Structure, function and interaction partners of the traffic ATPase (PilF) and the secretin (PilQ) in a bacterial DNA transport machinery

• Applicant: Professorin Dr. Beate Averhoff
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2012 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 211427752

The acquisition of novel genetic determinants facilitates microorganisms to colonize diverse niches. In extremophilic bacteria, gene transfer has been recognized as a major force in adaptation and diversification, and inter-domain gene flow has been widely reported. The thermophilic bacterium Thermus thermophilus possesses a DNA transporter which mediates uptake of free DNA from members of all domains of life. In the first funding period we have performed a thorough structure/function analysis of two key players of the DNA transporter, the motor ATPase PilF and the secretin PilQ, both of which form complex assemblies. To get insights into the structure of the PilF complex, we tried to crystallize the complex. This approach was successfull and recently, the first crystals were obtained These experiments will be continued with the aim to obtain a high resolution X-ray structure. PilF is interacting with a membrane base but its interaction partners are unknown. We found that the ATPase activity of PilF is stimulated by PilM and N, and both proteins also co-elute with PilF, indicating a PilF-PilM-PilN interaction. We will verify this hypothesis by purifying heteropolymeric PilFMN assemblies and analyze their structure and interaction by biochemical and structural means. PilF not only functions as a motor ATPase, but unexpectedly, also binds DNA. We will elaborate on this finding in more detail and analyze its significance in the process of DNA transport. A high resolution structure of PilQ is not available. In the last funding period, we obtained the first crystals of the PilQ complex, and X-ray analyses will now be performed to get first high resolution insights into PilQ. We established a procedure to purify the PilQ complex and provided its first EM structure. It has a unique structure comprising of a cone, a cup and six stacked rings. Deletion analyses led to the identification of domains of PilQ that built the six rings and unravelled their function in transformation and piliation. Next, we aim to elucidate the minimal number of rings required for complex assembly and transformation. The C-terminus of PilQ has a conserved domain which is present in many secretins thereby mediating binding to specific pilot proteins. We have obtained strong indications that PilQ interacts with PilW. This hypothesis will be verified, and the role of the C-terminal domain in this interaction will be determined. A ground-breaking observation was the detection of additional proteins on top of the cone of the PilQ complex that are apparently involved in DNA binding. These proteins will be identified and characterized, and their role in DNA binding analyzed. Recently, in collaboration with Vicki Gold (MPI of Biophysics, Frankfurt) we presented the first in situ structure of a PilQ complex by cryo-ET. Next, we aim to identify the proteins linked to the PilQ complex in situ.

• DFG Programme: Research Grants