Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

Paraskevi Gkeka,Zoe Cournia,Vasiliki Lazani,Savvas Christoforidis,Bogos Agianian,Thomas Evangelidis,Maria Pavlaki,Bert L De Groot

doi:10.1371/journal.pcbi.1003895

Abstract

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, 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 PIK3CA 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.

Highlights

The phosphatidylinositol 3-kinase alpha (PI3Ka) protein is involved in cellular processes vital for cancer progression, such as cell growth, proliferation, motility, survival, and metabolism [1]
We have modeled the full-length catalytic p110a subunit in the WT and H1047R mutant forms in order to gain insights into the overactivation mechanism of the commonlyexpressed H1047R mutant through Molecular Dynamics (MD) simulations and Functional Mode Analysis (FMA) and have used Surface Plasmon Resonance (SPR) experiments to validate our results
The overlap of the 2D projections of the trajectories on the first two eigenvectors indicates that the five independent simulations for the WT and the H1047R mutant proteins span the same or similar conformational phase space (Figure S3)

Summary

Introduction

The PI3Ka protein is involved in cellular processes vital for cancer progression, such as cell growth, proliferation, motility, survival, and metabolism [1]. Somatic mutations within the gene encoding p110a (PIK3CA) are frequently observed in a variety of human tumors, including breast, colon, endometrial cancers, and glioblastomas [3]. These mutations are scattered over the length of p110a but two hotspots account for nearly 80% of them: an H1047R substitution close to the C-terminus and a cluster of three charge-reversal mutations (E542K, E545K, Q546K) in the helical domain of p110a [4]. According to structural and functional studies, these two hot spot mutations act synergistically, but independently [8,9,10]

Methods

Results

Conclusion