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PMID: 11158168 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating.

The Journal of general physiology ·Vol. 117 ·No. 2 ·2001-02-00 ·Pages 165-80

Cortes DM, Cuello LG, Perozo E

Abstract

The molecular architecture of the NH(2) and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5-24 and 121-160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (DeltaHo(-1)), accessibility parameters (PiO(2) and PiNiEdda), and inter-subunit spin-spin interaction (Omega) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH(2) terminus of KcsA forms an alpha-helix anchored at the membrane-water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40-50 A towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.

MeSH Terms
Bacterial Proteins Crystallization Cytoplasm/metabolism Ion Channel Gating/physiology Liposomes Magnetic Resonance Spectroscopy Potassium Channels/chemistry,genetics,metabolism Protein Structure, Secondary/physiology Protein Structure, Tertiary/physiology Spin Labels
Chemicals
Bacterial Proteins Liposomes Potassium Channels Spin Labels prokaryotic potassium channel
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Cortes D M
Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906, USA.
Cuello L G
Perozo E
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Article Info
Journal
The Journal of general physiology
Abbr.
J Gen Physiol
ISSN
0022-1295
DOI
10.1085/jgp.117.2.165
Published
2001-02-00
Pages
165-80
Language
English
Region
United States
NLM ID
2985110R
PMCID
PMC2217246
Subset
IM
Grants
NIGMS NIH HHS · R01 GM057846 · United States
NIGMS NIH HHS · GM54690 · United States
NIGMS NIH HHS · GM57846 · United States
External Links
PubMed source DOI: 10.1085/jgp.117.2.165 PMC Full Text View journal details
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