Molecular insights into pyruvate-sensing of the LytS/LytTR-type BtsS/BtsR signaling cascade of Escherichia coli

Qiu, Jin (2023): Molecular insights into pyruvate-sensing of the LytS/LytTR-type BtsS/BtsR signaling cascade of Escherichia coli. Dissertation, LMU München: Fakultät für Biologie

Abstract

LytS/LytTR-type histidine kinase/response regulator systems regulate pathogen-specific mechanisms during infection of human or plant hosts. Escherichia coli has two LytS/LytTR systems, BtsS/BtsR and YpdA/YpdB. The histidine kinase BtsS is a high-affinity sensor for extracellular pyruvate and interacts with response regulator BtsR to activate the expression of btsT, which encodes a symporter for pyruvate and proton. This thesis describes new insights into the molecular mechanism of how pyruvate binding triggers the signal transduction process. It was experimentally shown that BtsS is a seven-helix receptor, with its N-terminus located in the periplasm. Using a screening assay based on site-directed mutagenesis, the pyruvate-binding site was identified within the membrane-spanning domains of BtsS. It is a small cavity, and pyruvate forms interactions with the side chains of arginine 72, arginine 99, cysteine 110, and serine 113, located in the transmembrane helices III, IV and V, respectively. The interactions between pyruvate and these four amino acids were further confirmed by molecular dynamics simulation studies. In addition, three other amino acids in BtsS were found to be important for structure and signal transduction. Arginine 192 (helix VII) plays a role in the interaction with BtsR. Serine 25 (helix I) is important for conformational dynamics, as BtsS variants in which Serine 25 has beenreplaced by alanine or valine maintain the signal transduction system in an ON state. Cysteine 15 is likely involved in the formation of a disulfide bridge, but this is not essential for signal transduction in vivo under the conditions tested. In previous studies, the autophosphorylation activity of BtsS was not detectable. Here, for the first time, BtsS was shown to have very low autophosphorylation activity in the presence of Mg2+-ATP, which was approximately 10-fold higher when Mn2+-ATP was used. The predicted phosphorylation site at histidine 382 was confirmed. Moreover, the autokinase activity of BtsS could be stimulated in the presence of pyruvate. Measurements of in vitro autophosphorylation activity were used as a parameter to determine the molecular effects of individual amino acid replacements in BtsS, and the effects resulted not only in massive changes in activity levels but also in pyruvate-independent activity for many variants. The elucidation of the pyruvate-binding site of BtsS opened the possibility of redesigning BtsS into a sensor of pyruvate-like ligands such as lactate, which can be used as a biosensor in medicine. The required workflow and a high-throughput screening system were established.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: bacterial sensing and signal transduction, receptor, membrane protein, stimulus perception
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Dezember 2023
• 1. Bericht­erstatter:in: Jung, Kirsten