The transmembrane domain of the T4SS coupling protein TrwB and its role in protein–protein interactions

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function. © 2013 Elsevier B.V.
This work was supported with funds from the Spanish Ministerio de Educación y Ciencia (grant no. BFU2007-62062), from MICINN (grant no. BFU2010-22103) and from LSHM-CT-2005_019023 (European VI Framework Program). R.L.S. was a postdoctoral scientist supported by CSIC I3P and Basque Government postdoctoral fellowships. A.J.V. was a postdoctoral scientist supported by the University of the Basque Country. S.A-A. was a pre-doctoral student supported by the Basque Government. B.U-U. was supported by a University of the Basque Country pre-doctoral fellowship. Work in the F.C. laboratory was supported by grant 282004/FP7-HEALTH.2011.2.3.1-2 (European VII Framework Program).