The Three Musketeers: The Molecular Basis of PI3Kα Activation in the Presence of HRas and the Cell Membrane

Abstract

The central focus of this study is PI3Kα, a key cell signalling, peripheral membrane binding protein, implicated in various cancers. PI3Kα is explored through atomistic and CGMD simulations to unravel its dynamic interactions with lipid-anchored HRas in the presence of a model cell membrane, which increases the membrane binding of PI3Kα thus enhancing its kinase activity. PI3Kα regulates cell growth and survival, functioning as a lipid kinase that phosphorylates PI(4,5)P2 to generate lipid secondary messenger PI(3,4,5)P3 in the plasma membrane. One of the main highlights of this study is the inclusion of a membrane in the simulations, which until now has not been incorporated in other MD studies on PI3Kα, even those examining the interactions of PI3Kα and HRas. Using Martini CG simulations enables the analysis of large systems like PI3Kα-HRas and the membrane over extended timescales, effectively reaching around 30 μs. This approach allows for a detailed exploration of membrane properties, such as lipid redistribution and curvature. The first part of this study (Chapter 2) reveals how HRas affects the interactions of PI3Kα with the membrane, particularly with PIP2, resulting in a membrane orientation of the protein that favours catalysis. The absence of HRas leads to altered PI3Kα membrane binding patterns, particularly involving the RBD. The results also show PI3Kα has a strong preference for binding to PI(4,5)P2 in the membrane, with lipid occupancy and residence times modulated by HRas. Additionally, PI3Kα binding to the membrane induces negative curvature and PI(4,5)P2 clustering, independent of HRas presence. The second part of this study (Chapter 3) uncovers significant interactions between PI3Kα and HRas, including key salt-bridges, but does not observe a consistent β-sheet interface as previously reported. Instead, the results suggest a transient nature of this interface, potentially influenced by the membrane. The third part of this study (Chapter 4) explores the predicted structure of a complete PI3Kα complex, which has not been experimentally resolved up to date, comprising all p85α domains in addition to p110α, revealing insights into the stability and dynamics of the domains. The anticorrelated motion between p110α and p85α and the dynamic interactions within p85α are highlighted, suggesting potential regulatory mechanisms. Overall, the results presented in this thesis provide insight into how the interaction of PI3Kα with the membrane and with HRas are affected by the presence of HRas and the membrane, as well as how the membrane is affected by HRas binding, and how the domains of the p85α subunit of PI3Kα may regulate activity of the catalytic p110α subunit.