An engineered right-handed coiled coil domain imparts extreme thermostability to the KcsA channel.
KcsA, a potassium channel from Streptomyces lividans, was the first ion channel to have its transmembrane domain structure determined by crystallography. Previously we have shown that its C-terminal cytoplasmic domain is crucial for the thermostability and the expression of the channel. Expression was almost abolished in its absence, but could be rescued by the presence of an artificial left-handed coiled coil tetramerization domain GCN4. In this study, we noticed that the handedness of GCN4 is not the same as the bundle crossing of KcsA. Therefore, a compatible right-handed coiled coil structure was identified from the Protein Data Bank and used to replace the C-terminal domain of KcsA. The hybrid channel exhibited a higher expression level than the wild-type and is extremely thermostable. Surprisingly, this stable hybrid channel is equally active as the wild-type channel in conducting potassium ions through a lipid bilayer at an acidic pH. We suggest that a similar engineering strategy could be applied to other ion channels for both functional and structural studies.
Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Canada.
Zhiguang
Z
Yuchi
Victor P T
VP
Pau
Bridget X
BX
Lu
Murray
M
Junop
Daniel S C
DS
Yang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D001483:Base Sequence; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; C107790:prokaryotic potassium channel