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

获得新的遗传决定因素有助于微生物在不同的生态位中定植。在嗜极细菌中，基因转移已被认为是适应和多样化的主要力量，基因域间流动已被广泛报道。嗜热细菌拥有一个DNA转运体，它介导从所有生命领域的成员摄取游离DNA。在第一个资助期，我们对DNA转运体的两个关键参与者，运动atp酶PilF和分泌素PilQ进行了彻底的结构/功能分析，两者都形成复杂的组装体。为了深入了解PilF复合物的结构，我们试图使该复合物结晶。这种方法是成功的，最近，获得了第一批晶体。这些实验将继续进行，目的是获得高分辨率的x射线结构。PilF与膜基相互作用，但其相互作用伙伴是未知的。我们发现PilF的atp酶活性受到PilM和N的刺激，并且这两种蛋白也与PilF共洗脱，表明PilF-PilM- piln相互作用。我们将通过纯化异聚PilFMN组件来验证这一假设，并通过生化和结构手段分析它们的结构和相互作用。PilF不仅具有运动atp酶的功能，而且出乎意料的是，它还能结合DNA。我们将详细阐述这一发现，并分析其在DNA运输过程中的意义。没有高分辨率的PilQ结构。在上一个资助期内，我们获得了PilQ复合物的第一个晶体，现在将进行x射线分析，以获得对PilQ的第一个高分辨率见解。我们建立了纯化PilQ复合物的程序，并提供了其首个EM结构。它有一个独特的结构，由一个锥体，一个杯子和六个堆叠的环组成。缺失分析确定了构建六个环的PilQ结构域，并揭示了它们在转化和调解中的功能。接下来，我们的目标是阐明复杂组装和转换所需的最小环数。PilQ的c端有一个保守结构域，存在于许多分泌蛋白中，从而介导与特定先导蛋白的结合。我们已经获得了强有力的迹象表明PilQ与PilW相互作用。这一假设将得到验证，并确定c端结构域在这种相互作用中的作用。一项突破性的观察是在PilQ复合体的锥体上发现了额外的蛋白质，这些蛋白质显然与DNA结合有关。这些蛋白质将被鉴定和表征，并分析它们在DNA结合中的作用。最近，在与Vicki Gold（法兰克福生物物理学MPI）的合作中，我们通过冷冻- et提出了第一个原位结构的PilQ复合物。接下来，我们的目标是原位鉴定与PilQ复合物相连的蛋白质。

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.