Publication: Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA
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Date
2004-11-05
Authors
Molina Gallego, María Luisa ; Encinar Hidalgo, José Antonio ; Barrera Olivares, Francisco Nicolás ; Fernández Ballester, Gregorio ; Riquelme Pino, Gloria ; González Ros, José Manuel
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Publisher
American Chemical Society
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DOI
https://doi.org/10.1021/bi048889+
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info:eu-repo/semantics/article
Description
© 2004 American Chemical Society. This document is the Published version of a Published Work that appeared in final form in Biochemistry. To access the final edited and published work see https://doi.org/10.1021/bi048889+
Abstract
KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of 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) Science280, 69−77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Δ125−160 and Δ120−160, as well as 1−125 KcsA obtained from chymotrypsin cleavage of full-length 1−160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120−124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.
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Citation
Biochemistry 2004, Vol. 43, N. 47, pp. 14924–14931
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