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子有助于微生物在不同的生态位中定居。在极端细菌中，基因转移已被认为是适应和多样化的主要力量，并且域间基因流动已被广泛报道。嗜热菌Thermus thermophilus具有DNA转运蛋白，其介导从所有生命领域的成员摄取游离DNA。在第一个资助期内，我们对DNA转运蛋白的两个关键参与者进行了彻底的结构/功能分析，即马达ATP酶PilF和分泌素PilQ，两者都形成了复杂的组件。为了深入了解PilF复合物的结构，我们试图使复合物结晶。这种方法是成功的，最近，获得了第一个晶体。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与膜基底相互作用，但其相互作用伙伴未知。我们发现PilF的ATP酶活性受PilM和N的刺激，并且这两种蛋白质也与PilF共同作用，表明PilF-PilM-PilN相互作用。我们将通过纯化杂聚PilFMN组件来验证这一假设，并通过生物化学和结构手段分析它们的结构和相互作用。PilF不仅作为马达ATP酶发挥作用，而且出乎意料地还结合DNA。我们将更详细地阐述这一发现，并分析其在DNA转运过程中的意义。PilQ的高分辨率结构不可用。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，并提供了其第一个EM结构。它有一个独特的结构，包括一个圆锥体，一个杯子和六个堆叠的环。缺失分析导致PilQ结构域的鉴定，这些结构域构建了六个环，并解开了它们在转化和毛化中的功能。接下来，我们的目标是阐明复杂的组装和转换所需的环的最小数量。PilQ的C-末端具有存在于许多分泌素中的保守结构域，从而介导与特异性先导蛋白的结合。我们已经获得了PilQ与PilW相互作用的强烈迹象。这一假设将得到验证，并将确定C-末端结构域在这种相互作用中的作用。一个突破性的观察是在PilQ复合物的锥体顶部检测到明显参与DNA结合的其他蛋白质。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（MPI of Biophysics，法兰克福）的合作中，我们通过冷冻ET提出了PilQ复合物的第一个原位结构。接下来，我们的目标是原位鉴定与PilQ复合物连接的蛋白质。

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.