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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

Residue pairs observed in cross-linking experiments20212223.Inward-facing crystal structure (PDB: 4M1M-A) (A), initial structure of outward-facing Pgp (B) and equilibrated outward-facing Pgp with MgATP (C) are shown. Pgp is shown as cartoon representation with half1 in pink and half2 in ice blue. The highlighted residues are drawn as spheres in various colors: L328-L971 (L332-L975 in human Pgp) in red, V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) in gray, N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) in green and D173-N816 (D177-N820 in human Pgp) in blue. MgATP is drawn in spheres colored by elements as H in white, N in blue, C in cyan, O in red and P in tan. In the satellite panels of (C), Cα of highlighted residues are drawn as spheres and side chains are drawn as licorice colored by elements. Cα distances are labeled in black dash. The distances in A and B are single measurements and the distances in C are averages of the last 50 ns equilibrate trajectories over triplicate simulations.
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f1: Residue pairs observed in cross-linking experiments20212223.Inward-facing crystal structure (PDB: 4M1M-A) (A), initial structure of outward-facing Pgp (B) and equilibrated outward-facing Pgp with MgATP (C) are shown. Pgp is shown as cartoon representation with half1 in pink and half2 in ice blue. The highlighted residues are drawn as spheres in various colors: L328-L971 (L332-L975 in human Pgp) in red, V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) in gray, N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) in green and D173-N816 (D177-N820 in human Pgp) in blue. MgATP is drawn in spheres colored by elements as H in white, N in blue, C in cyan, O in red and P in tan. In the satellite panels of (C), Cα of highlighted residues are drawn as spheres and side chains are drawn as licorice colored by elements. Cα distances are labeled in black dash. The distances in A and B are single measurements and the distances in C are averages of the last 50 ns equilibrate trajectories over triplicate simulations.

Mentions: Inter-TM packing and helix registry of outward-facing Pgp initial model and equilibrated models was validated by previous disulfide crosslinking experiments based on double cysteine mutation (5–7 Å Cu-phenanthroline spacer). The following pairs were previously observed to form disulfide bonds and are mapped to atomic structures in Figure 1: L328-L971 (L332-L975 in human Pgp) between TM6 and TM1220; V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) between TM2 and TM1121; N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) between TM5 and TM822; pairs between TM3 and TM9 as D173-N816 (D177-N820 in human Pgp)23 and L171-N816 (L175 and N820 in human Pgp)29. Most residue pairs maintain direct contact between inward-facing and outward-facing conformations except for the three conformation-sensitive pairs L328-L971, D173-N816 and L171-N816. The Cα distance of residue pair L328-L971 is 12.4 Å apart in the inward-facing crystal structure (PDB: 4M1M; Figure 1A) but is 19.8 Å in the outward-facing conformation due to the opening of the drug pocket to the outer leaflet (Figure 1B). The formation of a disulfide bond would lock the protein in the inward-facing conformation, which explains the inhibition of verapamil-stimulated ATPase activity20. The two Cα of D173-N816 are 19.9 Å apart in the inward-facing crystal structure (Figure 1A) but are much closer in the outward-facing model with a Cα distance of ~8.0 Å. The side chains are in direct contact in the outward-facing model preventing the two Cα from closer approach, but would favor disulfide bond formation when mutated to Cys-Cys and crosslinked with Cu-phenanthroline as previously described23 (Figure 1B). The equilibrated structure of Pgp-MgATP showed little change in the pairwise distances except for L328-L971 due to partial closing of ECL1-ECL3.


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

Pan L, Aller SG - Sci Rep (2015)

Residue pairs observed in cross-linking experiments20212223.Inward-facing crystal structure (PDB: 4M1M-A) (A), initial structure of outward-facing Pgp (B) and equilibrated outward-facing Pgp with MgATP (C) are shown. Pgp is shown as cartoon representation with half1 in pink and half2 in ice blue. The highlighted residues are drawn as spheres in various colors: L328-L971 (L332-L975 in human Pgp) in red, V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) in gray, N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) in green and D173-N816 (D177-N820 in human Pgp) in blue. MgATP is drawn in spheres colored by elements as H in white, N in blue, C in cyan, O in red and P in tan. In the satellite panels of (C), Cα of highlighted residues are drawn as spheres and side chains are drawn as licorice colored by elements. Cα distances are labeled in black dash. The distances in A and B are single measurements and the distances in C are averages of the last 50 ns equilibrate trajectories over triplicate simulations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Residue pairs observed in cross-linking experiments20212223.Inward-facing crystal structure (PDB: 4M1M-A) (A), initial structure of outward-facing Pgp (B) and equilibrated outward-facing Pgp with MgATP (C) are shown. Pgp is shown as cartoon representation with half1 in pink and half2 in ice blue. The highlighted residues are drawn as spheres in various colors: L328-L971 (L332-L975 in human Pgp) in red, V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) in gray, N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) in green and D173-N816 (D177-N820 in human Pgp) in blue. MgATP is drawn in spheres colored by elements as H in white, N in blue, C in cyan, O in red and P in tan. In the satellite panels of (C), Cα of highlighted residues are drawn as spheres and side chains are drawn as licorice colored by elements. Cα distances are labeled in black dash. The distances in A and B are single measurements and the distances in C are averages of the last 50 ns equilibrate trajectories over triplicate simulations.
Mentions: Inter-TM packing and helix registry of outward-facing Pgp initial model and equilibrated models was validated by previous disulfide crosslinking experiments based on double cysteine mutation (5–7 Å Cu-phenanthroline spacer). The following pairs were previously observed to form disulfide bonds and are mapped to atomic structures in Figure 1: L328-L971 (L332-L975 in human Pgp) between TM6 and TM1220; V129-G935 (V133-G939 in human Pgp) and C133-A931 (C137-R935 in human Pgp) between TM2 and TM1121; N292-G770 (N296-G774 in human Pgp) and G296-F766 (G300-F770 in human Pgp) between TM5 and TM822; pairs between TM3 and TM9 as D173-N816 (D177-N820 in human Pgp)23 and L171-N816 (L175 and N820 in human Pgp)29. Most residue pairs maintain direct contact between inward-facing and outward-facing conformations except for the three conformation-sensitive pairs L328-L971, D173-N816 and L171-N816. The Cα distance of residue pair L328-L971 is 12.4 Å apart in the inward-facing crystal structure (PDB: 4M1M; Figure 1A) but is 19.8 Å in the outward-facing conformation due to the opening of the drug pocket to the outer leaflet (Figure 1B). The formation of a disulfide bond would lock the protein in the inward-facing conformation, which explains the inhibition of verapamil-stimulated ATPase activity20. The two Cα of D173-N816 are 19.9 Å apart in the inward-facing crystal structure (Figure 1A) but are much closer in the outward-facing model with a Cα distance of ~8.0 Å. The side chains are in direct contact in the outward-facing model preventing the two Cα from closer approach, but would favor disulfide bond formation when mutated to Cys-Cys and crosslinked with Cu-phenanthroline as previously described23 (Figure 1B). The equilibrated structure of Pgp-MgATP showed little change in the pairwise distances except for L328-L971 due to partial closing of ECL1-ECL3.

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