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Lee, S. (2009). Multidrug efflux transporter AcrB structure: mechanism-unclear salt bridge changes and cytoplasmic drug transport. PHILICA.COM Article number 165.

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Multidrug efflux transporter AcrB structure: mechanism-unclear salt bridge changes and cytoplasmic drug transport

Sang Hee Leeconfirmed user (Drug Resistance Proteomics Laboratory, Department of Biological Sciences, Myongji University)

Published in bio.philica.com

Abstract
Multidrug resistance caused by export proteins (e.g., P-glycoprotein and AcrB) is a serious problem in the chemotherapy of cancer (Gottesman, 2002) as well as in the antibiotic treatment of numerous different infections (Li and Nikaido, 2004). As a result of a heated competition, the new structures of AcrB have been determined independently by two different research groups. In all two structures, both two groups have defined the structural changes that constitute the basis of substrate transport by AcrB and have proposed a novel transport mechanism. However, I have found three different points (unclear mechanisms) between Swiss (Seeger et al., 2006) and Japanese (Murakami et al., 2006) groups’ reports, although the primary sequence of AcrB from Swiss group is 100% identical to that from Japanese group. Furthermore, I propose that the substrates collected at the central cavity from the cytoplasm or inner leaflet of cytoplasmic membrane are transported out of cell as follows: (a) from the central cavity to the entrance, (b) from the entrance to the periplasm, and then (c) from the periplasm to the vestibule. And then (d) the subsequent transport processes accord with those via the functional rotation transport mechanism (Seeger et al., 2006; Murakami et al., 2006).

Article body

 

Multidrug efflux transporter AcrB structure: mechanism-unclear salt bridge changes and cytoplasmic drug transport

 

Sang Hee Lee*

 

Drug Resistance Proteomics Laboratory

Department of Biological Sciences

Myongji University

San 38-2 Namdong, Yongin

Gyeonggido, 449-728, Republic of Korea

*Corresponding author. E-mail: sangheelee@mju.ac.kr

 

 

Abstract

 

Multidrug resistance caused by export proteins (e.g., P-glycoprotein and AcrB) is a serious problem in the chemotherapy of cancer (Gottesman, 2002) as well as in the antibiotic treatment of numerous different infections (Li and Nikaido, 2004). As a result of a heated competition, the new structures of AcrB have been determined independently by two different research groups. In all two structures, both two groups have defined the structural changes that constitute the basis of substrate transport by AcrB and have proposed a novel transport mechanism. However, I have found three different points (unclear mechanisms) between Swiss (Seeger et al., 2006) and Japanese (Murakami et al., 2006) groups' reports, although the primary sequence of AcrB from Swiss group is 100% identical to that from Japanese group. Furthermore, I propose that the substrates collected at the central cavity from the cytoplasm or inner leaflet of cytoplasmic membrane are transported out of cell as follows: (a) from the central cavity to the entrance, (b) from the entrance to the periplasm, and then (c) from the periplasm to the vestibule. And then (d) the subsequent transport processes accord with those via the functional rotation transport mechanism (Seeger et al., 2006; Murakami et al., 2006).

 

 

Materials and Methods

 

Comparison of binding pocket and salt bridge between two AcrB trimer structures was performed via the program QUANTA. Figures were made using PyMOL (http://pymol.sourceforge.net/). Other data and figures were adapted from Seeger et al. (2006) and Murakami et al. (2006).

 

 

Results and Discussions

 

