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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 approximately 8 kT, are identified close to the L3 loop in the most constricted region of the pore. A dissociation constant for phosphate of 6 microM 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.

SUBMITTER: Pongprayoon P

PROVIDER: S-EPMC2789165 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The kinetics of sodium-stimulated phosphate flux and phosphate-stimulated sodium flux in human red cells have been previously described (Shoemaker, D.G., C.A. Bender, and R.B. Gunn. 1988. J. Gen. Physiol. 92:449-474). However, despite the identification of multiple isoforms in three gene families (Timmer, R.T., and R.B. Gunn. 1998. Am. J. Physiol. Cell Physiol. 274:C757-C769), the molecular basis for the sodium-phosphate cotransporter in erythrocytes is unknown. Most cells express multiple isoforms, thus disallowing explication of isoform-specific kinetics and function. We have found that erythrocyte membranes express one dominant isoform, hBNP-1, to which the kinetics can thus be ascribed. In addition, because the erythrocyte Na-PO(4) cotransporter can also mediate Li-PO(4) cotransport, it has been suggested that this transporter functions as the erythrocyte Na-Li exchanger whose activity is systematically altered in patients with bipolar disease and patients with essential hypertension. To determine the molecular basis for the sodium-phosphate cotransporter, we reasoned that if the kinetics of phosphate transport in a nucleated erythroid-like cell paralleled those of the Na-activated pathway in anucleated erythrocytes and yet were distinct from those known for other Na-PO(4) cotransporters, then the expressed genes may be the same in both cell types. In this study, we show that the kinetics of sodium phosphate cotransport were similar in anuclear human erythrocytes and K562 cells, a human erythroleukemic cell line. Although the erythrocyte fluxes were 750-fold smaller, the half-activation concentrations for phosphate and sodium and the relative cation specificities for activation of (32)PO(4) influx were similar. Na-activation curves for both cell types showed cooperativity consistent with the reported stoichiometry of more than one Na cotransported per PO(4). In K562 cells, external lithium activation of phosphate influx was also cooperative. Inhibition by arsenate, K(I) = 2.6-2.7 mM, and relative inhibition by amiloride, amiloride analogs, phosphonoformate, and phloretin were similar. These characteristics were different from those reported for hNaPi-3 and hPiT-1 in other systems. PCR analysis of sodium-phosphate cotransporter isoforms in K562 cells demonstrated the presence of mRNAs for hPiT-1, hPiT-2, and hBNP-1. The mRNAs for hNaPi-10 and hNaPi-3, the other two known isoforms, were absent. Western analysis of erythrocytes and K562 cells with isoform-specific antibodies detected the presence of only hBNP-1, an isoform expressed in brain neurons and glia. The similarities in the kinetics and the expression of only hBNP-1 protein in the two cell types is strong evidence that hBNP-1 is the erythrocyte and K562 cell sodium-phosphate cotransporter.

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

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