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PMID:18539740
| Citation |
Arechaga, I, Peña, A, Zunzunegui, S, del Carmen Fernández-Alonso, M, Rivas, G and de la Cruz, F (2008) ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system. J. Bacteriol. 190:5472-9 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2493248 Online version:10.1128/JB.00321-08 |
| Keywords |
Adenosine Triphosphatases/isolation & purification; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Conjugation, Genetic; Image Processing, Computer-Assisted; Kinetics; Macromolecular Substances; Membrane Transport Proteins/isolation & purification; Membrane Transport Proteins/metabolism; Microscopy, Electron; Plasmids; Sequence Homology, Amino Acid; Ultracentrifugation |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0016887: ATPase activity |
ECO:0000314: |
F |
Fig. 2 - ATPase activity was monitored by decrease of NADH absorbance at 340 nm. Fig. 3 - Kinetic analysis of ATPase activity |
complete | ||||
| GO:0009291: unidirectional conjugation |
ECO:0000315: |
P |
Wild type TrwK showed a conjugation frequencies 4x10^-1 where a deletion in TrwK showed a conjugation frequency of 4x10^-6. This value is closer to the negative control of 4x10^-7 showing that TrwK is needed for conjugation. |
complete | ||||
|
NOT |
GO:0016021: integral to membrane |
ECO:0000314: |
C |
TrwK does not behave as an integral membrane protein(Paragraph) + Table 2 Researchers found that the addition of detergent and phospholipids didn't affect the solubility of TrwK in solution from which they infered that unlike homologs, TrwK was not an integral membrane protein.
|
complete | |||
|
enables |
GO:0016887: ATPase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
|
NOT|part_of |
GO:0016021: integral component of membrane |
ECO:0000314: direct assay evidence used in manual assertion |
C |
Seeded From UniProt |
complete | |||
|
involved_in |
GO:0009291: unidirectional conjugation |
ECO:0000315: mutant phenotype evidence used in manual assertion |
P |
Seeded From UniProt |
complete | |||
| GO:0016887: ATPase activity |
ECO:0000314: |
F |
Figure 2. TrwK ATP hydrolase activity was monitored by the decrease of NADH absorbance at 340 nm |
complete | ||||
|
enables |
GO:0016887: ATPase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
See also
References
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