Structure and Assembly of the Contractile Tail of Bacteriophage

本研究的目的是从分子和原子水平上阐明噬菌体作为模型系统的组装机制和功能。我们获得的主要结果如下：在阐明噬菌体T4收缩尾巴结构转化机制方面最重要的进展来自于与普渡大学Michael Rossmann的研究小组的合作。Rossmann团队的Leiman通过低温EM的3D图像重建，成功地确定了收缩尾巴的低分辨率结构。亚基的相同高分辨结构可以与收缩构象的电子密度相适应的事实表明，亚基本身并没有显著地改变其构象，但在基板构象变化过程中改变了基板上的相对位置。利用尾部的扩展和收缩图像以及内插，我们能够得到一个比较详细的…模型更多的是亚基重排发生的方式。GP11与GP12的结构域III强结合，结构改变前的短尾纤维与GP10的结构域IV结合。在基板“六角形”到“星形”构象变化过程中起关键作用的蛋白质是GP10。感染后，当GP10绕纵轴旋转时，曾经与GP12结合的GP11现在结合到近端的长尾纤维gp34上，短尾纤维从基板的底部延伸出来。其次，我们成功地阐明了尾部溶菌酶(Gp5)的结构，其中351^&lt；st&gt；位的氨基酸被7种不同的氨基酸取代。其中一个S351L的裂解被完全抑制，并被表达、纯化和结晶，用于X射线衍射。S351L突变体的结构表明，裂解位点高度暴露，这是噬菌体成熟过程中有效消化所必需的。由于gp5的结构与任何已知的蛋白酶都不相似，这种切割很可能不是自催化的，而是大肠杆菌蛋白酶的结果。突变结构进一步表明，来自邻近亚基的抑制肽在切割前已经位于底物识别位置。较少

The purpose of the present study is to elucidate the mechanism of assembly and function of bacteriophage as a model system in terms of its molecular and atomic level. The major results we obtained were the following :The most important progress in elucidating the mechanism of the structural transformation of the contractile tail of phage T4 came from the collaboration with Michael Rossmann's group at Purdue. Leiman in Rossmann's group succeeded in determining the low resolution structure of the contracted tail by 3D image reconstruction from cryo-EM. The fact that the same high resolution structure of the subunits can be fitted into the electron density of the contracted conformation indicates that the subunits themselves do not change their conformation significantly, but change the relative positions in the baseplate during conformational change of the baseplate. Using the extended and contracted images of the tail and interpolation, we were able obtain a reasonably detailed model of … More how the subunit rearrangement takes place. Gp11 strongly binds to the domain III and the N-terminus of gp12 which is the short tail fiber binds to domain IV of gp10 before structural change. The protein which plays a key role during the "hexagon" to "star" conformational change of the baseplate is gp10. Upon infection, as gp10 rotates around the longitudinal axis, gp11 which had been bound to gp12 now binds to gp34, the proximal long tail fiber, and the short tail fiber extends out from the bottom of the baseplate.Secondly, we have succeeded in elucidating the tail lysozyme (gp5) structure, where the amino acid at 351^<st> position was replaced with seven different amino acids. One of them, S351L, of which the cleavage was completely suppressed, was expressed, purified and crystallized for X-ray diffraction. The structure of the S351L mutant showed that the cleavage site was highly exposed, as would be required for efficient digestion during phage maturation. Since the structure of gp5 does not resemble any known protease, the cleavage is most likely not autocatalytic, but the result of an E.coli protease. The mutant structure further demonstrates that the inhibition peptide from the neighboring subunit is already at the substrate recognition site prior to cleavage. Less