Relaxosome NMR studies

Bacterial conjugation is the unidirectional transfer of single-stranded DNA of conjugative plasmids or chromosome-encoded conjugative elements from a donor to a recipient cell via direct cell to cell contact. This kind of horizontal gene transfer is commonly used by bacteria for exchanging genetic information and is an important driving force for the evolution of bacteria, archaea and unicellular eukaryotes. Conjugative DNA transfer is for example the main mechanism for the spread of antibiotic resistance genes. All of the proteins necessary for nucleic acid transfer are encoded by the donor plasmid. Most of the proteins involved in the conjugation process are encoded in genes located in the tra (transfer) region. It includes proteins which form the relaxsosome, a multicomponent nucleoprotein complex which prepares the DNA for transfer. Thereby the DNA is cut by the relaxase domain of TraI and unwound by the helicase domain of TraI. Subsequently, it is transferred through the inner membrane pore protein TraD. The binding of DNA to TraI is facilitated through the binding of auxiliary proteins TraY, TraM and IHF to DNA. The relaxosome interacts with the membrane protein TraD via TraM. Specific interactions between auxiliary proteins and DNA not only enhance the specificity but also the efficiency of the relaxase and even promote local melting of dsDNA. Not much is currently known about how the auxiliary proteins enhance the DNA binding specificity. Within this project we will investigate the structure of the auxiliary protein TraY. To understand the biological function not only the structure but also the interaction with DNA is important. Therefore the structure or interaction of TraY with DNA will be studied. In addition we will be looking at structural changes of the C-terminal tetramerization domain of TraM as a function of pH and how such changes affect the binding to TraD. Deciphering the pH dependent binding of TraM to TraD might help in the hunt for the elusive "mating signal" which inititates bacterial conjugation upon the formation of cell to cell contact. For these structural studies we will use relaxation enhancements exerted by a paramagnetic environment (PREs) to probe the distances of nuclei from the protein surfaces. The PREs will be used together with conventional structural restraints in an alternative structure calculation algorithm which will be further developed within this project.

Bacterial conjugation is the unidirectional transfer of DNA between bacteria through cell to cell contact. It constitutes a major mechanism for bacterial evolution and the spread of antibiotic resistance. In this process, the relaxosome (a protein-nucleic acid complex) attaches to the coupling protein TraD, which then transfers the DNA or a DNA-protein complex through the membranes of Gram negative bacteria. During this project we have found that bacterial conjugation in the F plasmid family, in particular the docking of the relaxosome to the transfer machinery is regulated by the mainly unstructured C-terminal domain of the hexameric coupling protein TraD. This regulatory domain of TraD catches the relaxosome protein TraM in a two-step jellyfish mechanism. First, one of the six unstructured TraD tentacles binds per tetramer of TraM. Subsequently, TraM binds to TraD in a 1:1 ratio, thus it is sucked in by the TraD tentacles. The intrinsically disordered nature of this domain allows it to search a larger volume for its binding partners. The only structure found in the C-terminal domain of TraD is an alpha-helical region, which binds to the ATPase TraC. The role of this binding mechanism is currently under investigation. Therefore, in contrast to the structure-function paradigm, the most disordered domain of the coupling protein is the major regulatory switch. A stretch of only ~60 C-terminal residues combines two major functional events in bacterial conjugation and mediates interactions with at least two other proteins. In addition the structure of the TSA domain of TraI a combined relaxase and helicase of the relaxosom was determined through a collaboration and we found that this domain does not interact with the C-terminal regulatory domain of TraD.