Nemoto Mai, Mio Kazuhiro, Kanamaru Shuji, Arisaka Fumio
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4359-B39 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
J Bacteriol. 2008 May;190(10):3606-12. doi: 10.1128/JB.00095-08. Epub 2008 Mar 7.
KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.
KVP40是一种与T4相关的噬菌体，由386个开放阅读框（ORF）组成，宿主范围广泛。在此，我们对KVP40基因组中编码的两种蛋白质，即gp5和ORF334进行了过表达、纯化及生物物理特性分析。通过基于同源性的比较，将KVP40与其特征更明确的姐妹噬菌体T4进行比较，以评估这两种KVP40蛋白质的功能。KVP40 gp5在N端和C端结构域与T4 gp5具有显著同源性。与T4 gp5不同，KVP40 gp5缺乏内部溶菌酶结构域。与T4 gp5一样，发现KVP40 gp5在溶液中形成同源三聚体。与之形成鲜明对比的是，KVP40 ORF334与T4相关噬菌体的任何已知蛋白质均无显著同源性。发现KVP40 ORF334在溶液中与KVP40 gp5形成异源六聚体，其方式与T4 gp5和gp27蛋白之间的相互作用几乎相同。对KVP40 gp5 - ORF334复合物的电子显微镜图像分析表明，其尺寸与T4 gp5 - gp27结构非常相似。基于我们的生物物理特性分析以及基因组位置信息，我们提出ORF334是T4 gp27的直系同源物，并且它在gp5和噬菌体基板之间起连接物的作用。
