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Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 958Article in journal (Refereed) Published
Abstract [en]

The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homodimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019. Vol. 10, article id 958
Keywords [en]
T4SS, accessory factor, conjugation, relaxosome, X-ray crystallography, protein structural and functional analysis
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-159103DOI: 10.3389/fmicb.2019.00958ISI: 000467035600001OAI: oai:DiVA.org:umu-159103DiVA, id: diva2:1316399
Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2019-06-13Bibliographically approved

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Rehman, SaimaSchmitt, AndreasLassinantti, LenaBerntsson, Ronnie P. -A.
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