Substrate binding pocket

First, two different structures of the substrate binding pocket are identified. Seeger et al. (2006) of Swiss group define the structural changes that constitute the basis of substrate transport by AcrB and propose a novel functional rotation transport mechanism different from previous functional interpretation (Murakami et al., 2002) based on the symmetric R32 crystal form of AcrB trimer. Seeger et al. (2006) further suggest that subtle changes in the transmembrane part (TM4 and TM10) produce the large conformational changes in the pore domain ultimately resulting in multidrug efflux. However, Murakami et al. (2006) have already reported the same functionally rotating mechanism as the peristaltic pump mechanism proposed by Seeger et al. (2006). Murakami et al. (2006) described crystal structures of AcrB with and without substrates (drugs) but Seeger et al. (2006) showed only those without substrates. Thus, Seeger et al. (2006) cannot show that there are some polar residues (e.g., N274 and Q176) in the substrate binding pocket and that different sets of residues in this pocket are used for binging of the different kinds of substrates. Yu et al. (2003) have shown that binding of substrate triggered a 1° rigid-body rotation in each AcrB monomer. Therefore, it is not reasonable that without the presentation of data showing multi-site binding of substrates, Seeger et al. (2006) propose that the specific binding of substrates in the hydrophobic (substrate binding) pocket of T monomer (binding protomer) occurs. In actuality, the comparison of the structure of the substrate binding pocket solved by Seeger et al. (2006) with that (without substrates) by Murakami et al. (2006) reveals good overall alignment (about 0.6 Å RMSD). 
 
Changes of salt bridges
Second, two different changes of salt bridges are identified (Figure 1). Seeger et al. (2006) stated that they observed that in the only O monomer (extrusion protomer), K940 formed a salt bridge with D407, and this subtle change might produce the large conformational changes in the pore domain of AcrB. That is, K940 side chain reoriented away from D408 and toward D407 in O conformation. 
 
Figure 1. Comparison of salt bridge formation in TM domain between AcrB trimer (a) from Seeger et al. (2006) and AcrB trimer (b) from Murakami et al. (2006). In the left panel (a), structural (salt bridge formation) changes in the putative proton translocation site were shown. Conserved residues D407, D408 (TM4) and K940 (TM10) in the three monomers (L-loose conformation, blue; T-tight conformation, yellow; O-open conformation, red) are depicted with 2Fo-Fc electron density maps contoured at 0.5 σ (L) or 1 σ (T and O) as viewed from the cytoplasm. In the L and T monomers, the same conformation is observed, whereas in the O monomer, K940 forms a salt bridge with D407. This interaction seems to be stabilized by hydrogen bonding of T978 (TM11). To restore the geometry as it appears in the L monomer, proton uptake is anticipated. In the right panel (b), structure with a slab (~23 Å) of the transmembrane domain viewed from the periplasmic side was shown. The side chains of three functionally essential charged residues-D407, D408 and K940-and functionally important residue T978 are shown in a ball-and-stick representation. Colors of three protomers (Access, Binding and Extrusion) are green, blue and red, respectively. Roman numerals indicate the transmembrane helix numbers (IV, TM4; X, TM10; XI, TM11). In the transmembrane region of the access protomer (L monomer) and the binding protomer (T monomer), the K940 residue was coordinated by salt bridges with D407 and D408, but in the extrusion protomer (O monomer), K940 was turned nearly 45° toward T978 of TM11 and the salt bridges were abolished.
 
Contrary to the opinions expressed by Seeger et al. (2006), Murakami et al. (2006) stated that in the transmembrane region of the access protomer (L monomer) and the binding protomer (T monomer), the K940 residue was coordinated by salt bridges with D407 and D408 (Murakami et al., 2002 and 2006), but in the extrusion protomer (O monomer), K940 was turned nearly 45° toward T978 of TM11 and the salt bridges were abolished (Murakami et al., 2006). These salt bridge changes would be central to proton binding and release (Murakami et al., 2002; Guan and Nakae, 2001). However, it remains to be ascertained which changes of salt bridges in the transmembrane part of AcrB produce the conformational changes in pore domain. 
 
