Conversion of OprO into an OprP-like Channel by Exchanging Key Residues in the Channel Constriction

Abstract

Under phosphate-limiting conditions, the channels OprP and OprO are induced and expressed in the outer membrane of Pseudomonas aeruginosa. Despite their large homology, the phosphate-specific OprP and the diphosphate-specific OprO pores show structural differences in their binding sites situated in the constriction region. Previously, it was shown that the mutation of amino acids in OprP (Y62F and Y114D) led to an exchange in substrate specificity similar to OprO. To support the role of these key amino acids in the substrate sorting of these specific channels, the reverse mutants for OprO (F62Y, D114Y, and F62Y/D114Y) were created in this study. The phosphate and diphosphate binding of the generated channels was studied in planar lipid bilayers. Our results show that mutations of key residues indeed reverse the substrate specificity of OprO to OprP and support the view that just a few strategically positioned amino acids are mainly responsible for its substrate specificity.

Fosmidomycin transport through the phosphate‐specific porins OprO and OprP of Pseudomonas aeruginosa

“…The inhibition constant for fosmidomycin‐mediated block of OprO‐conductance was in this case 24 ± 5 1/ M (average of three titration experiments), which means that the affinity of fosmidomycin for the binding site inside OprO was smaller than for that in OprP. This was also the results of phosphate binding to OprO as compared to that to OprP (Ganguly et al., 2017; Modi et al., 2015).…”

“…Former studies on OprP and OprO were often performed at pH 6, but their single‐channel conductance was also characterized by varying the pH (Benz et al., 1993; Benz & Hancock, 1987; Ganguly et al., 2017). We decided to analyze the interaction of OprO and OprP with fosmidomycin in 10 mM HEPES pH 7.4 and at an ionic strength of 0.1 M KCl because both conditions have a higher physiological relevance.…”

“…The single‐channel conductance was 151 ± 8.9 pS and 166 ± 12 pS for OprP and OprO, respectively (see Figures 2a and 3, upper panels). This has to be compared with 160 pS and 240 pS, respectively, for OprP and OprO at pH 6, which means that the higher pH let to some decrease of the single‐channel conductance, probably due to the decrease of the protonation states of the amino acids localized within the binding sites of the channels (Benz & Hancock, 1987; Ganguly et al., 2017). Then, we added fosmidomycin to both sides of the membrane such that its final concentration was 5 mM and measured the single‐channel conductance for OprP again.…”

“…A comparison of the histograms indicated clearly that the effect of 20 mM fosmidomycin on pore conductance was much higher for OprP than for OprO. This may be caused by the closely spaced binding site for phosphate in OprP as compared to OprO (Ganguly et al, 2017). Table 1 shows a summary of all single-channel measurements with OprP and OprO at 0, 5, 10, and 20 mM fosmidomycin.…”

“…This means that the charge of fosmidomycin does obviously not play a major role in the interaction with the binding sites in OprP and OprO. It seems moreover that the closely spaced binding site in OprP is responsible for its higher affinity for fosmidomycin (Ganguly et al, 2017;Modi et al, 2015). The wider constriction of OprO is definitely the reason that fosmidomycin should be able to flip better in order to permeate this pore (Golla et al 2020).…”