Structural basis of SPP1 bacteriophage infectivity

细菌病毒（噬菌体或噬菌体）是生物圈中数量最多的生物实体。大多数已知的噬菌体具有在感染期间作为基因组递送到宿主细胞质中的管道的尾部。这些病毒的主要结构特征是众所周知的，包括一个二十面体的头部（衣壳），它使基因组（线性双链DNA）免受危险环境的影响，一个长的柔性非收缩性的尾巴连接到衣壳，和一个专门的分子吸附装置位于自由端的尾巴。该装置对于噬菌体感染性是必不可少的，因为它检测宿主细胞表面的特异性受体。一旦受体被识别，噬菌体就会附着在细菌细胞壁上，形成一个穿过细胞膜的通道。尾巴紧紧地粘在细胞膜上，这样基因组就可以直接进入宿主细胞。噬菌体吸附装置与细菌细胞膜的相互作用诱导信号，该信号沿尾部沿着传递到噬菌体头部，在该处该信号刺激位于尾部和头部之间的连接器的开口。连接器作为一个阀门，以保持DNA锁定在衣壳。连接器的打开导致DNA释放。这一过程的生化分析提供了信息，但是，目前还不清楚信号如何通过尾部传播，以及哪些噬菌体系统组件控制结构构象变化。我们的研究已经证明了广泛的结构重排在内壁的SPP 1噬菌体的尾管，然而，它仍然是未知的事件的顺序诱导DNA释放。我们认为，吸附装置-受体相互作用触发了构象转换，该转换以多米诺骨牌式级联方式沿着1600 μ m长的螺旋尾部传播，到达头-尾连接器。这导致连接器的打开，最终导致DNA通过尾管从头部进入宿主细胞。为了验证这一假设，我们需要记录受体结合后尾部结构中发生的结构变化，直到基因组成功地从噬菌体颗粒中释放出来。在这种类型的研究中，噬菌体SPP 1是一个独特的模型，因为SPP 1特异性受体已被鉴定和纯化。在体外可以控制DNA从噬菌体颗粒中排出的过程，并且可以测试时间依赖性。由于噬菌体是巨大的不对称大分子系统，电子显微镜（EM）结合图像分析是首选的方法。现代样品制备方法允许捕获噬菌体的结构构象变化，因此电子显微镜结合生物化学和生物物理方法将使我们能够观察不同状态的噬菌体。对两个突变体尾部结构的分析将阐明尾管中亚基之间的相互作用系统，时间分辨实验将启发信号传播的基础。将使用单颗粒不对称方法和断层摄影术在DNA喷射之前和之后定位噬菌体衣壳内的连接器。噬菌体组分的已知或预测的原子结构的对接将允许理解信号传播和衣壳门功能背后的结构原理。伯克贝克学院的晶体学院拥有该项目所需的EM，计算机设施和软件包。在2006年，我们已经获得了设备赠款，提供FEI 300千电子伏FEG显微镜（Polara），将在2007年秋季安装。该显微镜将配备允许自动数据收集的软件Leginon。需要更大的数据集来提高分析的可靠性。统计方法是在E.奥尔洛娃和H. Saibil允许分析异构数据集。

Bacterial viruses (bacteriophages or phages) are the most populated biological entity in the Biosphere. Most known bacteriophages have tails that serve as a pipeline at genome delivery into the host cytoplasm during infection. The main structural features of these phages are well known and include an icosahedral head (capsid) that keeps the genome (linear ds DNA) safe from a hazardous environment, a long flexible non-contractile tail attached to the capsid, and a specialised molecular adsorption apparatus located on the free end of the tail. This apparatus is essential for the phage infectivity as it detects a specific receptor in the host cell surface. Once the receptor has been recognised, the phage affixes itself to the bacterial cell wall and forms a channel through the cell membrane. The tail sticks to the cell membrane tightly so that the genome can be delivered straight into the host cell. The interaction of the phage adsorption device with the bacterial cell membrane induces a signal that is transmitted along the tail to the phage head, where the signal stimulates the opening of the connector located between the tail and the head. The connector serves as a valve to keep DNA locked in the capsid. Opening of the connector leads to the DNA release. Biochemical analysis of this process has provided information; however, it is still unclear how the signal propagates through the tail and which phage system components control structural conformational changes. Our study has demonstrated extensive structural rearrangements in the internal wall of the tail tube of SPP1 bacteriophage, however, it remains unknown what sequence of events induces DNA release. We propose that the adsorption device-receptor interaction triggers a conformational switch, which is propagated in a domino-like cascade along the 1600 Å-long helical tail to reach the head-to-tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube. To test this hypothesis we need to document the structural changes that occur in the tail structure after receptor binding until the genome is successfully released from the phage particle. In this type of study bacteriophage SPP1 is a unique model since the SPP1 specific receptor has been identified and purified. The process of DNA ejection from phage particles in vitro could be controlled and time dependence can be tested. Since bacteriopaghes are huge asymmetrical macromolecular systems, electron microscopy (EM) in combination with image analysis is the method of choice. Modern methods of sample preparation allow structural conformational changes in phages to be captured and, therefore electron microscopy in combination with biochemical and biophysical methods would allow us to observe the phage in different states. Analysis of two mutant tail structures will clarify a system of interactions between subunits in the tail tube and time resolving experiments will enlighten a basis of the signal propagation. A single particle asymmetrical approach and tomography will be used to localize the connector within the phage capsid before and after DNA ejection. Docking of known or predicted atomic structures of the phage components will allow understanding of structural principles behind signal propagation and function of the capsid gate. The School of Crystallography at Birkbeck College has the EM, computer facilities and software packages required for the project. In 2006 year we have obtained an equipment grant that is providing an FEI 300 keV FEG microscope (Polara), that will be installed in autumn 2007. This microscope will be equipped with the software Leginon that allows automated data collection. Larger data sets are required to improve the reliability of analysis. Statistical approaches developed in the EM groups of Dr. E. Orlova and Prof. H. Saibil allow analysis of heterogeneous data sets.