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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

Comparison of the pseudo-symmetric halves of mouse-Pgp.Structures of the four parts (two TMDs and two NBDs) of the mouse-Pgp are separately demonstrated in parallel for comparison. The structures 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. Psuedosysmetric regions with distinctive residue type composition are in squares for Half1 (A-F) and Half2 (A′-F′).
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f2: Comparison of the pseudo-symmetric halves of mouse-Pgp.Structures of the four parts (two TMDs and two NBDs) of the mouse-Pgp are separately demonstrated in parallel for comparison. The structures 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. Psuedosysmetric regions with distinctive residue type composition are in squares for Half1 (A-F) and Half2 (A′-F′).

Mentions: In contrast to the symmetric Sav1866 template, mouse Pgp has evolved with a high degree of asymmetry between its two "halves", which share only 59.4% sequence similarity. More specifically, 77.3% similarity lies between two NBDs and only 43.5% similarity between two halves of TMDs (Figure 2). The differences in the primary sequence result in the asymmetry of chemical properties between pseudo symmetric residues and patches. In general, charged residues (K, R, E, D) are highly hydrophilic with the largest entropy profiles whereas hydrophobic residues prefer to pack together and stabilize each other in the aqueous phase. Therefore, the analysis of different chemical properties based on the residue types of each region can help explain their different dynamics (Figure 2). Specifically, TM1 and TM2 (Figure 2 A) are 5-residues longer and with more charged residues than their pseudo symmetric domains TM6 and TM7 (Figure 2 A′). The intracellular end of TM3 (Figure 2B) has more charged residues than TM9 (Figure 2 B′). These features render larger mobility of half1 than half2 at TMDs. Even though the ATP-binding residues are conserved between site 1 and site 2 (Figure 3), their surrounding residues are substantially different. WA1 and WB1 are surrounded by more charged residues (Figure 2 D and E) than WA2 and WB2 (Figure 2 D′and E′). Whereas LSGGQ2 is surrouded by more charged residues (Figure 2 C and F′) than that of LSGGQ1 (Figure 2 C′and F). In sum, our observations reveal a more hydrophilic site 1 (WA1 + WB1 + LSGGQ2) than site 2 (WA2 + WB2 + LSGGQ1) and indicate different thermodynamics and kinetics between the two sites, consistent with multiple findings in the literature373839. A previous MD study40 was performed using the inward-facing conformation with ATP bound at each of the Walker A motifs but with both LSGGQ motifs completely dissociated. Even though the study was done at the near-opposite conformation and different ATP binding environment compared to our study, asymmetric structural dynamics between two NBDs was also observed. The asymmetry between the two halves of Pgp must therefore play a major role in the overall function and mechanism of the transporter.


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

Pan L, Aller SG - Sci Rep (2015)

Comparison of the pseudo-symmetric halves of mouse-Pgp.Structures of the four parts (two TMDs and two NBDs) of the mouse-Pgp are separately demonstrated in parallel for comparison. The structures 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. Psuedosysmetric regions with distinctive residue type composition are in squares for Half1 (A-F) and Half2 (A′-F′).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Comparison of the pseudo-symmetric halves of mouse-Pgp.Structures of the four parts (two TMDs and two NBDs) of the mouse-Pgp are separately demonstrated in parallel for comparison. The structures 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. Psuedosysmetric regions with distinctive residue type composition are in squares for Half1 (A-F) and Half2 (A′-F′).
Mentions: In contrast to the symmetric Sav1866 template, mouse Pgp has evolved with a high degree of asymmetry between its two "halves", which share only 59.4% sequence similarity. More specifically, 77.3% similarity lies between two NBDs and only 43.5% similarity between two halves of TMDs (Figure 2). The differences in the primary sequence result in the asymmetry of chemical properties between pseudo symmetric residues and patches. In general, charged residues (K, R, E, D) are highly hydrophilic with the largest entropy profiles whereas hydrophobic residues prefer to pack together and stabilize each other in the aqueous phase. Therefore, the analysis of different chemical properties based on the residue types of each region can help explain their different dynamics (Figure 2). Specifically, TM1 and TM2 (Figure 2 A) are 5-residues longer and with more charged residues than their pseudo symmetric domains TM6 and TM7 (Figure 2 A′). The intracellular end of TM3 (Figure 2B) has more charged residues than TM9 (Figure 2 B′). These features render larger mobility of half1 than half2 at TMDs. Even though the ATP-binding residues are conserved between site 1 and site 2 (Figure 3), their surrounding residues are substantially different. WA1 and WB1 are surrounded by more charged residues (Figure 2 D and E) than WA2 and WB2 (Figure 2 D′and E′). Whereas LSGGQ2 is surrouded by more charged residues (Figure 2 C and F′) than that of LSGGQ1 (Figure 2 C′and F). In sum, our observations reveal a more hydrophilic site 1 (WA1 + WB1 + LSGGQ2) than site 2 (WA2 + WB2 + LSGGQ1) and indicate different thermodynamics and kinetics between the two sites, consistent with multiple findings in the literature373839. A previous MD study40 was performed using the inward-facing conformation with ATP bound at each of the Walker A motifs but with both LSGGQ motifs completely dissociated. Even though the study was done at the near-opposite conformation and different ATP binding environment compared to our study, asymmetric structural dynamics between two NBDs was also observed. The asymmetry between the two halves of Pgp must therefore play a major role in the overall function and mechanism of the transporter.

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