Arechaga Ignacio, Peña Alejandro, Zunzunegui Sandra, del Carmen Fernández-Alonso María, Rivas Germán, de la Cruz Fernando
Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC, CSIC-UC-IDICAN, Santander, Spain.
J Bacteriol. 2008 Aug;190(15):5472-9. doi: 10.1128/JB.00321-08. Epub 2008 Jun 6.
Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.
IV型分泌系统（T4SS）介导DNA和蛋白质底物向靶细胞的转移。由接合质粒R388编码的TrwK是VirB4家族的成员，该家族包含T4SS中最大且最保守的蛋白质。有人认为VirB4是一种ATP酶，参与为菌毛组装和底物转运提供能量。然而，关于VirB4 ATP水解酶活性的实验证据相互矛盾。在此，我们证明TrwK在没有其潜在大分子底物和其他T4SS组分的情况下能够在体外水解ATP。已经对其ATP酶活性的动力学参数进行了表征。通过分析超速离心和电子显微镜研究了TrwK的寡聚化状态，并分析了其对ATP酶活性的影响。结果表明，TrwK的六聚体形式是催化活性状态，这与结构相关的蛋白质TrwB（接合偶联蛋白）非常相似。
