Exploring a Non-ATP Pocket for Potential Allosteric Modulation of PI3Kα

Allosteric modulators offer a novel approach for kinase inhibition because they target less conserved binding sites compared to the active site; thus, higher selectivity may be obtained. PIK-108, a known pan phosphoinositide 3-kinase (PI3K) inhibitor, was recently detected to occupy a non-ATP binding site in the PI3Kα C-lobe. This newly identified pocket is located close to residue 1047, which is frequently mutated in human cancers (H1047R). In order to assess the interactions, stability, and any possible allosteric effects of this inhibitor on PI3Kα, extensive molecular dynamics (MD) simulations in aqueous solution were performed for the wild type (WT) human, WT murine, and H1047R human mutant PI3Kα proteins with PIK-108 placed in both catalytic and non-ATP sites. We verify the existence of the second binding site in the vicinity of the hotspot H1047R PI3Kα mutation through binding site identification and MD simulations. PIK-108 remains stable in both sites in all three variants throughout the course of the simulations. We demonstrate that the pose and interactions of PIK-108 in the catalytic site are similar in the murine WT and human mutant forms, while they are significantly different in the case of human WT PI3Kα protein. PIK-108 binding in the non-ATP pocket also differs significantly among the three variants. Finally, we examine whether the non-ATP binding site is implicated in PI3Kα allostery in terms of its communication with the active site using principal component analysis and perform in vitro experiments to verify our hypotheses

Investigating the Structure and Dynamics of the <i>PIK3CA</i> Wild-Type and H1047R Oncogenic Mutant

<div><p>The <i>PIK3CA</i> gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110<i>α</i>, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in <i>PIK3CA</i> is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.</p></div

Functional mode analysis of the Cα RMSD of active site residues.

<p>(A/B) Scatter plots of the data versus the model using the cross-validation sets only. (C/D) The functional mode representing the kinase C- and N-lobe twisting motion for the WT and mutant proteins, respectively. Red: activation loop; yellow: catalytic loop; orange: P-loop; brown: hinge; magenta: adenine pocket; blue: affinity pocket (hydrophobic region I); black: specificity pocket.</p

Conformation of a second binding pocket in PI3Kα.

<p>(A) The first three cluster representatives from the WT trajectory in blue, green, and magenta, respectively. Dots represent predicted binding sites identified by the Q-SiteFinder server. The color of the dots corresponds to the respective cluster representative. (B) The first cluster representative from the trajectory is colored in green and aligned with the 4A55 crystal structure in cyan. His1047 is shown in cyan stick representation. The crystallized ligand of 4A55, PIK-108, is shown in magenta <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003895#pcbi.1003895-Hon1" target="_blank">[8]</a>. The predicted binding site by QSiteFinder appears in yellow dots and highly overlaps with the position of PIK-108 in the experimental structure.</p

Polar contacts close to the activation site, in (A) the WT and (B) H1047R p110α.

<p>The activation loop is colored orange, the catalytic loop yellow, and the P-loop in pink. Residues that form polar contacts are shown in licorice representation.</p

PI3Kα binding to PIP2-liposomes.

<p>Typical SPR sensograms depicting concentration dependent binding of WT p110α/p85α (A) and H1047R p110α(H1047R)/p85α (B) PI3Kα to liposomes. Signal from WT at 1.25 and 2 nM (A) was nil and is not shown for clarity. Vertical bars indicate beginning and end of injection. Data shown are referenced and corrected for bulk effect.</p

Polar contacts close to the mutation site, in (A) the WT and (B) H1047R p110α.

<p>The activation loop is colored orange, the catalytic loop yellow, and the P-loop in pink. Residues that form polar contacts are shown in licorice representation.</p

The electrostatic potential on the surface of the average conformation of the WT and H1047R p110α.

<p>The images depict membrane interaction areas of the WT (A) and H1047R mutant (B) first cluster representatives. The surface has been colored with a color scale from −7 eV to 7 eV, with red representing negative charge, white neutral and blue positive. The charge value corresponds to the solvent accessible surface of the protein, namely 1.4 Å far from the surface. These structures were derived from a cluster analysis including all the Cα carbon with a cutoff of 1.7 Å.</p

Proposed model of the overactivation mechanism of the <i>PIK3CA</i> mutant H1047R based on structural and dynamic differences with its WT counterpart.

<p>From left to right: The mutant protein accumulates positive charge in regions that contact the cell membrane and displays higher membrane binding affinity compared to the WT protein. The auto-inhibitory role of the C-terminal tail, which strictly controls the DRH motif to limit its access to the catalytic site, is abolished in the mutant protein due to loss of crucial intermolecular interactions. In the WT protein, His-917 of the DRH motif, points away from the active site thus preventing ATP hydrolysis more efficiently, while in the mutant PI3Kα structure, His917 points towards the active site, a conformation that is also observed in the structure of the active PI3Kγ.</p

Immunolocalisation of Endogenous and Overexpressed Rabankyrin-5 in NIH3T3 Cells

<div><p>Untransfected NIH3T3 cells or cells transfected with Rabankyrin-5 were labelled with antibodies to Rabankyrin-5 followed by 10 nm protein A gold.</p> <p>(A) Transfected cell showing labelling of a group of vesicular structures underlying the plasma membrane (pm).</p> <p>(B and C) In control (untransfected) cells, low but specific labelling for Rabankyrin-5 (arrowheads) is associated with compartments close to the pm. Scale bars represent 200 nm.</p></div