Tuning the Affinity of Anion Binding Sites in Porin Channels with Negatively Charged Residues: Molecular Details for OprP

Abstract

The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

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

“…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.…”

“…Either phosphate or diphosphate ions were subsequently placed at the mouth of the one of the monomers on the extracellular side for each porin. The monovalent form of the phosphate ion, H 2 PO 4 -, was investigated consistent with our previous studies (16)(17)(18), whereas the divalent form of the diphosphate ion, H 2 P 2 O 7 2was chosen as the most probable protonation state at pH 6. The systems were neutralized by addition of potassium ions and each system contained roughly 120,000 atoms.…”

“…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).…”

Exploring bacterial outer membrane barrier to combat bad bugs

“… 7 , 15 , 22 The method includes reconstitution of a single or multiple Omps into an artificial planar lipid bilayer and further uses transmembrane potential-driven ion current across the channel as a detection probe. 7 , 67 Using ion current as a probe specifically demonstrates very well-characterized electrophysiological properties of the Omps, 15 , 34 , 45 , 65 , 66 , 84 , 106 , 119 – 121 including size, 122 , 123 single-channel conductance, channel ion selectivity, 58 , 75 , 76 , 90 , 99 – 101 channel gating dynamics, and more. 47 , 95 , 109 Likewise, the size of Omps is a key factor defining transport through the channel.…”

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.…”