A Structural Study of Ion Permeation in OmpF Porin from Anomalous X‑ray Diffraction and Molecular Dynamics Simulations

OmpF, a multiionic porin from <i>Escherichia coli</i>, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br^– ions from four different crystal structures and compared with Mg²⁺ sites and Rb⁺ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin

A Structural Study of Ion Permeation in OmpF Porin from Anomalous X‑ray Diffraction and Molecular Dynamics Simulations

OmpF, a multiionic porin from <i>Escherichia coli</i>, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br^– ions from four different crystal structures and compared with Mg²⁺ sites and Rb⁺ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin

A Structural Study of Ion Permeation in OmpF Porin from Anomalous X‑ray Diffraction and Molecular Dynamics Simulations

OmpF, a multiionic porin from <i>Escherichia coli</i>, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br^– ions from four different crystal structures and compared with Mg²⁺ sites and Rb⁺ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin

A Structural Study of Ion Permeation in OmpF Porin from Anomalous X‑ray Diffraction and Molecular Dynamics Simulations

OmpF, a multiionic porin from <i>Escherichia coli</i>, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br^– ions from four different crystal structures and compared with Mg²⁺ sites and Rb⁺ sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin

Position of the first gating charge along the S4 segment (R294) in the open-activated conformation of Kv1

2 channel deduced from MD. The mesh plots encompass the volume occupied 60% of the time by the Cβ of residue 294 during the different MD simulations. The data shown includes the wild-type Kv1.2 channel (A, orange mesh), the Kv1.2 channel with R294H-A351H Zn bridge (B, gray mesh), the Kv1.2 channel with the R294H-D352H Zn bridge (C, cyan mesh), and the Kv1.2 channel with the R294H-D352H Zn bridge and the substitution D352G-E353S (D, blue mesh). On the left is a side view from membrane and on the right is a side view from the pore. As a reference, the S4 segment (sky blue ribbon) and the pore domain (pale green ribbon) from the crystal structure of the Kv1.2 channel (PDB id 2A79) are shown. Three K ions are shown in the pore as orange spheres (left). The Cβ of residue 294 and the Cβ of residue 351 from the Kv1.2 x-ray structure are shown as red and magenta spheres, respectively. The Cβ of the residue at the corresponding position in the x-ray structure of the Kv1.2-Kv2.1 chimera PDB id 2R9R (Q290) is shown as a green sphere (assuming the same position for the pore domain). To record the probability density of the Cβ of residue 294 from the MD, the pore domain (S5–S6) was oriented with respect to the x-ray structure for every snapshot, followed by increments of 90° rotations to superimpose the voltage sensor modules of the four subunits (), yielding four data points per snapshot. For the wild-type channel, one snapshot every 5 ps is included (total 20,000 points), for each mutant one snapshot every 2 ps from two trajectories is included (total 10,000 points).<p><b>Copyright information:</b></p><p>Taken from "Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K Channel of Known Structure"</p><p></p><p>The Journal of General Physiology 2008;131(6):549-561.</p><p>Published online Jan 2008</p><p>PMCID:PMC2391244.</p><p></p

R294H-D352H forms a lower affinity Zn site but is still able to promote the activated conformational state

(A and B) Normalized relationships from oocytes expressing (A) Kv1.2-R294H-D352H ( = 7) and (B) Kv1.2-D352H ( = 7) channels recorded in the absence (solid squares) and presence (open circles) of 1 μM Zn. (C) A plot of ΔV versus Zn concentration in oocytes expressing Kv1.2-R294H-D352H channels, where ΔV is the difference between the fitted V values obtained in the absence and presence of varying concentrations of Zn. The data were fitted with a hyperbolic function (solid curve) with a half-maximal concentration of 470 nM ( = 5–10).<p><b>Copyright information:</b></p><p>Taken from "Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K Channel of Known Structure"</p><p></p><p>The Journal of General Physiology 2008;131(6):549-561.</p><p>Published online Jan 2008</p><p>PMCID:PMC2391244.</p><p></p

Metal ion binding to R294H-A351H stabilizes the activated state after neutralization of acidic residues D352 and E353

(A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (left) and presence (right) of 1 μM Zn, using the protocol outlined in the Materials and methods. (B) Normalized relationships from oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (solid squares, 9) and presence (open circles, 9) of 1 μM Zn. (C) A plot of ΔV versus Zn concentration in oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels, where ΔV is the difference between the fitted V values obtained in the absence and presence of varying concentrations of Zn. The data were fitted with a hyperbolic function (solid curve) with a half-maximal concentration of 0.36 ± 0.18 μM ( = 4–8). (D and E) Normalized curves obtained in the absence (solid squares) and presence (open circles) of 1 μM Zn for (D) Kv1.2-R294H-D352G-E353S ( = 7) and (E) Kv1.2-A351H-D352G-E353S ( = 7).<p><b>Copyright information:</b></p><p>Taken from "Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K Channel of Known Structure"</p><p></p><p>The Journal of General Physiology 2008;131(6):549-561.</p><p>Published online Jan 2008</p><p>PMCID:PMC2391244.</p><p></p

R294H-A351H forms a high affinity Zn binding site that reduces current magnitude

(A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H channels recorded in the absence (left) and presence (right) of 1 μM Zn, using the protocol outlined in the Materials and methods. Black arrowheads indicate where steady-state current was measured to generate and relationships. (B) Mean current–voltage relationships from oocytes expressing Kv1.2-R294H-A351H channels recorded in the absence (solid squares, 8) and presence (open circles, 8) of 1 μM Zn. (C) Normalized curves for Kv1.2-R294H-A351H obtained in the absence (black squares, = 8) and presence of various concentrations of Zn; 10 nM (gray triangles, = 7), 100 nM (open diamonds, = 7), and 1 μM (open circles, = 8) showing dose-dependent reduction in conductance with increasing concentrations of extracellular Zn. To demonstrate loss of current with Zn application, conductance () values obtained in either the absence or presence of Zn were normalized to the maximal value obtained in the absence of Zn. Data for control (no Zn) and 10 nM Zn were fitted with a single Boltzmann function, shown as solid black and gray curves respectively. (D and E) Normalized curves obtained in the absence (solid squares) and presence (open circles) of 1 μM Zn for (D) Kv1.2-R294H ( = 9) and (E) Kv1.2-A351H ( = 8). Single mutants showed no loss of current magnitude with application of 1 μM Zn; hence values were normalized to the maximal in each experimental condition (absence or presence of Zn).<p><b>Copyright information:</b></p><p>Taken from "Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K Channel of Known Structure"</p><p></p><p>The Journal of General Physiology 2008;131(6):549-561.</p><p>Published online Jan 2008</p><p>PMCID:PMC2391244.</p><p></p

Metal bridge formation slows channel closure signifying stabilization of the open-activated channel conformation

(A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (left) and presence (right) of 1 μM Zn. Bath solution contained 50 mM K in order to visualize inward tail currents. (B) Plot of time constant for deactivation versus voltage for channels recorded in the absence (solid squares) or presence (open circles) of 1 μM Zn ( = 7 each). Tail currents were fitted with a single exponential and the time constant of deactivation (τ, ms) plotted on a log scale against voltage.<p><b>Copyright information:</b></p><p>Taken from "Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K Channel of Known Structure"</p><p></p><p>The Journal of General Physiology 2008;131(6):549-561.</p><p>Published online Jan 2008</p><p>PMCID:PMC2391244.</p><p></p