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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 type comparison between mouse P-pg and Sav1866 at TMDs.Both proteins are drawn in cartoon: helices as tubes, beta-sheets as arrows, turn and coil as wires. The residues are colored by residue type: non-polar residues in white, polar-residues in green, basic residues in blue and acidic residues in red. There are only polar and non-polar residues in the drug-binding pocket (A) of mouse Pgp whereas there are 9 pairs (18 total) charged residues in the same region of Sav1866 (B). The counterpart residues in the mouse P-pg are shown in text blocks: mouse P-pg half1 in red, half2 in blue, Sav1866 both halves in green. These residues are drawn in spheres from top view (C) for mouse P-pg and (D) for Sav1866.
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f8: Residue type comparison between mouse P-pg and Sav1866 at TMDs.Both proteins are drawn in cartoon: helices as tubes, beta-sheets as arrows, turn and coil as wires. The residues are colored by residue type: non-polar residues in white, polar-residues in green, basic residues in blue and acidic residues in red. There are only polar and non-polar residues in the drug-binding pocket (A) of mouse Pgp whereas there are 9 pairs (18 total) charged residues in the same region of Sav1866 (B). The counterpart residues in the mouse P-pg are shown in text blocks: mouse P-pg half1 in red, half2 in blue, Sav1866 both halves in green. These residues are drawn in spheres from top view (C) for mouse P-pg and (D) for Sav1866.

Mentions: The Sav1866 crystal structure was used as the template for the initial structure of the outward-facing conformation of mouse Pgp. However, the homology modeling process was not complete until MD simulation produced thermodynamically favored conformations. This was due to the differences in the electrostatic and hydrophobic properties between Sav1866 and mouse Pgp. The partial closure of TMs was observed during equilibrium for all simulations for mouse-Pgp regardless of ATP-binding. Our starting structure based on Sav1866 is more likely to represent a high-energy short-lived transition state that is driven by ATP hydrolysis at the moment of drug ejection. Residue type analysis (Figure 8) and electrostatic potential (Figure S6) analysis on both mouse Pgp and Sav1866 explains why each protein favored inward-facing- and outward-facing- conformations, respectively, for crystallography. The analysis of residue type differences (Figure 8) reveal that only polar and non-polar residues are included in the drug-binding pocket (Figure 8A and C) of mouse Pgp whereas 9 pairs (18 total) of extra charged residues exist in the same region of Sav1866 (Figure 8B and D). The conterparts of the 18 charged residues in mouse Pgp are 4 aromatic residues, 5 hydrophobic residues and 9 neutral polar residues. The hydrophobicity of mouse Pgp in the drug-binding pocket is substantially larger than that of Sav1866. In addition, the position of the charged residues in Sav1866 would tend toward repulsion thereby promoting the outward-facing comformation with an open substrate pocket towards extracellular space (Figure 8D). The comparison in electrostatic potential shows an overall neutral region in mouse Pgp but highly charged region in Sav1866 at TMDs. In contrast, the interfaces between two NBDs in mouse Pgp (Figure S6 B1 and B2) are more charged than that of Sav1866 (Figure S6 B), which indicates more hydrophilic NBDs in mouse Pgp than Sav1866. Since the hydrophobic effect has a stronger attraction (~10 kcal/mol) than electrostatic interaction (~5 kcal/mol), mouse Pgp favors closed TMDs and open NBDs, whereas Sav1866 favors the exact opposite configurations. These differences imply that mouse Pgp and Sav1866 employ rather different transport mechanisms, particularly in regard to the role of water.


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

Pan L, Aller SG - Sci Rep (2015)

Residue type comparison between mouse P-pg and Sav1866 at TMDs.Both proteins are drawn in cartoon: helices as tubes, beta-sheets as arrows, turn and coil as wires. The residues are colored by residue type: non-polar residues in white, polar-residues in green, basic residues in blue and acidic residues in red. There are only polar and non-polar residues in the drug-binding pocket (A) of mouse Pgp whereas there are 9 pairs (18 total) charged residues in the same region of Sav1866 (B). The counterpart residues in the mouse P-pg are shown in text blocks: mouse P-pg half1 in red, half2 in blue, Sav1866 both halves in green. These residues are drawn in spheres from top view (C) for mouse P-pg and (D) for Sav1866.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Residue type comparison between mouse P-pg and Sav1866 at TMDs.Both proteins are drawn in cartoon: helices as tubes, beta-sheets as arrows, turn and coil as wires. The residues are colored by residue type: non-polar residues in white, polar-residues in green, basic residues in blue and acidic residues in red. There are only polar and non-polar residues in the drug-binding pocket (A) of mouse Pgp whereas there are 9 pairs (18 total) charged residues in the same region of Sav1866 (B). The counterpart residues in the mouse P-pg are shown in text blocks: mouse P-pg half1 in red, half2 in blue, Sav1866 both halves in green. These residues are drawn in spheres from top view (C) for mouse P-pg and (D) for Sav1866.
Mentions: The Sav1866 crystal structure was used as the template for the initial structure of the outward-facing conformation of mouse Pgp. However, the homology modeling process was not complete until MD simulation produced thermodynamically favored conformations. This was due to the differences in the electrostatic and hydrophobic properties between Sav1866 and mouse Pgp. The partial closure of TMs was observed during equilibrium for all simulations for mouse-Pgp regardless of ATP-binding. Our starting structure based on Sav1866 is more likely to represent a high-energy short-lived transition state that is driven by ATP hydrolysis at the moment of drug ejection. Residue type analysis (Figure 8) and electrostatic potential (Figure S6) analysis on both mouse Pgp and Sav1866 explains why each protein favored inward-facing- and outward-facing- conformations, respectively, for crystallography. The analysis of residue type differences (Figure 8) reveal that only polar and non-polar residues are included in the drug-binding pocket (Figure 8A and C) of mouse Pgp whereas 9 pairs (18 total) of extra charged residues exist in the same region of Sav1866 (Figure 8B and D). The conterparts of the 18 charged residues in mouse Pgp are 4 aromatic residues, 5 hydrophobic residues and 9 neutral polar residues. The hydrophobicity of mouse Pgp in the drug-binding pocket is substantially larger than that of Sav1866. In addition, the position of the charged residues in Sav1866 would tend toward repulsion thereby promoting the outward-facing comformation with an open substrate pocket towards extracellular space (Figure 8D). The comparison in electrostatic potential shows an overall neutral region in mouse Pgp but highly charged region in Sav1866 at TMDs. In contrast, the interfaces between two NBDs in mouse Pgp (Figure S6 B1 and B2) are more charged than that of Sav1866 (Figure S6 B), which indicates more hydrophilic NBDs in mouse Pgp than Sav1866. Since the hydrophobic effect has a stronger attraction (~10 kcal/mol) than electrostatic interaction (~5 kcal/mol), mouse Pgp favors closed TMDs and open NBDs, whereas Sav1866 favors the exact opposite configurations. These differences imply that mouse Pgp and Sav1866 employ rather different transport mechanisms, particularly in regard to the role of water.

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