The trypanosomatid protein kinase A activation mechanism - a target for structure-based inhibitor development

Wu, Qingping (2021): The trypanosomatid protein kinase A activation mechanism - a target for structure-based inhibitor development. Dissertation, LMU München: Fakultät für Biologie

Abstract

The Protein kinase A (PKA) is a universal serine/threonine kinase that is highly conserved from low unicellular eukaryotes to humans. PKA plays a pivotal role in regulating a plethora of physiological processes, including metabolism, apoptosis, cell cycle progression and differentiation. Although PKA has been widely studied both structurally and functionally in model eukaryotes, PKA in Trypanosoma brucei and other kinetoplastid parasites has surprisingly distinct properties. Previous work from the group showed that TbPKA is not activated by cAMP and does not bind cAMP. Instead, it can be regulated by nucleoside analogs and is activated by cold shock in vivo. Compared to the mammalian orthologs, the TbPKA holoenzyme is a heterodimer consisting of the single R and one of three C subunit isoforms, that are differentially expressed in the life cycle of the parasite. TbPKAR contains an unusual large N-terminal domain and an extended C-terminal α-subdomain (α-D helix) with unknown functions. Previous studies revealed a critical role of PKA for the survival and proliferation of T. brucei. In this thesis, we established a robust in vivo protein-protein interaction assay (NanoBiT) based on the complementary split NanoLuc to detect R-C association and dissociation. NanoBiT assay is proven to be highly sensitive, fast and convenient in operation and shows a wide dynamic range in measuring PKA activation. Unbiased compound screening by this assay has been achieved using membrane permeabilized trypanosomes, the resulting EC50 values are comparable with the radioactive in vitro kinase assay on purified kinase. Using this assay and mutagenesis, we found that without the unusual N-terminal domain, PKAR can hardly form holoenzyme with the C-subunit in vivo. However, the attempt to identify its interaction sites with the C-subunit based on a simulated holoenzyme model was not conclusive. Interestingly, we found the P-site (T205) phosphorylation is critical for PKA activation and plays an antagonistic role to the N-terminal domain in PKA activation. By gene manipulation, we found that PKAR devoid of the C-terminal α-D helix acts like a dominant-negative mutant that traps the C-subunits, and the host cells show severe growth and cytokinesis phenotype. A comprehensive investigation of the α-D helix using NanoBiT assay reveals a key residue Y485, its interaction with N438 and H440 of the B-site is critical for PKA activation. Mutation of the two interacting amino acids in the back of the B pocket confirmed the result. Combining this result and the analysis of the crystal structure of TbR bound to inosine, we concluded that the α-D helix is confining the ligand in the B pocket in a lid-like fashion and is important for ligand binding. We speculate that the movement of the lid upon ligand binding is initiating the conformation change required for activation. An attempt to design bulky inhibitors allosterically targeting PKAR by blocking the interactions of the α-D helix and mimicking the dominant-negative effect, was performed. The designed compounds were examined by computational docking. Although a potent inhibitor has not been obtained so far, nucleoside-related compounds were found with high binding affinity but low activation potency, indicating that the separation of binding affinity from activation potency is possible. These compounds can serve as the basis for further inhibitor development. More importantly, they provide a chemical tool for further exploration the TbPKA activation mechanism. Although the essentiality of PKAC1 catalytic activity and the possible redundancy of the PKAC subunits were reevaluated by reverse genetics in the thesis, the result is puzzling in comparison to previous studies. Interestingly, no difference was found in the activation of different PKA holoenzymes in the bloodstream and procyclic life cycle stages. To further investigate the functions and possible redundancy of the C subunit isoforms, a dominant-negative strategy using a SuperDN PKAR mutant was designed.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: PKA, trypanosoma, nucleoside, activation, structure-based drug design
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 14. Juli 2021
• 1. Bericht­erstatter:in: Boshart, Michael