Structural basis of SPP1 bacteriophage infectivity

细菌病毒(噬菌体或噬菌体)是生物圈中种群最多的生物实体。大多数已知的噬菌体都有尾巴，在感染期间将基因组输送到宿主细胞质时，尾巴起到管道的作用。这些噬菌体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)免受危险环境的伤害，附着在衣壳上的长而灵活的非收缩尾巴，以及位于尾部自由端的专门的分子吸附装置。这一装置对噬菌体的感染性至关重要，因为它检测宿主细胞表面的特定受体。一旦受体被识别，噬菌体就会将自己附着在细菌细胞壁上，并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送入宿主细胞。噬菌体吸附装置与细菌细胞膜的相互作用诱导了一个信号，该信号沿着尾部传输到噬菌体头部，其中该信号刺激位于尾部和头部之间的连接器的打开。这个连接器充当了一个阀门，将DNA锁在衣壳里。连接器的打开导致DNA的释放。对这一过程的生化分析已经提供了信息；然而，仍然不清楚信号如何通过尾巴传播，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SPP1噬菌体尾管内壁存在广泛的结构重排，然而，目前还不清楚是什么事件序列导致了DNA的释放。我们认为，吸附器件-受体的相互作用触发了一个构象开关，该开关沿着1600？长的螺旋尾巴以多米诺骨牌状的级联方式传播到头尾连接体。这导致连接器的打开，最终导致DNA从头部通过尾管进入宿主细胞。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SPP1是一个独特的模型，因为SPP1的特异性受体已经被鉴定和纯化。在体外，DNA从噬菌体颗粒中排出的过程是可以控制的，并且可以检验其时间依赖性。由于噬菌体是巨大的不对称大分子系统，电子显微镜(EM)与图像分析相结合是选择的方法。现代的样品制备方法可以捕捉噬菌体的结构构象变化，因此，电子显微镜与生化和生物物理方法相结合将使我们能够观察不同状态下的噬菌体。对两个突变的尾巴结构的分析将阐明尾管中亚单位之间的相互作用系统，时间分辨实验将为信号传播提供基础。将使用单颗粒不对称方法和断层扫描来定位DNA喷射前后噬菌体衣壳内的连接器。对接已知或预测的噬菌体成分的原子结构将允许理解信号传播背后的结构原理和衣壳门的功能。伯克贝克学院的结晶学院拥有该项目所需的EM、计算机设施和软件包。2006年，我们获得了一项设备拨款，将提供一台FEI 300kev FEG显微镜(Polara)，该显微镜将于2007年秋季安装。这台显微镜将配备Leginon软件，允许自动数据收集。需要更大的数据集来提高分析的可靠性。E.Orlova博士和H.Saibil教授的EM小组开发的统计方法允许分析不同种类的数据集。

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.