Molecular Mechanisms of Allosteric Inhibition in Cylic-Nucleotide Dependent Protein Kinases

Abstract

Allosteric inhibition of kinases provides high selectivity and potency due to lower evolutionary pressure in conserving allosteric vs. orthosteric sites. The former are regions distinct from the kinase active site, yet, when perturbed through allosteric effectors, induce conformational and/or dynamical changes that in turn modulate kinase function. Protein kinases involved in cyclic nucleotide signalling are important targets for allosteric inhibition due to their association with diseases, from infections to Cushing’s syndrome. This dissertation specifically focuses on elucidating the molecular mechanism of allosteric inhibition in the cAMP-dependent protein kinase (PKA) and the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG), which are targets for a generalized tumor predisposition commonly referred to as Carney Complex and for malaria, respectively. In chapters 2 and 3, we focus on the agonism-antagonism switch (i.e. allosteric pluripotency) observed as the phosphorothioate analog of cAMP, Rp-cAMPS (Rp), binds to PKA. Utilizing Nuclear Magnetic Resonance (NMR), Molecular Dynamics (MD) simulations and Ensemble Allosteric Model (EAM), we determined that two highly homologous cAMP-binding domains respond differently to Rp, giving rise to a conformational ensemble that includes excited inhibition-competent states. The free energy difference between this state and the ground inhibition-incompetent state is tuned to be similar to the effective free energy of association of the regulatory (R) and catalytic (C) subunits, leading to allosteric pluripotency depending on conditions that perturb the R:C affinity. The general significance of these results is a re-definition of the concept of allosteric target to include not only the isolated allosteric receptor, but also its metabolic and proteomic sub-cellular environment. In chapter 4, we utilize a mutant that silences allosteric pluripotency to reveal that the agonism-antagonism switch of PKA not only arises from the mixed response of tandem domains, but also from the mixed response of allosteric regions within a single domain that mediates interactions with Rp. In chapter 5, the allosteric inhibition of PfPKG associated with malaria is induced through base-modified cGMP-analogs and the underlying inhibitory mechanism is determined. We show that, when bound to a PfPKG antagonist, the regulatory domain of PfPKG samples a mixed intermediate state distinct from the native inhibitory and active conformations. This mixed state stabilizes key cGMP-binding regions, while perturbing the regions critical for activation, and therefore it provides an avenue to preserve high affinity, while promoting significant inhibition. Overall, in this thesis, previously elusive mechanisms of allosteric inhibition were elucidated through the combination of NMR, MD, and EAM methods. Through this integrated approach, we have unveiled an emerging theme of inhibitory ‘mixed’ states, either within a single domain or between domains, which offer a simple but effective explanation for functional allostery in kinases.