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Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

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Abstract

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening–closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.

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Acknowledgements

We are grateful to Sandra Barral and Stephanie Siegmund (University of Columbia, NY, USA) for assistance with statistical analysis and English editing of the manuscript, respectively.

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Author notes

  1. Delfina Larrea

    Present address: Department of Neurology, Columbia University Medical Center, New York, USA

  2. Héctor D. de Paz

    Present address: Molecular Microbiology Department, University Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain

  3. Inmaculada Matilla

    Present address: Cell Biology Unit, Children’s Medical Research Institute, Westmead, Australia

Authors and Affiliations

  1. Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain

    Delfina Larrea, Héctor D. de Paz, Inmaculada Matilla, Dolores L. Guzmán-Herrador, Fernando de la Cruz, Elena Cabezón & Matxalen Llosa

  2. Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (Universidad de Cantabria, CSIC, SODERCAN), Santander, Spain

    Delfina Larrea, Héctor D. de Paz, Inmaculada Matilla, Dolores L. Guzmán-Herrador, Fernando de la Cruz, Elena Cabezón & Matxalen Llosa

  3. Department of Biochemistry and Molecular Biophysics, Center for Computational Biology and Bioinformatics, Columbia University, New York, NY, 10032, USA

    Gorka Lasso

  4. Department of Systems Biology, Howard Hughes Medical Institute, Columbia University, New York, NY, 10032, USA

    Gorka Lasso

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  1. Delfina Larrea
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Correspondence to Matxalen Llosa.

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Funding

This work was supported by Grants BIO2013-46414-P, BFU2016-78521-R, and BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness to ML, EC, and FdlC, respectively. GL acknowledges funding from NIH Grants GM030518, S10OD012351, and S10OD021764. DL was a recipient of a predoctoral fellowship from CSIC (JAE-PRE). DLG-H and IM were recipients of predoctoral fellowships from the University of Cantabria.

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Larrea, D., de Paz, H.D., Matilla, I. et al. Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB. Mol Genet Genomics 292, 1037–1049 (2017). https://doi.org/10.1007/s00438-017-1331-3

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