AUTHOR=Bardelang Philip , Murray Ewan J. , Blower Isobel , Zandomeneghi Sara , Goode Alice , Hussain Rohanah , Kumari Divya , Siligardi Giuliano , Inoue Katsuaki , Luckett Jeni , Doutch James , Emsley Jonas , Chan Weng C. , Hill Philip , Williams Paul , Bonev Boyan B. 

TITLE=Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate

JOURNAL=Frontiers in Chemistry

VOLUME=Volume 11 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2023.1113885

DOI=10.3389/fchem.2023.1113885

ISSN=2296-2646

ABSTRACT=Virulence gene expression in the human pathogen, Staphylococcus aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB and is released into the extracellular environment.  Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and MD annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays, we provide experimental evidence to suggest that the AgrB N- and C- termini are located on the cytoplasmic side of the membrane and that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from E. coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD.  Conformational alteration of AgrB following exogenous provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. We obtained atomistic description of AgrB and AgrD and explored the stages of interaction, stability and substrate processing. We confirm the 6TMD membrane topology of AgrB and the existence of molecular complexes with AgrD in vitro and in vivo.