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

DOI: 10.3389/fchem.2023.1113885

Authors: Philip Bardelang (University of Nottingham) , Ewan J. Murray (University of Nottingham) , Isobel Blower (University of Nottingham) , Sara Zandomeneghi (University of Nottingham) , Alice Goode (University of Nottingham) , Rohanah Hussain (Diamond Light Source) , Divya Kumari (University of Nottingham) , Giuliano Siligardi (Diamond Light Source) , Katsuaki Inoue (Diamond Light Source) , Jeni Luckett (University of Nottingham) , James Doutch (ISIS Neutron and Muon Source) , Jonas Emsley (University of Nottingham) , Weng C. Chan (University of Nottingham) , Philip Hill (University of Nottingham) , Paul Williams (University of Nottingham) , Boyan B. Bonev (University of Nottingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Frontiers In Chemistry , VOL 11

State: Published (Approved)
Published: May 2023
Diamond Proposal Number(s): 5098 , 12923 , 13185 , 13634 , 15146

Abstract: Virulence gene expression in the human pathogen, S. 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. 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. 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 in Staphylococcus aureus, we provide experimental evidence 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 Escherichia 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 provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. 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.

Journal Keywords: quorum sensing; AgrB; AgrD; Staphylococcus aureus; membrane protein structure; molecular dynamics simulations; synchrotron radiation circular dichroism; small angle X-ray scattering

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B21-High Throughput SAXS , B23-Circular Dichroism

Added On: 06/05/2023 10:51

Documents:
fchem-11-1113885.pdf

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Biophysics Life Sciences & Biotech

Technical Tags:

Scattering Spectroscopy Small Angle X-ray Scattering (SAXS) Circular Dichroism (CD)