PDBsum entry: 2hjf

Metal transport

PDB id: 2hjf

Contents

Protein chains

219 a.a. *

212 a.a. *

103 a.a. *

Ligands

TBA

Metals

__K ×4

Waters ×11

* Residue conservation analysis

PDB id:

2hjf

Name:

Metal transport

Title:

Potassium channel kcsa-fab complex with tetrabutylammonium (tba)

Structure:

Antibody fragment heavy chain. Chain: a. Antibody fragment light chain. Chain: b. Voltage-gated potassium channel. Chain: c. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Streptomyces lividans. Organism_taxid: 1916

Biol. unit:

Dodecamer (from PDB file)

Resolution:

2.90Å R-factor: 0.225 R-free: 0.284

Authors:

J.D.Faraldo-Gomez,E.Kutluay,V.Jogini,Y.Zhao,L.Heginbotham, B.Roux

Key ref:

J.D.Faraldo-Gómez et al. (2007). Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations. J Mol Biol, 365, 649-662. PubMed id: 17070844 DOI: 10.1016/j.jmb.2006.09.069

Date:

30-Jun-06 Release date: 05-Dec-06

Protein chain

No UniProt id for this chain

Protein chain

No UniProt id for this chain

Protein chain

P0A334  (KCSA_STRLI) -  Voltage-gated potassium channel

Seq: 160 a.a.

Struc: 103 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 

• Cellular component: voltage-gated potassium channel complex (1 term)
• Biological process: potassium ion transport (1 term)
• Biochemical function: voltage-gated potassium channel activity (1 term)

DOI no: 10.1016/j.jmb.2006.09.069

J Mol Biol 365:649-662 (2007)

PubMed id: 17070844

Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations.

J.D.Faraldo-Gómez, E.Kutluay, V.Jogini, Y.Zhao, L.Heginbotham, B.Roux.

ABSTRACT

The mechanism of intracellular blockade of the KcsA potassium channel by tetrabutylammonium (TBA) is investigated through functional, structural and computational studies. Using planar-membrane electrophysiological recordings, we characterize the binding kinetics as well as the dependence on the transmembrane voltage and the concentration of the blocker. It is found that the apparent affinity of the complex is significantly greater than that of any of the eukaryotic K(+) channels studied previously, and that the off-rate increases with the applied transmembrane voltage. In addition, we report a crystal structure of the KcsA-TBA complex at 2.9 A resolution, with TBA bound inside the large hydrophobic cavity located at the center of the channel, consistent with the results of previous functional and structural studies. Of particular interest is the observation that the presence of TBA has a negligible effect on the channel structure and on the position of the potassium ions occupying the selectivity filter. Inspection of the electron density corresponding to TBA suggests that the ligand may adopt more than one conformation in the complex, though the moderate resolution of the data precludes a definitive interpretation on the basis of the crystallographic refinement methods alone. To provide a rationale for these observations, we carry out an extensive conformational sampling of an atomic model of TBA bound in the central cavity of KcsA, using the Hamiltonian replica-exchange molecular dynamics simulation method. Comparison of the simulated and experimental density maps indicates that the latter does reflect at least two distinct binding orientations of TBA. The simulations show also that the relative population of these binding modes is dependent on the ion configuration occupying the selectivity filter, thus providing a clue to the nature of the voltage-dependence of the binding kinetics.

Selected figure(s)

Figure 4.
Figure 4. Crystal structure of wild-type KcsA in complex with TBA and potassium. (a) The backbone of two of the four protein monomers, viewed from the plane of the membrane, alongside the binding sites for K^+ (magenta) and the electron density corresponding to TBA. The σ-weighted 2F[o]–F[c] maps are contoured at 2.5σ (protein), 3.5σ (ions) and 0.5σ (blocker), and drawn as a blue mesh. (b) A close-up view of the selectivity filter and the potassium-binding sites; the 2F[o]–F[c] maps are contoured at 1.5σ (protein) and 3.5σ (ions). (c) and (d) Electron density corresponding to TBA (contoured at 0.5σ), viewed down the 4-fold symmetry axis of the channel, or from the plane of the membrane. The molecular graphics in Figure 4, Figure 5, Figure 6 and Figure 7 were rendered with Pymol [http://pymol.sourceforge.net/]. Figure 4. Crystal structure of wild-type KcsA in complex with TBA and potassium. (a) The backbone of two of the four protein monomers, viewed from the plane of the membrane, alongside the binding sites for K^+ (magenta) and the electron density corresponding to TBA. The σ-weighted 2F[o]–F[c] maps are contoured at 2.5σ (protein), 3.5σ (ions) and 0.5σ (blocker), and drawn as a blue mesh. (b) A close-up view of the selectivity filter and the potassium-binding sites; the 2F[o]–F[c] maps are contoured at 1.5σ (protein) and 3.5σ (ions). (c) and (d) Electron density corresponding to TBA (contoured at 0.5σ), viewed down the 4-fold symmetry axis of the channel, or from the plane of the membrane. The molecular graphics in [3]Figure 4, [4]Figure 5, [5]Figure 6 and [6]Figure 7 were rendered with Pymol [http://pymol.sourceforge.net/].

Figure 7.
Figure 7. Representative configurations of TBA within the transmembrane cavity of KcsA, extracted from simulations SIM#3 and SIM#4. The configurations are ordered according to the tilt of the molecule relative to the plane of the membrane and to the position of the nitrogen atom of TBA along the axis of the channel (Z = 0 corresponds approximately to the location of the binding site for K^+ in the absence of TBA). For each tilt, the characteristic RMS deviation (in Å) with respect to the ideal, all-trans D[2d] geometry (Figure 5(a)) is provided, derived from an ensemble average. The number at the top corresponds to the C[8]N^+ core (which defines the conformational state, i.e. D[2d] versus S[4]); the number at the bottom corresponds to all non-hydrogen atoms in the molecule. Figure 7. Representative configurations of TBA within the transmembrane cavity of KcsA, extracted from simulations SIM#3 and SIM#4. The configurations are ordered according to the tilt of the molecule relative to the plane of the membrane and to the position of the nitrogen atom of TBA along the axis of the channel (Z = 0 corresponds approximately to the location of the binding site for K^+ in the absence of TBA). For each tilt, the characteristic RMS deviation (in Å) with respect to the ideal, all-trans D[2d] geometry ([3]Figure 5(a)) is provided, derived from an ensemble average. The number at the top corresponds to the C[8]N^+ core (which defines the conformational state, i.e. D[2d] versus S[4]); the number at the bottom corresponds to all non-hydrogen atoms in the molecule.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 365, 649-662) copyright 2007.

Figures were selected by an automated process.