Multiple binding modes of AKT on PIP$_3$-containing membranes

TL;DR

Four distinct membrane-binding modes that differ in the orientations and membrane contacts of the PH and kinase domains of AKT are identified, shedding light on how lipid recognition by the PH domain and the kinase domain of AKT cooperatively shape its membrane-bound conformations.

Abstract

The PI3K/AKT signaling pathway is triggered by recruitment of AKT to cellular membranes. Although AKT is a multidomain serine/threonine kinase composed of an N-terminal pleckstrin homology (PH) domain and a C-terminal kinase domain, how these domains cooperate to regulate AKT activation on membranes remains unclear at the molecular level. Here, using molecular dynamics simulations of full-length AKT on PIP$_3$-containing lipid bilayers, we identify four distinct membrane-binding modes that differ in the orientations and membrane contacts of the PH and kinase domains. In addition to PIP$_3$ binding to the PH domain, we observe specific PIP$_3$ interactions with basic residues in the kinase domain. In the most stable mode, PIP$_3$ interacts with both the canonical and a secondary binding site in the PH domain, while the kinase domain adopts an orientation in which the activation-loop phosphorylation site is exposed to the solvent. Interestingly, the populations of these binding modes depend on the PIP$_3$ concentration in the membrane, leading to changes in the preferred orientation of AKT. These findings shed light on how lipid recognition by the PH domain and the kinase domain of AKT cooperatively shape its membrane-bound conformations.

Figures (10)

• Figure 1: AKT binds to the lipid bilayer via its PH domain. (A) Schematic representation of AKT. (B) Distance between the COM of each domain and the lipid bilayer as a function of simulation time. The inset shows a snapshot from a representative simulation illustrating membrane-bound AKT. The PH domain and kinase domain are shown in light blue and orange, respectively. PIP$_3$ lipids in the membrane are highlighted. (C) Probability density functions of the distance between the COM of each domain and the lipid bilayer. The dashed line indicates the membrane surface (phosphate group).
• Figure 1: Simulations for rescaling protein--protein interactions to reproduce the compactness of full-length AKT. (A) Relationship between the protein--protein interaction scaling parameter $\lambda$ and the radius of gyration $R_g$. (B) Structural comparison between the crystal structure (PDB ID: 4GV1, cyan) and the AlphaFold2-predicted model (pink). (C) Simulation system used for the rescaling procedure. (D--F) Time series of $R_g$ at $\lambda = 91\%$ (D), $92\%$ (E), and $93\%$ (F).
• Figure 2: Membrane-binding modes and orientations of AKT on the lipid bilayer. (A) Definition of the orientation angles. $\theta$ is defined as the angle between the bilayer normal and the $\alpha$-helix (T92--E116) in the PH domain (left), and $\phi$ is defined as the angle between the bilayer normal and the $\alpha$-helix (R328--C344) in the kinase domain (right). (B) Normalized density maps for the PH domain (left) and the kinase domain (right) shown as a function of the orientation angle and the $z$-component of the domain COM--bilayer distance. (C) Representative orientations of the PH domain (left) and the kinase domain (right). PIP$_3$ lipids in the membrane are highlighted, with their oxygen atoms shown in red. (D) PCA analysis of the AKT orientation. (E) Four binding modes identified by DBSCAN clustering in the PCA space.
• Figure 2: Time series of the distance between the domain COM and the lipid bilayer. (A) PH domain. (B) Kinase domain.
• Figure 3: Residues critical for PIP$_3$ binding across the four binding modes. Normalized contacts were calculated using data from each cluster. For normalization, the number of contacts between a given residue and phosphate headgroups was divided by the maximum number of contacts observed within each cluster. Hydrophobic, basic, acidic, and other residues are shown in yellow, blue, red, and white, respectively.