Molecular analysis of the Escherichia coli glutathione-gated potassium efflux system, KefC
Potassium is the major intracellular cation of the bacterium Escherichia coli and is accumulated at high levels in order to maintain turgor pressure. Regulation of potassium is achieved by a number of uptake and efflux systems. The efflux of potassium is regulated by five different systems, two of which are KefB and KefC. The KefB and KefC systems play a major role in projection of cells against the toxicity of electrophiles (e.g. N-ethylmaleimide and methylglyoxal) and these proteins exhibit strong similarity at most levels. Both systems consist of a channel protein (KefB and KefC) and an ancillary protein (KefG and KefF) that is required for full activity. The two systems are maintained in a closed state by glutathione (GSH). Both systems are activated by GSH adducts, which are formed by the reaction between GSH and an electrophile. They differ in their response to methylglyoxal, with only KefB being strongly activated by this electrophile. The KefC protein comprises of two major domains (each of which have sub-domains)- an N-terminal membrane domain and a C-terminal “soluble” domain, conducted by a Q-linker. The linker sequence is a predominantly hydrophilic with a large number of acidic residues. This thesis presents mutational studies on the linker sequence that show the linker length and residue position are important for full activity of the KefC system. Within the N-terminal domain of KefC a conserved sequence (HALESDIEP) has been identified that is important for channel regulation and gating. Further experiments show that this region interacts with specific residues within the two sub-domains of the carboxy-terminal domain. The C- terminal domain consists of a KTN and a SAM domain, residues in both these sub-domains are confirmed to play a role in channel gating and regulation via interactions with GSH and the HALESDIEP sequence. Cross-linking studies show the linker region to be highly flexible also the KefC channel protein is shown to form oligomers.