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Stepwise substrate translocation mechanism revealed by free energy calculations of doxorubicin in the multidrug transporter AcrB.

Zuo Z, Wang B, Weng J, Wang W - Sci Rep (2015)

Bottom Line: Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them.Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states.Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.

ABSTRACT
AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.

No MeSH data available.


Related in: MedlinePlus

Energetics of DOX translocation in the binding protomer.(a) The PMF profile of DOX translocation along the reaction coordinate witherror bars. The two deep energy minima correspond to DOX binding at the PBP andDBP. (b) A representative snapshot of DOX bound near the entrancecorresponding to the shallow energy minimum atRC = 13.6 Å. DOX (orange for carbon atoms) and theresidues (yellow for carbon atoms) interacting with it are drawn as a ball-stickmodel. Their oxygen and nitrogen atoms are colored in red and blue, respectively.The hydrogen bonds are represented by blue dashed lines and the PC-loop ishighlighted in magenta. The Cα atoms of residues Val557 andAsn871 are represented by green spheres. (c) A representative snapshot ofDOX bound in the DBP. The switch-loop is highlighted in magenta. (d) Topview of DOX (yellow for carbon atoms) bound in the PBP. The DOX dimer (grey forcarbon atoms) bound at the PC1/PC2 cleft in the crystal structure (PDB ID: 4DX7)is shown by superimposing the access protomer of 4DX7 with the binding protomer ofthe snapshot in (e). (e) A representative snapshot of DOX bound inthe PBP(RC = 26.5 ~ 28.5 Å). ThePC-loop and the switch-loop are highlighted in magenta.
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f2: Energetics of DOX translocation in the binding protomer.(a) The PMF profile of DOX translocation along the reaction coordinate witherror bars. The two deep energy minima correspond to DOX binding at the PBP andDBP. (b) A representative snapshot of DOX bound near the entrancecorresponding to the shallow energy minimum atRC = 13.6 Å. DOX (orange for carbon atoms) and theresidues (yellow for carbon atoms) interacting with it are drawn as a ball-stickmodel. Their oxygen and nitrogen atoms are colored in red and blue, respectively.The hydrogen bonds are represented by blue dashed lines and the PC-loop ishighlighted in magenta. The Cα atoms of residues Val557 andAsn871 are represented by green spheres. (c) A representative snapshot ofDOX bound in the DBP. The switch-loop is highlighted in magenta. (d) Topview of DOX (yellow for carbon atoms) bound in the PBP. The DOX dimer (grey forcarbon atoms) bound at the PC1/PC2 cleft in the crystal structure (PDB ID: 4DX7)is shown by superimposing the access protomer of 4DX7 with the binding protomer ofthe snapshot in (e). (e) A representative snapshot of DOX bound inthe PBP(RC = 26.5 ~ 28.5 Å). ThePC-loop and the switch-loop are highlighted in magenta.

Mentions: The calculated PMF profile is shown in Fig. 2a, the zero point ofwhich is set at the DBP. The convergence of the ABF simulations was examined in severalaspects. First, the final PMF profile is barely influenced by further extension of thesimulation time by 10 ns in each window (Figure S1a, Supporting information), and the estimated standard errors are allbelow 1.0 kcal/mol (Fig. 2a). On the other hand, theaverage forces, i.e. the first derivative of free energy are continuous (Figure S1b, Supporting information). These analysesdemonstrate that the simulation is well converged.


Stepwise substrate translocation mechanism revealed by free energy calculations of doxorubicin in the multidrug transporter AcrB.

Zuo Z, Wang B, Weng J, Wang W - Sci Rep (2015)

Energetics of DOX translocation in the binding protomer.(a) The PMF profile of DOX translocation along the reaction coordinate witherror bars. The two deep energy minima correspond to DOX binding at the PBP andDBP. (b) A representative snapshot of DOX bound near the entrancecorresponding to the shallow energy minimum atRC = 13.6 Å. DOX (orange for carbon atoms) and theresidues (yellow for carbon atoms) interacting with it are drawn as a ball-stickmodel. Their oxygen and nitrogen atoms are colored in red and blue, respectively.The hydrogen bonds are represented by blue dashed lines and the PC-loop ishighlighted in magenta. The Cα atoms of residues Val557 andAsn871 are represented by green spheres. (c) A representative snapshot ofDOX bound in the DBP. The switch-loop is highlighted in magenta. (d) Topview of DOX (yellow for carbon atoms) bound in the PBP. The DOX dimer (grey forcarbon atoms) bound at the PC1/PC2 cleft in the crystal structure (PDB ID: 4DX7)is shown by superimposing the access protomer of 4DX7 with the binding protomer ofthe snapshot in (e). (e) A representative snapshot of DOX bound inthe PBP(RC = 26.5 ~ 28.5 Å). ThePC-loop and the switch-loop are highlighted in magenta.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595977&req=5

f2: Energetics of DOX translocation in the binding protomer.(a) The PMF profile of DOX translocation along the reaction coordinate witherror bars. The two deep energy minima correspond to DOX binding at the PBP andDBP. (b) A representative snapshot of DOX bound near the entrancecorresponding to the shallow energy minimum atRC = 13.6 Å. DOX (orange for carbon atoms) and theresidues (yellow for carbon atoms) interacting with it are drawn as a ball-stickmodel. Their oxygen and nitrogen atoms are colored in red and blue, respectively.The hydrogen bonds are represented by blue dashed lines and the PC-loop ishighlighted in magenta. The Cα atoms of residues Val557 andAsn871 are represented by green spheres. (c) A representative snapshot ofDOX bound in the DBP. The switch-loop is highlighted in magenta. (d) Topview of DOX (yellow for carbon atoms) bound in the PBP. The DOX dimer (grey forcarbon atoms) bound at the PC1/PC2 cleft in the crystal structure (PDB ID: 4DX7)is shown by superimposing the access protomer of 4DX7 with the binding protomer ofthe snapshot in (e). (e) A representative snapshot of DOX bound inthe PBP(RC = 26.5 ~ 28.5 Å). ThePC-loop and the switch-loop are highlighted in magenta.
Mentions: The calculated PMF profile is shown in Fig. 2a, the zero point ofwhich is set at the DBP. The convergence of the ABF simulations was examined in severalaspects. First, the final PMF profile is barely influenced by further extension of thesimulation time by 10 ns in each window (Figure S1a, Supporting information), and the estimated standard errors are allbelow 1.0 kcal/mol (Fig. 2a). On the other hand, theaverage forces, i.e. the first derivative of free energy are continuous (Figure S1b, Supporting information). These analysesdemonstrate that the simulation is well converged.

Bottom Line: Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them.Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states.Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.

ABSTRACT
AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.

No MeSH data available.


Related in: MedlinePlus