Lin, Hong-Ting (2008) Characterisation of the MacA/MacB/TolC tripartite pump that confers resistance to macrolides in E. coli. Doctoral thesis, Durham University.
Gram-negative bacteria possess tripartite pumps, composed of a membrane fusion protein (MFP), an inner membrane protein (IMP) and an outer membrane protein (OMP), to transport drugs across the inner and outer membranes. The plasmid encoding MacA, MacB and TolC confers resistance to the macrolide erythromycin in the host E. coli cell Kam3, indicating the three proteins are assembled and actively functional. MFPs are believed to have an important role in the stabilizing the pump complex; intriguingly, we found that the MFP MacA not only interacts directly with the IMP MacB and the OMP TolC, but regulates the function of MacB, apparently increasing its affinity for both ATP and erythromycin. As MacB hydrolyzes ATP there is a burst in phosphate production that is symptomatic of the reaction being rate- limited by product release; but the burst disappeared in the presence of MacA. Since MacA caused only a marginal increase in the k(_cat), but a significant decrease in the Km, for the steady-state ATPase activity, this suggests that the disappearance of the phosphate burst is due to a decrease in the rate of hydrolysis, rather than an increase in the rate of product release. This kinetic behaviour indicates that MacA promotes and stabilizes the ATP-binding form of the transporter. MacA regulates the activity of MacB via its ẞ-strand domain since S. aureus MacA, which lacks the coiled coil structure that is present in E. coli MacA and believed to be involved in the interaction with TolC, was able to abolish the Pi burst catalysed by MacB, in direct analogy with the effect of E. coli MacA on MacB. Analytical ultracentrifugation, mass spectrometry and atomic force microscopy indicated that MacB forms dimers, in analogy to ABC- transporters that require a pair of NBDs to bind ATP. Our data suggests a direct role for MacA in facilitating the delivery of drugs by MacB to TolC: by enhancing the binding of drugs to MacB and stabilizing the reorientation of MacB to the outward- facing conformation.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:28|