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Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.

Pan L, Aller SG - Sci Rep (2015)

Bottom Line: Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access.The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket.Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1025 18th Street South, Birmingham, AL, 35205 USA.

ABSTRACT
P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

No MeSH data available.


Related in: MedlinePlus

Effect of ATP on conformation dynamics and stability.The MgATP, K429/K1072 (2xWalker A), and S1173/S528 (2xLSGGQ) are drawn in licorice with H in white, N in blue, C in cyan, O in red, P in gold and Mg2+ in pink. The Cα of the ATP binding residues are drawn as green spheres, pairwise distances are drawn in dashed lines (E-G). H-bonds <2.5 Å are in solid black lines and H-bonds >2.5 Å are in dashed lines (E1, E2, F1, F2, G1 and G2). Orange arrows point to the directions of conformational change (F1, F2 and G1). The protein is shown in cartoon with half1 in pink and half2 in ice blue. (A) site 1 residue pair K429(WA)-S1173(LSGGQ) and (C) site 2 residue pair K1072(WA)-S528(LSGGQ) from the three apo-Pgp simulations. The same residue pairs in the three simulations of Pgp-MgATP are shown in (B) (site 1) and (D) (site 2). Conformation 1, “double-tight occluded” (E) (equilibrated structure from run3) consists of ATP in a tight sandwich at site 1 (E1) and site 2 (E2) with the K-S distance of ~9.4 Å. Conformation 2, “double-loose occluded” (F) (equilibrated structure from run2) is at loose sandwich between Walker A and LSGGQ with the K-S distance of ~11.1 Å due to the shift of ATP(Oγ)-S528/S1173(HG) to ATP(Oα)-S528/S1173(HG) at both site (F1, F2). This conformation has a slightly tilted Walker A to the intracellular direction and tilted LSGGQ to the membrane direction for both sites. Conformation 3, “loose-tight occluded” (G) (equilibrated structure from run1) adopts detached LSGGQ on site 1 (G1) with a K-S distance of 12.2 Å and the ATP locked site 2 (G2) with a K-S distance of ~9.4 Å.
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f5: Effect of ATP on conformation dynamics and stability.The MgATP, K429/K1072 (2xWalker A), and S1173/S528 (2xLSGGQ) are drawn in licorice with H in white, N in blue, C in cyan, O in red, P in gold and Mg2+ in pink. The Cα of the ATP binding residues are drawn as green spheres, pairwise distances are drawn in dashed lines (E-G). H-bonds <2.5 Å are in solid black lines and H-bonds >2.5 Å are in dashed lines (E1, E2, F1, F2, G1 and G2). Orange arrows point to the directions of conformational change (F1, F2 and G1). The protein is shown in cartoon with half1 in pink and half2 in ice blue. (A) site 1 residue pair K429(WA)-S1173(LSGGQ) and (C) site 2 residue pair K1072(WA)-S528(LSGGQ) from the three apo-Pgp simulations. The same residue pairs in the three simulations of Pgp-MgATP are shown in (B) (site 1) and (D) (site 2). Conformation 1, “double-tight occluded” (E) (equilibrated structure from run3) consists of ATP in a tight sandwich at site 1 (E1) and site 2 (E2) with the K-S distance of ~9.4 Å. Conformation 2, “double-loose occluded” (F) (equilibrated structure from run2) is at loose sandwich between Walker A and LSGGQ with the K-S distance of ~11.1 Å due to the shift of ATP(Oγ)-S528/S1173(HG) to ATP(Oα)-S528/S1173(HG) at both site (F1, F2). This conformation has a slightly tilted Walker A to the intracellular direction and tilted LSGGQ to the membrane direction for both sites. Conformation 3, “loose-tight occluded” (G) (equilibrated structure from run1) adopts detached LSGGQ on site 1 (G1) with a K-S distance of 12.2 Å and the ATP locked site 2 (G2) with a K-S distance of ~9.4 Å.

Mentions: In order to better demonstrate the conformational effects in NBDs upon MgATP binding, Cα distance between the pairs K429-S1173 (site 1) and K1072-S528 (site 2) were monitored over time (Figure 5). In apo-Pgp, both ATP-binding site 1 and site 2 (Figure 5A and C) demonstrated constant fluctuations between these residue pairs. However, site 1 showed a larger flexibility than that of site 2. The residue distance at site 1 also increased up to 20 Å whereas the residue distance at site 2 remained below 12 Å. A larger separation of NBDs at site 1 compared to site 2 was a consistently observed feature. On the other hand, MgATP-binding markedly stabilized the residue pairs at both site 1 (Figure 5B) and site 2 (Figure 5D) with nearly no fluctuation observed. Three distinct meta-stable states were revealed at the binding regions (Figure 5 E-G).


Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.

Pan L, Aller SG - Sci Rep (2015)

Effect of ATP on conformation dynamics and stability.The MgATP, K429/K1072 (2xWalker A), and S1173/S528 (2xLSGGQ) are drawn in licorice with H in white, N in blue, C in cyan, O in red, P in gold and Mg2+ in pink. The Cα of the ATP binding residues are drawn as green spheres, pairwise distances are drawn in dashed lines (E-G). H-bonds <2.5 Å are in solid black lines and H-bonds >2.5 Å are in dashed lines (E1, E2, F1, F2, G1 and G2). Orange arrows point to the directions of conformational change (F1, F2 and G1). The protein is shown in cartoon with half1 in pink and half2 in ice blue. (A) site 1 residue pair K429(WA)-S1173(LSGGQ) and (C) site 2 residue pair K1072(WA)-S528(LSGGQ) from the three apo-Pgp simulations. The same residue pairs in the three simulations of Pgp-MgATP are shown in (B) (site 1) and (D) (site 2). Conformation 1, “double-tight occluded” (E) (equilibrated structure from run3) consists of ATP in a tight sandwich at site 1 (E1) and site 2 (E2) with the K-S distance of ~9.4 Å. Conformation 2, “double-loose occluded” (F) (equilibrated structure from run2) is at loose sandwich between Walker A and LSGGQ with the K-S distance of ~11.1 Å due to the shift of ATP(Oγ)-S528/S1173(HG) to ATP(Oα)-S528/S1173(HG) at both site (F1, F2). This conformation has a slightly tilted Walker A to the intracellular direction and tilted LSGGQ to the membrane direction for both sites. Conformation 3, “loose-tight occluded” (G) (equilibrated structure from run1) adopts detached LSGGQ on site 1 (G1) with a K-S distance of 12.2 Å and the ATP locked site 2 (G2) with a K-S distance of ~9.4 Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389535&req=5

f5: Effect of ATP on conformation dynamics and stability.The MgATP, K429/K1072 (2xWalker A), and S1173/S528 (2xLSGGQ) are drawn in licorice with H in white, N in blue, C in cyan, O in red, P in gold and Mg2+ in pink. The Cα of the ATP binding residues are drawn as green spheres, pairwise distances are drawn in dashed lines (E-G). H-bonds <2.5 Å are in solid black lines and H-bonds >2.5 Å are in dashed lines (E1, E2, F1, F2, G1 and G2). Orange arrows point to the directions of conformational change (F1, F2 and G1). The protein is shown in cartoon with half1 in pink and half2 in ice blue. (A) site 1 residue pair K429(WA)-S1173(LSGGQ) and (C) site 2 residue pair K1072(WA)-S528(LSGGQ) from the three apo-Pgp simulations. The same residue pairs in the three simulations of Pgp-MgATP are shown in (B) (site 1) and (D) (site 2). Conformation 1, “double-tight occluded” (E) (equilibrated structure from run3) consists of ATP in a tight sandwich at site 1 (E1) and site 2 (E2) with the K-S distance of ~9.4 Å. Conformation 2, “double-loose occluded” (F) (equilibrated structure from run2) is at loose sandwich between Walker A and LSGGQ with the K-S distance of ~11.1 Å due to the shift of ATP(Oγ)-S528/S1173(HG) to ATP(Oα)-S528/S1173(HG) at both site (F1, F2). This conformation has a slightly tilted Walker A to the intracellular direction and tilted LSGGQ to the membrane direction for both sites. Conformation 3, “loose-tight occluded” (G) (equilibrated structure from run1) adopts detached LSGGQ on site 1 (G1) with a K-S distance of 12.2 Å and the ATP locked site 2 (G2) with a K-S distance of ~9.4 Å.
Mentions: In order to better demonstrate the conformational effects in NBDs upon MgATP binding, Cα distance between the pairs K429-S1173 (site 1) and K1072-S528 (site 2) were monitored over time (Figure 5). In apo-Pgp, both ATP-binding site 1 and site 2 (Figure 5A and C) demonstrated constant fluctuations between these residue pairs. However, site 1 showed a larger flexibility than that of site 2. The residue distance at site 1 also increased up to 20 Å whereas the residue distance at site 2 remained below 12 Å. A larger separation of NBDs at site 1 compared to site 2 was a consistently observed feature. On the other hand, MgATP-binding markedly stabilized the residue pairs at both site 1 (Figure 5B) and site 2 (Figure 5D) with nearly no fluctuation observed. Three distinct meta-stable states were revealed at the binding regions (Figure 5 E-G).

Bottom Line: Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access.The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket.Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1025 18th Street South, Birmingham, AL, 35205 USA.

ABSTRACT
P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

No MeSH data available.


Related in: MedlinePlus