S C

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Fig. 30: Activation of Akt by PIP3. The conformational changes in Akt1 upon PIP3 binding mapped by HDX-MS. Protection of the PH domain (blue) and exposure of the substrate-binding cleft of the kinase domain (red) accompany PIP3 binding.

obvious question of how they can be phosphorylated by Akt. While some studies have proposed that Akt could disengage from the plasma membrane in an active conformation in order to reach such substrates, this would imply the uncoupling of Akt activity from the very signal that activated it. This is now known not to be the case. With the help of a llama-derived nanobody against Akt, the crystal structure of the auto-inhibited conformation of Akt1 was determined from data collected at macromolecular crystallography beamline ID23-2, and the solution structure by small- angle X-ray scattering (SAXS) data taken at beamline

BM29, revealing the interface between its PH and kinase domains at near-atomic resolution. The structure revealed that the entire PIP3-binding site is buried in intramolecular contacts with the kinase domain, blocking both PIP3 binding by the PH domain and substrate binding to the kinase domain [2]. Primarily held together by water-mediated hydrogen bonds (Figure 29), a conformational equilibrium between open and closed states appears to regulate PIP3 binding to the PH domain [3]. PIP3 binding relieves the inhibitory interaction, leading to exposure of the regulatory sites for phosphorylation and the consequent acquisition

Fig. 29: Auto-inhibition of Akt by its PH domain. The interface between the PH and kinase domains is mediated by a hydrogen bond network involving several ordered water molecules.