[Paper Review] Multiple binding modes of AKT on PIP$_3$-containing membranes

Yuki Nakagaki, Eiji Yamamoto|arXiv (Cornell University)|Jan 29, 2026

PI3K/AKT/mTOR signaling in cancer0 citations

TL;DR

The study uses coarse-grained MD simulations to reveal four distinct membrane-binding modes of full-length AKT on PIP3-containing membranes, showing cooperative PH and kinase domain interactions and PIP3-dependent mode switching.

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.

Motivation & Objective

• Understand how the PH and kinase domains of AKT cooperate to bind PIP3-containing membranes.
• Characterize the orientations and membrane contacts of full-length AKT on membranes.
• Identify distinct membrane-binding modes and how PIP3 concentration affects mode preference.
• Map residue-level PIP3 interactions across binding modes to reveal binding-site usage.

Proposed method

• Perform coarse-grained molecular dynamics simulations of full-length AKT on a PIP3-containing lipid bilayer using Martini 2.2 force field with reduced protein–protein LJ interactions (scaled to 93%).
• Place AKT ~15 nm above the membrane and run ten 50 μs production runs to observe binding.
• Define membrane-binding orientation angles theta and phi from PH and kinase domain helices and construct two-dimensional density maps.
• Apply PCA followed by DBSCAN clustering to identify distinct membrane-binding modes.
• Compute residue-level PIP3 contacts within 0.7 nm to map binding sites and analyze PIP3 density around AKT for each mode.

Experimental results

Research questions

• RQ1 What are the plausible membrane-bound orientations of full-length AKT on PIP3-containing membranes?
• RQ2 How do the PH and kinase domains interact with PIP3 and with each other across different orientations?
• RQ3 How does PIP3 concentration influence binding mode preference and activation-ready orientations?
• RQ4 Which AKT residues participate in PIP3 binding across modes and how are canonical and secondary PH domain sites utilized?
• RQ5 Can the observed binding modes reveal mechanisms by which AKT activation sites (Thr308, Ser473) become solvent-exposed?

Key findings

• AKT predominantly binds the membrane via its PH domain, with a strong peak at a 3.8 nm COM distance and a secondary peak at 7.0 nm.
• Four membrane-binding modes (Clusters I–IV) exist with distinct PH/kinase orientations, where Cluster I shows PH upright and kinase N-/C-lobe contacting the membrane.
• In the most stable mode, PIP3 engages both canonical and secondary PH sites while the activation loop is exposed for phosphorylation sites Thr308 and Ser473.
• PIP3 interactions also occur with positively charged residues in the kinase domain, though these contacts are more diffuse and weaker than PH-domain contacts.
• Increasing PIP3 concentration increases kinase-domain PIP3 contacts and stabilizes orientations that favor exposure of activation sites across multiple clusters; at low PIP3, PH-domain contacts dominate and kinase contacts diminish.
• Under reduced PIP3 (two PIP3 per leaflet), the kinase-domain stabilization diminishes and PH-domain canonical/secondary site binding remains critical for AKT membrane association.