Role of the Central Arginine R133 toward the Ion Selectivity of the Phosphate Specific Channel OprP: Effects of Charge and Solvation

Abstract

The outer membrane porin OprP of Pseudomonas aeruginosa forms a highly specific phosphate selective channel. This channel is responsible for the high-affinity uptake of phosphate ions into the periplasmic space of the bacteria. A detailed investigation of the structure-function relationship of OprP is inevitable to decipher the anion and phosphate selectivity of this porin in particular and to broaden the present understanding of the ion selectivity of different channels. To this end we investigated the role of the central arginine of OprP, R133, in terms of its effects in selectivity and ion transport properties of the pore. Electrophysiological bilayer measurements and free-energy molecular dynamics simulations were carried out to probe the transport of different ions through various R133 mutants. For these mutants, the change in phosphate binding specificity, ion conduction, and anion selectivity was determined and compared to previous molecular dynamic calculations and electrophysiological measurements with wild-type OprP. Molecular analysis revealed a rather particular role of arginine 133 and its charge, while at the same time this residue together with the network of other residues, namely, D94 and Y114, has the ability to dehydrate the permeating ion. These very specific features govern the ion selectivity of OprP.

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

“…We hypothesize that Arg-33/Arg-255 in TM1 of FEX plays a similar role in transport of the negatively charged fluoride ion. Interestingly, arginines are required for transporting anions through other proteins (59,60). It is possible that the positive charge provided by Arg-33/Arg-255 is part of an electrostatic well for fluoride ions entering the channel.…”

Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa

“…The black lipid membrane assay has been previously described in detail (17). In brief: a membrane was formed from a 1% (w/v) solution of diphytanoylphoshatidylcholine (DiPhPC, Avanti Polar Lipids, Alabaster, AL) in n-decane (Fluka Sigma-Aldrich, Seelze, Germany) over a 0.4 mm 2 aperture, between two aqueous compartments in a Teflon cell.…”

“…The output signal of the amplifier was monitored with a digital oscilloscope and a stripchart recorder (Rikadenki Electronics, Freiburg, Germany). Titration experiments were carried out to measure the inhibition of chloride conductance by phosphate binding to the binding sites, as previously described in detail with OprP wild-type and its mutants (16,17). Details are given in the Supporting Material.…”

“…Among these substrate-selective channels is OprP, a phosphate-selective porin that is induced under phosphate starvation conditions and facilitates the high-affinity acquisition of phosphate ions that are important for Pseudomonas growth and proliferation (7). A crystal structure of OprP is available (13) and detailed investigations have been carried out to understand the phosphate specificity of this interesting channel using bilayer electrophysiological measurements (6,14,15) and computational molecular dynamics (MD) simulations (16)(17)(18)(19).…”

Molecular Simulations of Gram-Negative Bacterial Membranes: A Vignette of Some Recent Successes

“…The understanding of ion binding in OprP was further improved as Modi et al (50,51) investigated the role of two other charged residues. Specifically, the roles of residues D94 and R133 were explored via a combination of simulations and experiments.…”