptional response in leaves 24 hours later on the whole genome level by RNA-sequencing analyses in relation to control-treated (H2O) plants. Col-0 plants were additionally treated with doses of 10 µmol Pip in order to directly compare the transcriptional responses of the foliage between Pip and NHP. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21°C and 18°C, respectively. Experiments were performed with 5-week-old, naive plants exhibiting a uniform appearance. Treatments with NHP and Pip were essentially performed as detailed in Hartmann et al. (2018, Cell 173, 456–469). In brief, 10 ml of a 1 mM aqueous solution of NHP or Pip (equates to a dose of 10 µmol) were pipetted onto the soil of individually cultivated plants. 10 ml of water applied in the way served as control treatments. In total, three biologically independent, replicate experiments were performed. In each experiment, 18 full-grown leaves from 6 different plants were pooled 24 hours after the respective treatments for one biological replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained.

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Structure of autoinhibited Akt1 reveals mechanism of PIP 3 -mediated activation

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