title: Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating.
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.
authors.affiliation: Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Cortes
authors.forename: L G
authors.initials: LG
authors.lastname: Cuello
authors.forename: E
authors.initials: E
authors.lastname: Perozo
mesh_terms: D001426:Bacterial Proteins; D003460:Crystallization; D003593:Cytoplasm / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013113:Spin Labels
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D015221:Potassium Channels; D013113:Spin Labels; C107790:prokaryotic potassium channel