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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

Conformational changes upon DOX binding in the PBP.(a) Variation of the average distance between the centers of mass ofsubdomains PC1 and PC2 along the RC with error bars. The PC1-PC2 distances in thecrystal structure are indicated by the horizontal dashed lines. The PBP and DBPregions are highlighted with the yellow and orange shaded bands, respectively.(b) Time evolution of the distance between the centers of mass of PC1and PC2 subdomains with DOX bound in the PBP (green) or in the DBP (blue) in the50-ns unbiased trajectories. (c) Variation of the average radius ofgyration of the DBP against the RC with error bars.
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f3: Conformational changes upon DOX binding in the PBP.(a) Variation of the average distance between the centers of mass ofsubdomains PC1 and PC2 along the RC with error bars. The PC1-PC2 distances in thecrystal structure are indicated by the horizontal dashed lines. The PBP and DBPregions are highlighted with the yellow and orange shaded bands, respectively.(b) Time evolution of the distance between the centers of mass of PC1and PC2 subdomains with DOX bound in the PBP (green) or in the DBP (blue) in the50-ns unbiased trajectories. (c) Variation of the average radius ofgyration of the DBP against the RC with error bars.

Mentions: Along with the DOX binding at the PBP, significant conformational changes of thetranslocation pathway have been identified. The most prominent conformational changeis the relative motion between subdomains PC1 and PC2. The PC1/PC2 cleft closedremarkably with the average distance between the centers of mass of PC1 and PC2reduced by ~4 Å with respect to the crystal structure (Fig. 3a), which occludes the translocation tunnel toward thePC1/PC2 cleft entrance. To validate this conformational change, we performed two50-ns unbiased MD simulations with DOX initially bound inside the PBP or DBP,respectively. In the PBP-bound system, the simulation showed a similar DOXorientation in the PBP as that in the ABF trajectory. At the same time, the closingmotion of the PC1/PC2 cleft was observed, with the separation between PC1 and PC2decreasing from 31 to 27.5 Å in 50 ns (Fig.3b). In contrast, the PC1-PC2 distance fluctuated around30 Å in the DBP-bound system, and seldom fell below29 Å (Fig. 3b). Thus, the unbiased simulationsare well consistent with the ABF simulations by showing that DOX binding at the PBPof binding protomer induces the closure of PC1/PC2 cleft. In the previous MDsimulation studies of AcrB, the PC1/PC2 cleft motions were also observed although inthe absence of any substrate26. These opening and closing motionsindicate the intrinsic conformational flexibility of the cleft, whereas oursimulations further demonstrate that DOX binding can regulate the conformation of thePC1/PC2 cleft by stabilizing it in either open or closed state depending on thelocation of DOX. In the crystal structures of AcrB with substrates bound at the PBPof access protomer, similar closing motion of the PC1/PC2 cleft, however, was notobserved2369. This may be attributed to the different stateof protomer or to the differences in the residues involved in substrate binding. Thesubstrates often contact with more lateral region of the PC1/PC2 cleft in crystalstructures, but DOX gets into the innermost part of the cleft in our simulation(Fig. 2d). The closure of the PC1/PC2 cleft as DOX residesin PBP is reminiscent of the X-ray structures of the Zn(II)/proton antiporter ZneA, amember of the heavy metal efflux subfamily of RND pumps, in which the periplasmiccleft closes in the presence of Zn2+ at the proximal site (equivalentto the PBP in AcrB)33. In analogy with ZneA, the closure of thePC1/PC2 cleft in AcrB may also be important for preventing the backflow of substrateduring translocation.


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)

Conformational changes upon DOX binding in the PBP.(a) Variation of the average distance between the centers of mass ofsubdomains PC1 and PC2 along the RC with error bars. The PC1-PC2 distances in thecrystal structure are indicated by the horizontal dashed lines. The PBP and DBPregions are highlighted with the yellow and orange shaded bands, respectively.(b) Time evolution of the distance between the centers of mass of PC1and PC2 subdomains with DOX bound in the PBP (green) or in the DBP (blue) in the50-ns unbiased trajectories. (c) Variation of the average radius ofgyration of the DBP against the RC with error bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Conformational changes upon DOX binding in the PBP.(a) Variation of the average distance between the centers of mass ofsubdomains PC1 and PC2 along the RC with error bars. The PC1-PC2 distances in thecrystal structure are indicated by the horizontal dashed lines. The PBP and DBPregions are highlighted with the yellow and orange shaded bands, respectively.(b) Time evolution of the distance between the centers of mass of PC1and PC2 subdomains with DOX bound in the PBP (green) or in the DBP (blue) in the50-ns unbiased trajectories. (c) Variation of the average radius ofgyration of the DBP against the RC with error bars.
Mentions: Along with the DOX binding at the PBP, significant conformational changes of thetranslocation pathway have been identified. The most prominent conformational changeis the relative motion between subdomains PC1 and PC2. The PC1/PC2 cleft closedremarkably with the average distance between the centers of mass of PC1 and PC2reduced by ~4 Å with respect to the crystal structure (Fig. 3a), which occludes the translocation tunnel toward thePC1/PC2 cleft entrance. To validate this conformational change, we performed two50-ns unbiased MD simulations with DOX initially bound inside the PBP or DBP,respectively. In the PBP-bound system, the simulation showed a similar DOXorientation in the PBP as that in the ABF trajectory. At the same time, the closingmotion of the PC1/PC2 cleft was observed, with the separation between PC1 and PC2decreasing from 31 to 27.5 Å in 50 ns (Fig.3b). In contrast, the PC1-PC2 distance fluctuated around30 Å in the DBP-bound system, and seldom fell below29 Å (Fig. 3b). Thus, the unbiased simulationsare well consistent with the ABF simulations by showing that DOX binding at the PBPof binding protomer induces the closure of PC1/PC2 cleft. In the previous MDsimulation studies of AcrB, the PC1/PC2 cleft motions were also observed although inthe absence of any substrate26. These opening and closing motionsindicate the intrinsic conformational flexibility of the cleft, whereas oursimulations further demonstrate that DOX binding can regulate the conformation of thePC1/PC2 cleft by stabilizing it in either open or closed state depending on thelocation of DOX. In the crystal structures of AcrB with substrates bound at the PBPof access protomer, similar closing motion of the PC1/PC2 cleft, however, was notobserved2369. This may be attributed to the different stateof protomer or to the differences in the residues involved in substrate binding. Thesubstrates often contact with more lateral region of the PC1/PC2 cleft in crystalstructures, but DOX gets into the innermost part of the cleft in our simulation(Fig. 2d). The closure of the PC1/PC2 cleft as DOX residesin PBP is reminiscent of the X-ray structures of the Zn(II)/proton antiporter ZneA, amember of the heavy metal efflux subfamily of RND pumps, in which the periplasmiccleft closes in the presence of Zn2+ at the proximal site (equivalentto the PBP in AcrB)33. In analogy with ZneA, the closure of thePC1/PC2 cleft in AcrB may also be important for preventing the backflow of substrateduring translocation.

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