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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‚ÄČkcal/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

Stepwise substrate translocation during the access to binding transition in thefunctional rotating mechanism of AcrB.(a,d) Side and top views of the access protomer with DOX bound atthe lateral PC1/PC2 cleft in the crystal structure 4DX7 (with one of the DOXsremoved). DOX is represented by VDW mode and colored in orange. In the side view,the PC1 and PC1 subdomains are shown in VDW model and colored in ice blue.(b,e) Side and top views of the binding protomer with DOX boundin the PBP observed in the ABF simulations. (c,f) Side and top viewsof the binding protomer with DOX bound in the DBP in the crystal structure 4DX7.(g) A cartoon diagram showing substrate translocation and conformationalchanges during the access to binding transition of AcrB.
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f4: Stepwise substrate translocation during the access to binding transition in thefunctional rotating mechanism of AcrB.(a,d) Side and top views of the access protomer with DOX bound atthe lateral PC1/PC2 cleft in the crystal structure 4DX7 (with one of the DOXsremoved). DOX is represented by VDW mode and colored in orange. In the side view,the PC1 and PC1 subdomains are shown in VDW model and colored in ice blue.(b,e) Side and top views of the binding protomer with DOX boundin the PBP observed in the ABF simulations. (c,f) Side and top viewsof the binding protomer with DOX bound in the DBP in the crystal structure 4DX7.(g) A cartoon diagram showing substrate translocation and conformationalchanges during the access to binding transition of AcrB.

Mentions: To sum up, our work provides the missing link in the translocation mechanism of AcrB byshowing that substrate can form stable binding in the PBP of binding protomer. Thebinding also accompanies significant conformational changes, giving an intermediatestate between the access and binding states. The findings entail a more detailedstepwise mechanism for substrate translocation (Fig. 4). Thetranslocation process starts with the attachment of substrate to the lateral PC1/PC2cleft in the access protomer (Fig. 4a,d). During thetransformation from the access state to the binding state, the substrate achievesoptimal binding inside the PBP with high affinity, and the PC1/PC2 cleft closes toprevent backflow of substrate (Fig. 4b,e). Transition to the DBPis readily to occur with a relatively low energy barrier. As the substrate forms stablebinding in the DBP, the PC1/PC2 cleft re-opens, giving the binding state as observed inmany asymmetric AcrB structures (Fig. 4c,f). This stepwise processof substrate translocation conforms the peristaltic pump mechanism, showing that theconformational flexibility of the porter domain is prerequisite for substrate bindingand transportation, and the functional rotating involves more intermediate states otherthan the access, binding and extrusion states (Fig. 4g). It is,however, also worth noting that substrates with different properties than DOX could givevery different free energy profiles of translocation24 and requiredifferent kinds of conformational changes during the functional rotation, which are allinteresting topics in the future studies.


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)

Stepwise substrate translocation during the access to binding transition in thefunctional rotating mechanism of AcrB.(a,d) Side and top views of the access protomer with DOX bound atthe lateral PC1/PC2 cleft in the crystal structure 4DX7 (with one of the DOXsremoved). DOX is represented by VDW mode and colored in orange. In the side view,the PC1 and PC1 subdomains are shown in VDW model and colored in ice blue.(b,e) Side and top views of the binding protomer with DOX boundin the PBP observed in the ABF simulations. (c,f) Side and top viewsof the binding protomer with DOX bound in the DBP in the crystal structure 4DX7.(g) A cartoon diagram showing substrate translocation and conformationalchanges during the access to binding transition of AcrB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Stepwise substrate translocation during the access to binding transition in thefunctional rotating mechanism of AcrB.(a,d) Side and top views of the access protomer with DOX bound atthe lateral PC1/PC2 cleft in the crystal structure 4DX7 (with one of the DOXsremoved). DOX is represented by VDW mode and colored in orange. In the side view,the PC1 and PC1 subdomains are shown in VDW model and colored in ice blue.(b,e) Side and top views of the binding protomer with DOX boundin the PBP observed in the ABF simulations. (c,f) Side and top viewsof the binding protomer with DOX bound in the DBP in the crystal structure 4DX7.(g) A cartoon diagram showing substrate translocation and conformationalchanges during the access to binding transition of AcrB.
Mentions: To sum up, our work provides the missing link in the translocation mechanism of AcrB byshowing that substrate can form stable binding in the PBP of binding protomer. Thebinding also accompanies significant conformational changes, giving an intermediatestate between the access and binding states. The findings entail a more detailedstepwise mechanism for substrate translocation (Fig. 4). Thetranslocation process starts with the attachment of substrate to the lateral PC1/PC2cleft in the access protomer (Fig. 4a,d). During thetransformation from the access state to the binding state, the substrate achievesoptimal binding inside the PBP with high affinity, and the PC1/PC2 cleft closes toprevent backflow of substrate (Fig. 4b,e). Transition to the DBPis readily to occur with a relatively low energy barrier. As the substrate forms stablebinding in the DBP, the PC1/PC2 cleft re-opens, giving the binding state as observed inmany asymmetric AcrB structures (Fig. 4c,f). This stepwise processof substrate translocation conforms the peristaltic pump mechanism, showing that theconformational flexibility of the porter domain is prerequisite for substrate bindingand transportation, and the functional rotating involves more intermediate states otherthan the access, binding and extrusion states (Fig. 4g). It is,however, also worth noting that substrates with different properties than DOX could givevery different free energy profiles of translocation24 and requiredifferent kinds of conformational changes during the functional rotation, which are allinteresting topics in the future studies.

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‚ÄČkcal/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