噬菌体P22是Podoviridae超家族的原型成员。自1952年发现以来,P22已成为噬菌体转导的范例和二十面体病毒衣壳组装的模型。这里,我们描述了P22尾部装置(gp1,gp4,gp10,gp9和gp26)的完整结构以及P22喷射蛋白(gp7,gp20和gp16)的潜在位置和组织,使用低温EM局部重建确定,基因敲除,和生化分析。我们发现尾部装置存在两个等价的构象,相对于衣壳旋转~6°。门静脉探针在两种构象中都与涂层亚基进行独特的接触,解释12:5对称失配。尾部围绕六角形尾部轮毂(gp10)组装,折叠成一个中断的β螺旋桨,其特征是顶端插入域。尾部集线器在近端连接到十二聚体门静脉蛋白和头对尾适配器(gp4),远端三聚体尾针(GP26),并横向连接到不对称地连接到gp10插入域的六个三聚体尾标(gp9)。缺乏喷射蛋白gp7或gp20的P22突变体的Cryo-EM分析以及纯化的重组蛋白的生化分析表明,gp7和gp20通过门静脉蛋白桶形成与尾部装置相关的分子复合物。我们确定了从尾尖到尾针的推定信号转导途径,由尾部枢纽中的三个柔性环介导,这解释了脂多糖(LPS)如何足以在体外触发P22DNA的喷射。
Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted β-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.