Characterization of the molecular role of PcoB in copper efflux in Caulobacter crescentus

Student thesis: Doc typesDoctor of Sciences

Abstract

Living organisms face a dual role of heavy metals: essential as cofactors for certain proteins but toxic in excess. Maintaining a delicate balance in heavy metal concentrations is crucial for cellular well-being. In response to toxic levels of copper (Cu), C. crescentus, a dimorphic aquatic bacterium employs a bi-modal strategy called "fight or flight." The motile swarmer cell flights away from the Cu stress, while the sessile stalked cell engages in Cu detoxification using the PcoAB system (Lawareé et al., 2016). The molecular processes facilitating Cu efflux through PcoB remain poorly understood, especially the mechanisms enabling Cu export at the outer membrane (OM) in the absence of hydrolysable energy sources (such as ATP, GTP) or ion gradients.
The 3D structure prediction of PcoB reveals a compact β-barrel and a disordered N-terminal (N-term) domain. Experimental characterization using circular dichroism spectroscopy (CD) aligns with bioinformatic predictions, classifying PcoB as an intrinsically disordered protein (IDP).
Canonical efflux pumps involve tripartite complexes; however, a pull-down assay aimed at identifying partners for PcoB, capable of forming such an organization, fails to reveal any significant candidates. Drawing inspiration from an entropy-driven transport mechanism observed in Bacteroidetes, we propose a hypothesis suggesting that the disordered N-term domain of PcoB creates a pocket for Cu ions, facilitating their efflux via an energy-independent mechanism.
The deletion of the N-term domain highlights its crucial role in Cu tolerance. Replacing the N-term domains of C. crescentus with the poorly conserved domain from E. coli restored the WT phenotype, indicating that PcoB’s function is not strictly ‘sequence dependent’. A minimum set of His residues within the N-term domain is required for the function hinting at their potential role in Cu coordination. Additionally, we emphasized a potential interaction between PcoA and PcoB mediated by the PcoB's N-term domain. indeed, the stability of PcoA and PcoB is compromised in mutants lacking this N-term domain.
Additionally, we unexpectedly shed light on an intriguing aspect of PcoB. The protein exhibits a poorly conserved Sec-like sequence at its N-term extremity that is not essential for its secretion into the periplasmic environment and the OM. Indeed, PcoB lacking this predicted signal peptide (SP) is fully functional in terms of Cu tolerance and is present in the periplasmic + OM fraction, challenging conventional translocation assumptions.
Date of Award12 Feb 2024
Original languageEnglish
Awarding Institution
  • University of Namur
SponsorsCERUNA
SupervisorJean-Yves Matroule (Supervisor), Xavier De Bolle (President), Catherine Michaux (Jury), Françoise Jacob-Dubuisson (Jury) & Marianne Ilbert (Jury)

Keywords

  • bacteria
  • Caulobacter Crescentus
  • copper
  • copper efflux
  • intrinsic disordered protein
  • energy-less mediated efflux

Attachment to an Research Institute in UNAMUR

  • NARILIS

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