The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors.
Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.
Tato
Irantzu
I
Departamento de Biología Molecular, Universidad de Cantabria (UC) e Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), 39011 Santander, Spain.
Matilla
Inmaculada
I
Arechaga
Ignacio
I
Zunzunegui
Sandra
S
de la Cruz
Fernando
F
Cabezon
Elena
E
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D003227:Conjugation, Genetic / Q000502:physiology; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; C006094:R388; D012097:Repressor Proteins; C061260:RuvB protein, Bacteria; C119757:TrwA protein, E coli; C403125:trwB protein, E coli; D013762:Tetrahydrofolate Dehydrogenase; C071059:Holliday junction DNA helicase, E coli; D006180:Proton-Translocating ATPases; D004265:DNA Helicases