From Canonical to Non-Canonical Allosteric Effectors

Abstract

Allostery regulates protein function by enabling communication between two remote sites. Understanding how allosteric perturbations regulate biological function provides new therapeutic strategies for drug discovery. Allosteric regulators include both canonical and non-canonical effectors, and in this thesis, we will explore both types of allosteric modulators at atomic resolution. To this end, we relied on solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations, in combination with other biophysical approaches. After a brief introduction to allostery in chapter one, chapter two focuses on the canonical allosteric effector cGMP, which is a full agonist of the Plasmodium falciparum cGMP dependent protein kinase (PfPKG), a target for antimalaria drugs. We detected a crucial activation pathway of PfPKG domain D (PfD) occurring through a compact intermediate. Through the comparative analysis of cGMP-bound active ensembles and cGMP-bound inactive intermediate conformers, we also differentiated key cGMP-PfD contacts required for binding vs. allosteric activation, as needed for the rational design of PfPKG allosteric inhibitors. Chapter three extends the study of allosteric effectors to noncanonical amyloid fibrils. The alpha synuclein (αS) E46K mutation enhances aggregation and triggers early-onset Parkinson’s disease (PD). We showed that E46K also promotes early-stage monomer-monomer self-association and late-stage monomer-fibril interactions through additional recognition sites absent in wt αS. Furthermore, we show that the E46K fibrils allosterically disrupt intramolecular interactions in monomers driving a shift toward more extended monomer conformers. Chapter four explores potential new allosteric effectors that are unique to extracellular biological fluids, i.e. cerebrospinal fluid (CSF) and plasma. Our data revealed that the interactomes of αS reconstituted in extracellular fluids markedly differ from those in intracellular environments. In CSF, αS binds to extracellular vesicles through an unconventional recognition mechanism involving the entire αS sequence, including the C-terminal region that is typically disengaged from lipid membranes. We also observed that the interactions of αS with extracellular vesicles are detuned by HSA in plasma and by methionine oxidation of αS, providing critical insight into the regulation of αS interactions in extracellular fluids. Overall, in this thesis we explored how protein allosteric effectors modify functional free energy landscapes from both structural and dynamical perspectives.