The treatment and management of such infections has been compounded by the emergence of multi-antibiotic resistant strains such as MRSA (methicillin resistant Staphylococcus aureus) in both hospital and community settings highlighting the urgent need for novel anti-infective agents ( Turner et al., 2019 Cheung et al., 2021). Staphylococcus aureus is an important human pathogen capable of causing a broad range of mild to severe infections from, for example, wound, blood-borne and respiratory infections to exotoxin-mediated diseases such as scalded skin and toxic shock syndromes ( Rasigade et al., 2014 Balasubramanian et al., 2017 Cheung et al., 2021). An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. 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. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. 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. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. Virulence gene expression in the human pathogen, S. 5School of Pharmacy, Biodiscovery Institute, University of Nottingham, Nottingham, United Kingdom.4ISIS Neutron and Muon Source, Rutherford Appleton Laboratory, Harwell Oxford, Didcot, United Kingdom.3Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, Oxfordshire, United Kingdom.2School of Biosciences, University of Nottingham, Loughborough, United Kingdom.1Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.Chan 5, Philip Hill 2, Paul Williams 1* † and Boyan B. Murray 1, Isobel Blower 1,2, Sara Zandomeneghi 1, Alice Goode 1, Rohanah Hussain 3, Divya Kumari 1, Giuliano Siligardi 3, Katsuaki Inoue 3, Jeni Luckett 1, James Doutch 4, Jonas Emsley 5, Weng C.
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