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.
Project description:Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 ?M.
Project description:The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.
Project description:Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) >> Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) >> Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.
Project description:Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that (31)P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the (31)P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.
Project description:KcsA is a proton-activated, voltage-modulated K(+) channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 A, as well as that of its isolated C-terminal domain at 2.6 A. The structure of the full-length KcsA-Fab complex reveals a well-defined, 4-helix bundle that projects approximately 70 A toward the cytoplasm. This bundle promotes a approximately 15 degree bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA-Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.
Project description:Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 ?. The structure reveals that the activation gate expands about 20 ?, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K(+) ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.
Project description:KcsA is a tetrameric K(+) channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p(perm)) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p(perm) and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.
Project description:In K(+) channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K(+) channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K(+) channels.
Project description:We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.
Project description:The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.