Cytoplasmic drug transport
Finally, two different interpretations on how the substrates collected at the central cavity from the cytoplasm or inner leaflet of cytoplasmic membrane are transported out of cell are identified. Seeger et al. (2006) stated that substrate transport from the membrane domain to the periplasmic tunnel might be via a lateral pathway, including the TM8/TM9 groove and the AcrA and AcrB interface. The TM8/TM9 groove was the transmembrane groove that was shallow at the cytoplasmic end and deep at the periplasmic end. Substrates (e.g., minocycline and doxorubicin action sites of which are cytoplasmic parts) located in the cytoplasm or inner leaflet of cytoplasmic membrane might be transported across the membrane through the groove and then the transported substrates might be collected in the central cavity via the disordered region of the top TM8 (Murakami et al., 2002). However, Seeger et al. (2006) stated that access of the collected substrates from the central cavity to the funnel of TolC docking domain was prohibited by the small diameter of the AcrB trimer center pore. Although Murakami et al. (2006) described the central cavity, how the substrates collected at the central cavity from the cytoplasm or inner leaflet of cytoplasmic membrane are transported out of cell is still unclear. The central cavity was connected with the three entrances (three interfaces of each monomer) opened to the periplasm and the lateral grooves (vestibules) were near the entrances (Murakami et al., 2006; Yu et al., 2003). If the interpretations of Murakami et al. (2002 and 2006) would be correct, I could not rule out the probability that the collected substrates may freely pass as follows (Figure 2): (i) from the central cavity to the entrance, (ii) from the entrance to the periplasm, and then (iii) from the periplasm to the vestibule. And then the subsequent transport processes accord with those via the functional rotation transport mechanism (Seeger et al. 2006; Murakami et al. 2006). One of the most direct ways to test the probability is to perform some in situ imaging experiments using labeled substrates. Indeed, extensive studies will be needed to understand the cytoplasmic substrates (drugs) transport, through a combination of structural, biochemical and cell biological approaches.
 
 
Figure 2. New interpretation on how the substrates collected at the central cavity from the cytoplasm or inner leaflet of cytoplasmic membrane are transported out of cell. Ribbon representation viewed from the side parallel to the membrane plane. The three AcrB protomers are individually coloured as in Figure 1b. The minocycline molecule is shown in CPK (Corey-Pauling-Koltun) representation, with the position of the C, N and O atoms indicated by yellow, blue and red balls, respectively. The extra-membrane (periplasmic) headpiece is at the top and the transmembrane region is at the bottom. The pathway of the substrate transportation from the cytoplasm out of cell is indicated by red dash-dot arrows. I propose that the substrates collected at the central cavity from the cytoplasm (a, or inner leaflet of cytoplasmic membrane) are transported out of cell as follows: from the central cavity (b) to the entrance (c), from the entrance to the periplasm (d) and then from the periplasm to the vestibule (e). And then the subsequent transport processes from the vestibule out of cell accord with those via the functional rotation transport mechanism (Seeger et al. 2006; Murakami et al. 2006).
 
 
Acknowledgments
 
I acknowledge the financial supports of the National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008-313-C00790), the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Government (MEST) and the Driving Force Project for the Next Generation of Gyeonggi Provincial Government in Republic of Korea. 
 
 
References
 
1. Gottesman, M.M. (2002). Mechanisms of cancer drug resistance. Annual Review of Medicine, 53, 615-627.
2. Li, X.Z. & Nikaido, H. (2004). Efflux-mediated drug resistance in bacteria Drugs, 64, 159-204.

3. Seeger, M.A., Schiefner, A., Eicher, T., Verrey, F., Diederichs, K. & Pos, K.M. (2006). Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science, 313, 1295-1298. (opened 1 September 2006)

4. Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T. & Yamaguchi, A. (2006). Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature, 443, 173-179. (published online 16 August 2006).

5. Murakami, S., Nakashima, R., Yamashita, E. & Yamaguchi, A. (2002). Crystal structure of bacterial multidrug efflux transporter AcrB. Nature, 419, 587-593.

6. Yu, E.W., McDermott, G., Zgurskaya, H.I., Nikaido, H. & Koshland, D.E. Jr. (2003). Science 300, 976-980.

7. Guan, L. & Nakae, T. Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa. (2001). Journal of Bacteriology, 183, 1734-1739.

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This Article was published on 29th August, 2009 at 08:07:03 and has been viewed 5335 times.

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The full citation for this Article is:
Lee, S. (2009). Multidrug efflux transporter AcrB structure: mechanism-unclear salt bridge changes and cytoplasmic drug transport. PHILICA.COM Article number 165.


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