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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

Root-mean-square fluctuation (RMSF) per residue at equilibrium.The RMSFs are measured using the Cα of each residue from trajectories of the last 50 ns simulations and are averaged over triplicate simulations for both apo-Pgp in blue and Pgp-MgATP in red. 2D cartoon representatives of the secondary structures are in pink. Domains are shown in the same sequence order as that of the RMSF (helix as spring, coil and turn as straight line, beta sheet as arrow). The Figures of the two pseudo-symmetric halves (half1: residue 40-626, half2: residue 688-1271) are displayed in parallel for comparison. Labeled regions are region A (ECL1), region A′ (ECL4), region B (IH1), region B′ (IH3), region C (A-loop1), region C′ (A-loop2), region D (WA1), region D′ (WA2), region E (LSGGQ1), region E′ (LSGGQ2), region F (WB1), region F′ (WB2). In apo-Pgp, ATP-binding site 1(B + C + D + E′ + F) is more flexible than site 2 (B′ + C′ + D′ + E + F′).
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f4: Root-mean-square fluctuation (RMSF) per residue at equilibrium.The RMSFs are measured using the Cα of each residue from trajectories of the last 50 ns simulations and are averaged over triplicate simulations for both apo-Pgp in blue and Pgp-MgATP in red. 2D cartoon representatives of the secondary structures are in pink. Domains are shown in the same sequence order as that of the RMSF (helix as spring, coil and turn as straight line, beta sheet as arrow). The Figures of the two pseudo-symmetric halves (half1: residue 40-626, half2: residue 688-1271) are displayed in parallel for comparison. Labeled regions are region A (ECL1), region A′ (ECL4), region B (IH1), region B′ (IH3), region C (A-loop1), region C′ (A-loop2), region D (WA1), region D′ (WA2), region E (LSGGQ1), region E′ (LSGGQ2), region F (WB1), region F′ (WB2). In apo-Pgp, ATP-binding site 1(B + C + D + E′ + F) is more flexible than site 2 (B′ + C′ + D′ + E + F′).

Mentions: The root-means-square fluctuation (RMSF) (Figure 4) reveals asymmetric dynamics of apo-Pgp in equilibrated states (Figure 4, blue line). The results suggested that MgATP-binding reduced flexibility and introduced more symmetry to the protein dynamics (Figure 4, red line). In general, apo-Pgp has larger RMSF than Pgp-MgATP across all regions. Certain locations are particularly noteworthy: ECL1 (Figure 4, peak A) has the largest RMSF of the entire structure for both apo-Pgp (3.5 Å) and Pgp-MgATP (2.8 Å). ECL4 (Figure 4, peak A′) has the biggest RMSF of the TMs in half2. In apo-Pgp, ATP-binding site 1 (Figure 4 peak B, peak C, peak D, peak E′ and peak F) is more flexible than site 2 (Figure 4 peak B′, peak C′, peak D′, peak E and peak F′). Specifically, MgATP-binding reduces the flexibility of all the ATP-binding signature motifs at NBDs: Walker A (WA1: G423NSGCGK429; WA2: G1067SSGCGK1072), Walker B (WB1:I547LLLDE552; WB2:I1192LLLDE1197), LSGGQ (LSGGQ 1: L527SGGQ531; LSGGQ2: L1172SGGQ1176), A-loop (A-loop1: Y397PSR400; A-loop2: Y1040PTR1043). In addition, ATP-binding also reduced the flexibility of TMDs at various levels, which reveals an allosteric effect of nucleotide binding to the TMDs.


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

Pan L, Aller SG - Sci Rep (2015)

Root-mean-square fluctuation (RMSF) per residue at equilibrium.The RMSFs are measured using the Cα of each residue from trajectories of the last 50 ns simulations and are averaged over triplicate simulations for both apo-Pgp in blue and Pgp-MgATP in red. 2D cartoon representatives of the secondary structures are in pink. Domains are shown in the same sequence order as that of the RMSF (helix as spring, coil and turn as straight line, beta sheet as arrow). The Figures of the two pseudo-symmetric halves (half1: residue 40-626, half2: residue 688-1271) are displayed in parallel for comparison. Labeled regions are region A (ECL1), region A′ (ECL4), region B (IH1), region B′ (IH3), region C (A-loop1), region C′ (A-loop2), region D (WA1), region D′ (WA2), region E (LSGGQ1), region E′ (LSGGQ2), region F (WB1), region F′ (WB2). In apo-Pgp, ATP-binding site 1(B + C + D + E′ + F) is more flexible than site 2 (B′ + C′ + D′ + E + F′).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Root-mean-square fluctuation (RMSF) per residue at equilibrium.The RMSFs are measured using the Cα of each residue from trajectories of the last 50 ns simulations and are averaged over triplicate simulations for both apo-Pgp in blue and Pgp-MgATP in red. 2D cartoon representatives of the secondary structures are in pink. Domains are shown in the same sequence order as that of the RMSF (helix as spring, coil and turn as straight line, beta sheet as arrow). The Figures of the two pseudo-symmetric halves (half1: residue 40-626, half2: residue 688-1271) are displayed in parallel for comparison. Labeled regions are region A (ECL1), region A′ (ECL4), region B (IH1), region B′ (IH3), region C (A-loop1), region C′ (A-loop2), region D (WA1), region D′ (WA2), region E (LSGGQ1), region E′ (LSGGQ2), region F (WB1), region F′ (WB2). In apo-Pgp, ATP-binding site 1(B + C + D + E′ + F) is more flexible than site 2 (B′ + C′ + D′ + E + F′).
Mentions: The root-means-square fluctuation (RMSF) (Figure 4) reveals asymmetric dynamics of apo-Pgp in equilibrated states (Figure 4, blue line). The results suggested that MgATP-binding reduced flexibility and introduced more symmetry to the protein dynamics (Figure 4, red line). In general, apo-Pgp has larger RMSF than Pgp-MgATP across all regions. Certain locations are particularly noteworthy: ECL1 (Figure 4, peak A) has the largest RMSF of the entire structure for both apo-Pgp (3.5 Å) and Pgp-MgATP (2.8 Å). ECL4 (Figure 4, peak A′) has the biggest RMSF of the TMs in half2. In apo-Pgp, ATP-binding site 1 (Figure 4 peak B, peak C, peak D, peak E′ and peak F) is more flexible than site 2 (Figure 4 peak B′, peak C′, peak D′, peak E and peak F′). Specifically, MgATP-binding reduces the flexibility of all the ATP-binding signature motifs at NBDs: Walker A (WA1: G423NSGCGK429; WA2: G1067SSGCGK1072), Walker B (WB1:I547LLLDE552; WB2:I1192LLLDE1197), LSGGQ (LSGGQ 1: L527SGGQ531; LSGGQ2: L1172SGGQ1176), A-loop (A-loop1: Y397PSR400; A-loop2: Y1040PTR1043). In addition, ATP-binding also reduced the flexibility of TMDs at various levels, which reveals an allosteric effect of nucleotide binding to the TMDs.

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