Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate

Abstract

The outer membrane protein OprP from Pseudomonas aeruginosa forms a phosphate selective pore. To understand the mechanism of phosphate permeation and selectivity, we used three simulation techniques [equilibrium molecular dynamics simulations, steered molecular dynamics, and calculation of a potential of mean force (PMF)]. The PMF for phosphate reveals a deep free energy well midway along the OprP channel. Two adjacent phosphate-binding sites (W1 and W2), each with a well depth of Ϸ8 kT, are identified close to the L3 loop in the most constricted region of the pore. A dissociation constant for phosphate of 6 M is computed from the PMF, within the range of reported experimental values. The transfer of phosphate between sites W1 and W2 is correlated with changes in conformation of the sidechain of K121, which serves as a ''charged brush'' to facilitate phosphate passage between the two subsites. OprP also binds chloride, but less strongly than phosphate, as calculated from a Cl ؊ PMF. The difference in affinity and hence selectivity is due to the ''Lys-cluster'' motif, the positive charges of which interact strongly with a partially dehydrated phosphate ion but are shielded from a Cl ؊ by the hydration shell of the smaller ion. Our simulations suggest that OprP does not conform to the conventional picture of a channel with relatively flat energy landscape for permeant ions, but rather resembles a membrane-inserted binding protein with a high specificity that allows access to a centrally located binding site from both the extracellular and the periplasmic spaces.free energy profile ͉ potential of mean force ͉ anion selectivity ͉ hydration ͉ molecular dynamics simulation

Toward Understanding the Outer Membrane Uptake of Small Molecules by Pseudomonas aeruginosa

“…We therefore complemented the docking studies by performing 100-ns MD simulations for each of the four different OccD1-substrate combinations (WT-arginine, WT-glutamate, triple mutant-arginine, and triple mutant-glutamate), starting in each case from the lowest energy docking simulations (see "Experimental Procedures"). Previously, a similar MD approach was used to study phosphate binding to P. aeruginosa OprP, a phosphate channel that is not related to the Occ family (53). During the OccD1 wild type-arginine simulation, the carboxyl group initially faces away from Arg 410 in the basic ladder.…”

Free-Energy Calculations Reveal the Subtle Differences in the Interactions of DNA Bases with α-Hemolysin

“…28 The phosphate group alone is generally at unfavorable energies throughout the hemolysin barrel, compared with favorable energetics within OprP.…”

“…28 In general, the dimensions of the aHL barrel are such that the narrower regions provide greater stability for the DNA within the pore. Although there are no energetic barriers to base permeation, there are a number of regions that are more favorable than others.…”

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

“…Pongprayoon et al (48) reported an equilibrium and nonequilibrium atomistic MD investigation of the mechanism of phosphate permeation and selectivity in OprP. Notably, the potential-of-mean-force profile of phosphate permeation was calculated from umbrella sampling simulations.…”