PhD Defense | Molecular Basis for p85 Dimerization and Allosteric Ligand Recognition

Dec 06 2018 01:00 PM - Dec 06 2018 03:00 AM

By ​Safia S. Aljedani

The phosphatidylinositol-3-kinase α (PI3Kα) is a heterodimeric enzyme that is composed of a

p85α regulatory subunit and a p110α catalytic subunit. PI3Kα plays a critical role in cell

survival, growth and differentiation, and is the most frequently mutated pathway in human

cancers. The PI3Kα pathway is also targeted by many viruses, such as the human

immunodeficiency virus (HIV-1), the herpes simplex virus 1 (HSV-1) or the influenza A virus,

to create favourable conditions for viral replication. The regulatory p85α stabilizes the catalytic

p110α, but keeps it in an inhibited state. Various ligands can bind to p85α and allosterically

activate p110α, but the mechanisms are still ill-defined. Intriguingly, p85α also binds to, and

activates, the PTEN phosphatase, which is the antagonist of p110α. Previous studies indicated

that only p85α monomers bind to the catalytic p110α subunit, whereas only p85α dimers bind

to PTEN. These findings suggest that the balance of p85α monomers and dimers regulates the

PI3Kα pathway, and that interrupting this equilibrium could lead to disease development.

However, the molecular mechanism for p85α dimerization is controversial, and it is unknown

why PTEN only binds to p85α dimers, whereas p110α only binds to p85α monomers. Here we

set out to elucidate these questions, and to gain further understanding of how p85α ligands

influence p85α dimerization and promote activation of p110α. We first established a

comprehensive library of p85α fragments and protocols for their production and purification.

By combining biophysical and structural methods such as small angle X-ray scattering, X-ray crystallography, nuclear magnetic resonance, microscale thermophoresis, and chemical crosslinking, we investigated the contributions of all p85α domains to dimerization and ligand binding. Contrarily to the prevailing thought in the field, we find that p85α dimerization and

ligand recognition involves multiple domains, including those that directly bind to and inhibit

p110α. This finding allows us to suggest a molecular mechanism that links p85α dimerization

and allosteric p110α activation through ligands.