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Allosteric Regulatory Mechanisms of Bacterial RSH-enzymes

par Van Nerom, Katleen
Président du jury Vanhamme, Luc
Promoteur Garcia-Pino, Abel
Publication Non publié, 2022-02-02

Thèse de doctorat

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Résumé :

Long RSHs are enzymes that synthetize and hydrolyse a secondary messenger molecule, (p)ppGpp. This molecule acts as a global regulator targeting metabolic processes to ensure bacterial survival upon environmental challenges. Because (p)ppGpp is linked with bacterial virulence and resistance to antibiotics, a fundamental understanding of its regulation is key to develop novel antimicrobial treatments. Long RSH have two N-terminal domains, one for hydrolysis of (p)ppGpp (HD-domain) and one for synthesis of (p)ppGpp (SYNTH-domain), and four regulatory domains at the C-terminus that control catalysis by the N-terminal domains. A combination of structure determination and biochemical assays were used to study the long RSHs at the molecular level and determine the bases of the allosteric mechanisms that control the activities of the enzymes. The data demonstrate an allosteric conformational switch between the N-terminal catalytic domains of these enzymes that prevent futile catalytic cycles. Indeed, the data shows binding of substrates to one catalytic domain allosterically inhibits substrate-binding and catalysis by the opposing catalytic domain. Furthermore, feedback activation of (p)ppGpp-synthesis, the molecular basis of which was unknown, in context of amino-acid starvation was studied. The allosteric positive feedback regulation of (p)ppGpp-synthesis was shown to exploit the conformational switch between the two catalytic states to modulate both activities. An allosteric binding-pocket was located at the linker-region between the two catalytic domains at which (p)ppGpp-synthesis is auto-induced allosterically while (p)ppGpp-hydrolysis is inhibited. Lastly, the conformational landscape involved in the regulation of these enzymes was explored using Nanobodies to discover a previously undetected regulatory hotspot. Based on the conformational switch as well, this regulatory hotspot modulates catalytic activity allosterically. The newly discovered hotspot might be important to the autoregulation of long RSHs by its four regulatory domains that has been poorly understood for decades. This study uncovered three fundamental allosteric regulatory layers of the long RSHs that were previously unknown or poorly understood. It is the first step in the development of novel antimicrobial treatments that target (p)ppGpp-homeostasis, which is becoming more relevant as antibiotic resistance is occurring more frequently and is one of the most pressing problems to public health today.