Parker M H, Prevelige P E
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA.
Virology. 1998 Oct 25;250(2):337-49. doi: 10.1006/viro.1998.9386.
The first step in assembly of the bacteriophage P22 is the formation of a T=7 icosahedral "procapsid," the major components of which are the coat protein and an inner core composed of the scaffolding protein. Although not present in the mature virion, the scaffolding protein is required for procapsid assembly. Eleven amino-acid residues at the extreme carboxyl terminus of the scaffolding protein are required for binding to the coat protein, and upon deletion of these residues, approximately 20 additional residues become disordered. Sequence analysis and NMR data suggest that the 30 residues at the carboxyl terminus form a helix-loop-helix motif which is stabilized by interhelical hydrophobic interactions. This "coat protein recognition domain" presents an unusually high number of positively charged residues on one face, suggesting that electrostatic interactions between this domain and the coat protein may contribute to recognition and binding. We report here that high ionic strength (1 M NaCl) completely inhibited procapsid assembly in vitro. When scaffolding protein was added to empty procapsid "shells" of coat protein, 1 M NaCl partially inhibited the binding of scaffolding protein to the shells. This suggests that the positively charged coat protein recognition domain at the carboxyl terminus of the scaffolding protein binds to a negatively charged region on the coat protein. During DNA packaging, the scaffolding protein exits the procapsid; scaffolding protein exit is followed by the expansion of the procapsid into a mature capsid. Procapsid shells can be induced to undergo a similar expansion reaction in vitro by heating (45-70 degreesC); this process was also inhibited by 1 M NaCl. These results are consistent with a model in which negatively charged scaffold protein-binding domains in the coat proteins move apart during procapsid expansion; this relief of electrostatic repulsion could provide a driving force for expansion and subsequent maturation. High-salt concentrations would screen this repulsion, while packaging of DNA (a polyanion) in vivo may increase the instability of the procapsid enough to trigger its expansion.
噬菌体P22组装的第一步是形成T = 7二十面体“原衣壳”，其主要成分是衣壳蛋白和由支架蛋白组成的内核。尽管支架蛋白不存在于成熟病毒粒子中，但原衣壳组装需要它。支架蛋白极端羧基末端的11个氨基酸残基是与衣壳蛋白结合所必需的，缺失这些残基后，大约另外20个残基会变得无序。序列分析和核磁共振数据表明，羧基末端的30个残基形成一个螺旋-环-螺旋基序，该基序通过螺旋间疏水相互作用得以稳定。这个“衣壳蛋白识别结构域”在一面呈现出异常多的带正电荷残基，这表明该结构域与衣壳蛋白之间的静电相互作用可能有助于识别和结合。我们在此报告，高离子强度（1 M NaCl）在体外完全抑制原衣壳组装。当将支架蛋白添加到衣壳蛋白的空原衣壳“壳”中时，1 M NaCl部分抑制支架蛋白与壳的结合。这表明支架蛋白羧基末端带正电荷的衣壳蛋白识别结构域与衣壳蛋白上带负电荷的区域结合。在DNA包装过程中，支架蛋白从原衣壳中退出；支架蛋白退出后，原衣壳扩展为成熟衣壳。通过加热（45 - 70摄氏度）可在体外诱导原衣壳壳发生类似的扩展反应；该过程也受到1 M NaCl的抑制。这些结果与一个模型一致，即衣壳蛋白中带负电荷的支架蛋白结合结构域在原衣壳扩展过程中分开；这种静电排斥的缓解可为扩展及随后的成熟提供驱动力。高盐浓度会屏蔽这种排斥作用，而体内DNA（一种多阴离子）的包装可能会使原衣壳的不稳定性增加到足以触发其扩展的程度。
