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Philipps-Universität Marburg
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Abstract
The PhoQ/PhoP two-component system plays an important role in sensing and responding to various environmental signals, including magnesium limitation, low pH, and antimicrobial peptides. Upon sensing the stimuli, the histidine kinase PhoQ undergoes autophosphorylation. The phosphoryl group is then transferred to a highly conserved aspartate of PhoP. The phosphorylated PhoP can bind to the PhoP-box within the promoter region of target DNAs, thereby regulating numerous gene expressions. The small membrane protein MgrB, regulated by PhoQ/PhoP, can inhibit PhoQ histidine kinase activity, thus forming a negative feedback loop. However, the mechanism of this inhibition remains largely unknown. Moreover, small proteins have been discovered across all domains of life, and small membrane proteins constitute a significant portion of newly discovered small proteins. However, functional studies of small membrane proteins are challenging due to their size limitation and hydrophobic nature. To address these questions, in this thesis, the results not only elucidate the inhibitory mechanism between PhoQ and MgrB in vivo but also present a robust approach to generate functional small membrane proteins for discovering their function.
To elucidate this inhibition mechanism, the binding interface between PhoQ and MgrB was first identified using the chemical crosslinking approach, which was consistent with the AlphaFold2 model prediction. Subsequently, the crosslinking results showed that the binding of MgrB led to less crosslinking in the PhoQ transmembrane helix and linker region, indicating that MgrB pushed PhoQ transmembrane helices away from each other. The molecular dynamics simulation results also showed that the binding of MgrB led to the translocation of PhoQ's catalytic domain, which was necessary for PhoQ to bind adenosine triphosphate. These conformational changes ultimately resulted in an overall lowered PhoQ activity. Additionally, using combined approaches, our results revealed that MgrB appeared to mediate PhoQ sensing antimicrobial peptides under physiological magnesium conditions.
To generate small membrane proteins in a robust approach, several small membrane proteins were synthesized functionally, such as MgrB, up to a micro-molar scale using a combination of the cell-free system and lipid sponge droplets. Additionally, the cell-free system proved to be versatile, allowing for the synthesis of small membrane proteins from both eukaryotes and
prokaryotes. Furthermore, the synthesized small membrane proteins can be used for target discovery via in vitro co-immunoprecipitation.
Taken together, the results demonstrate the inhibitory mechanism of PhoQ by the small membrane protein MgrB, and provide new insights into uncovering the potential functions of small membrane proteins.
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Dates
Created: 2024Issued: 2025-02-26Updated: 2025-02-27
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
eng
Data types
DoctoralThesis
DDC-Numbers
570
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Jiang, Shan: Elucidate the mechanism of PhoQ inhibition by the small protein MgrB. : Philipps-Universität Marburg 2025-02-26. DOI: https://doi.org/10.17192/z2024.0218.