Yang Q, de Beer T, Woods L, Meyer J D, Manning M C, Overduin M, Catalano C E
Department of Pharmaceutical Sciences, Molecular Biology Program, University of Colorado Health Sciences Center, Denver 80262, USA.
Biochemistry. 1999 Jan 5;38(1):465-77. doi: 10.1021/bi981271d.
Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.
末端酶是来自噬菌体λ的一种酶，它是将病毒基因组插入空的原衣壳所必需的。这种酶在全酶复合物中由病毒蛋白gpNu1（20.4 kDa）和gpA（73.3 kDa）组成。目前关于末端酶组装到DNA上的模型表明，gpNu1与λ基因组中一个称为cosB的区域内的三个重复元件结合，这反过来又刺激了gpA二聚体在cosN亚位点的组装。这种预切口复合物是DNA包装所需的几种稳定核蛋白中间体中的第一个。我们注意到末端酶gpNu1亚基的一级氨基酸序列中有一个疏水区域，并假设该区域构成了在cosB处协同组装所需的蛋白质-蛋白质相互作用结构域，并且也负责观察到的分离蛋白的聚集行为。因此，我们构建了一个gpNu1突变体，其中这个疏水“结构域”已被删除，以检验这些假设。缺失突变蛋白gpNu1DeltaK完全可溶，与全长蛋白不同，没有聚集倾向；然而，通过凝胶渗透和沉降平衡分析确定，在所有检测的实验条件下，该蛋白都是二聚体。通过圆二色性和核磁共振光谱法，截短的蛋白具有二级和三级结构元件的折叠证据。虽然物理和生物学分析表明gpNu1DeltaK不与末端酶gpA亚基相互作用，但缺失突变体与含cos的DNA特异性结合。因此，我们构建了噬菌体λ末端酶gpNu1亚基的缺失突变体，它构成了该蛋白的一个高度可溶的DNA结合结构域。我们进一步提出，在赖氨酸100和脯氨酸141之间发现的疏水氨基酸定义了一个自缔合结构域，这是稳定核蛋白包装复合物组装所必需的，并且该蛋白的C末端尾巴定义了一个独特的gpA结合位点，负责末端酶全酶的形成。
