Influence of C-Terminal Protein Domains and Protein-Lipid Interactions on Tetramerization and Stability of the Potassium Channel KcsA
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- 作者：M. L. Molina ; J. A. Encinar ; F. N. Barrera ; G. Ferná ; ndez-Ballester ; G. Riquelme ; and J. M. Gonzá ; lez-Ros
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：November 30, 2004
- 年：2004
- 卷：43
- 期：47
- 页码：14924 - 14931
- 全文大小：390K
- 年卷期：v.43,no.47(November 30, 2004)
- ISSN：1520-4995
文摘
KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunitsaround a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portionof KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L.,Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77], the identification of the moleculardeterminant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminaldeletion channel mutants, KcsA 
125-160 and 120-160, as well as 1-125 KcsA obtained fromchymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminalsegment on the stability and tetrameric assembly of the channel protein. We found that the lack of thecytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairstetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modelingof KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogenbonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once theKcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociationinto subunits is not greatly influenced by whether the entire C-terminal domain continues being part ofthe protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminaldomain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsAtetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobicinteractions involving intramembrane protein domains.
125-160 and 120-160, as well as 1-125 KcsA obtained fromchymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminalsegment on the stability and tetrameric assembly of the channel protein. We found that the lack of thecytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairstetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modelingof KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogenbonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once theKcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociationinto subunits is not greatly influenced by whether the entire C-terminal domain continues being part ofthe protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminaldomain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsAtetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobicinteractions involving intramembrane protein domains.