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Báo cáo khoa học: An engineered right-handed coiled coil domain imparts extreme thermostability to the KcsA channel

KcsA, a potassium channel fromStreptomyces lividans, was the first ion channel to have its transmembrane domain structure determined by crystal-lography. 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. | ễFEBS Journal An engineered right-handed coiled coil domain imparts extreme thermostability to the KcsA channel Zhiguang Yuchi1 Victor P. T. Pau2 Bridget X. Lu1 Murray Junop1 and Daniel S. C. Yang1 1 Department of Biochemistry and BiomedicalSciences Faculty of Health Sciences McMaster University Hamilton Canada 2 Department of Biochemistry Temple University Schoolof Medicine Philadelphia PA USA Keywords chimeric channel coiled coil KcsA RHCC tetramerization domain Correspondence D. S. C. Yang Department of Biochemistry and Biomedical Sciences Faculty of Health Sciences McMaster University 1200 Main Street West Hamilton Ontario L8N 3Z5 Canada Fax 1 905 522 9033 Tel 1 905 525 9140 ext. 22455 E-mail yang@mcmaster.ca Received 10 June 2009 revised 14 July 2009 accepted 26 August 2009 doi 10.1111 j.1742-4658.2009.07327.x 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. Structured digital abstract MINT-7260032 kcsA uniprotkb P0A334 and kcsA uniprotkb P0A334 .