Structure and Assembly of the Contractile Tail of Bacteriophage

本研究的目的是从分子和原子水平阐明噬菌体作为模型系统的组装和功能机制。我们获得的主要结果如下：在阐明噬菌体T4的收缩尾的结构转化的机制的最重要的进展来自与迈克尔Rossmann的小组在普渡大学的合作。Rossmann小组的Leiman通过冷冻电镜的3D图像重建成功地确定了收缩尾部的低分辨率结构。亚基的相同高分辨率结构可以拟合到收缩构象的电子密度中的事实表明，亚基本身不显著改变其构象，但在基板的构象变化期间改变基板中的相对位置。使用尾部的扩展和收缩图像和插值，我们能够获得一个相当详细的模型， 关于我们 亚基重排是如何发生的在结构改变之前，Gp 11与结构域III强烈结合，并且作为短尾纤维的gp 12的N-末端与gp 10的结构域IV结合。在基板的“六边形”到“星星”构象变化过程中起关键作用的蛋白质是gp 10。感染后，由于gp 10围绕纵轴旋转，原先与gp 12结合的gp 11现在与gp 34结合，即近端的长尾纤维，短尾纤维从基板底部伸出。其次，我们成功地阐明了尾溶菌酶（gp 5）的结构，其中351^位的氨基酸<st>被7个不同的氨基酸取代。其中一个，S351 L，其中的切割被完全抑制，表达，纯化和结晶的X-射线衍射。S351 L突变体的结构显示切割位点高度暴露，这是噬菌体成熟期间有效消化所需的。由于gp 5的结构与任何已知的蛋白酶都不相似，因此切割很可能不是自催化的，而是大肠杆菌蛋白酶的结果。突变体结构进一步证明来自相邻亚基的抑制肽在切割之前已经在底物识别位点。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