Rabel Christian, Grahn A Marika, Lurz Rudi, Lanka Erich
Max-Planck-Institut für Molekulare Genetik, Abteilung Lehrach, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany.
J Bacteriol. 2003 Feb;185(3):1045-58. doi: 10.1128/JB.185.3.1045-1058.2003.
Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.
VirB4家族蛋白由接合质粒和IV型分泌系统编码，这些系统指定了与接合系统相关的大分子输出机制。VirB4蛋白的核心特征是核苷酸结合位点。在本研究中，我们探究了VirB4蛋白家族成员在一级结构上是否存在相似性，以及这些蛋白是否水解核苷酸。对VirB4蛋白家族的19个成员进行的多序列比对显示出显著的整体相似性。我们定义了四个共同基序和一个保守结构域。该蛋白家族的一个成员，质粒RP4的TrbE，通过定点诱变进行了遗传特征分析。trbE中的大多数突变导致其活性完全丧失，从而消除了菌毛产生、质粒特异性噬菌体的繁殖以及大肠杆菌中的DNA转移能力。对RP4 TrbE的可溶性衍生物和全长同源蛋白R388 TrwK的生化研究表明，VirB4蛋白家族的这些成员的纯化形式不水解ATP或GTP，并且在溶液中表现为单体。
