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Review
. 2021 May 12;121(9):5378-5416.
doi: 10.1021/acs.chemrev.0c00621. Epub 2020 Nov 19.

Structural and Functional Diversity of Resistance-Nodulation-Cell Division Transporters

Philip A Klenotic  1 , Mitchell A Moseng  1 , Christopher E Morgan  1 , Edward W Yu  1
Affiliations
Review

Structural and Functional Diversity of Resistance-Nodulation-Cell Division Transporters

Philip A Klenotic et al. Chem Rev. .

Abstract

Multidrug resistant (MDR) bacteria are a global threat with many common infections becoming increasingly difficult to eliminate. While significant effort has gone into the development of potent biocides, the effectiveness of many first-line antibiotics has been diminished due to adaptive resistance mechanisms. Bacterial membrane proteins belonging to the resistance-nodulation-cell division (RND) superfamily play significant roles in mediating bacterial resistance to antimicrobials. They participate in multidrug efflux and cell wall biogenesis to transform bacterial pathogens into "superbugs" that are resistant even to last resort antibiotics. In this review, we summarize the RND superfamily of efflux transporters with a primary focus on the assembly and function of the inner membrane pumps. These pumps are critical for extrusion of antibiotics from the cell as well as the transport of lipid moieties to the outer membrane to establish membrane rigidity and stability. We analyze recently solved structures of bacterial inner membrane efflux pumps as to how they bind and transport their substrates. Our cumulative data indicate that these RND membrane proteins are able to utilize different oligomerization states to achieve particular activities, including forming MDR pumps and cell wall remodeling machineries, to ensure bacterial survival. This mechanistic insight, combined with simulated docking techniques, allows for the design and optimization of new efflux pump inhibitors to more effectively treat infections that today are difficult or impossible to cure.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Phylogenetic Tree of RND transporters. Simplified diagram based on the SuperfamilyTree2program that analyzes proteins and generates a tree showing phylogenetic positions of members within the superfamily. Black boxes show the RND pumps that are highlighted in this review. AAPE – aryl-polyene pigments, EST – eukaryotic sterol transporter, HAE - hydrophobe/amphiphile efflux, HME, heavy metal efflux, NFE – nodulation factor exporter.
Figure 2.
Figure 2.
RND transport assembly. Cartoon representation depicting a trimeric RND transport system that spans both the inner and outer membranes of Gram-negative bacteria. Substrates such as antibiotics and other harmful biocides (yellow hexagons) that have entered the cell are shuttled from the periplasmic space to the exterior through a channel created by the coordinated assembly between the IMP (blue), MFP (red) and OMP (green) subunits.
Figure 3.
Figure 3.
General structure of trimeric RND efflux systems. (a) The assembled components of a tripartite efflux system (adapted from PDB ID 5O66) visualized in side view with the inner membrane pump IMP (dark blue), membrane fusion protein MFP (dark red) and outer membrane protein OMP (dark green). Subunits are color-coded by slight variations of the indicated color accordingly. (b) Top view of trimeric OMP visualized from above the outer membrane suface (green). (c) Top view through the MFP hexamer within the periplasm (red). (d) The trimeric IMP as viewed from the periplasmic inner membrane surface (blue).
Figure 4.
Figure 4.
Comparison of the AcrAB-TolC efflux system components in open and closed channel conformations. Subunits are color-coded by slight variations of the indicated color accordingly: AcrA (red), AcrB (blue) and TolC (green). (a) Structure of AcrAB-TolC efflux system in the open channel state visualized in side view (adapted from PDB ID 5O66). (b) Top view (top and middle panels) and side view (bottom panel) of AcrA-TolC interface in the ligand bound open channel state. (c) Structure of the apo closed channel state of the AcrAB-TolC pump visualized in side view (adapted from PDB ID 5V5S). (d) Top view (top and middle panels) and side view (bottom panel) of AcrA-TolC interface in apo closed channel state. (e-h) AcrB protomers viewed in their access, binding and extrusion states. Separate colors depict distinct subdomains. (e) Ribbon diagram of the AcrB protomers viewed from the inner membrane plane (adapted from PDB ID 5NC5). (f) Side view of the access state of the AcrB protomer with substrate (cyan pentagon) entering the periplasmic cleft. (g) Side view of the binding state with arrows indicating the closing of the periplasmic cleft and the transport of protons through the transmembrane domain. (h) Side view of the extrusion state with closed PN1/PC2 and PN2/PC1 subdomains and exported substrate.
Figure 5.
Figure 5.
Substrate binding sites and proton-relay network of the MtrDCR103 pump (adapted from PDB ID 6VKT). (a) Ribbon diagram of the trimeric MtrD CR103 pump viewed from the membrane plane with the access, binding and extrusion protomers colored dark red, dark green and dark blue, respectively. (b) The periplasmic multidrug binding sites of MtrD CR103 with the periplasmic cleft entrance site, proximal drug binding site and distal drug binding site in the first, second and third panels. Residues that are important for selectivity are represent as sticks (black). The F loop and G loop are colored green and yellow, respectively, in each panel. (c) The proton-relay network of the MtrD CR103 multidrug efflux pump with the residue involved in the proton transport process (yellow sticks). In the access state K948 forms a hydrogen bond with D406 in the first panel. In the second panel the binding state of MtrD CR103 protomer K948 forms hydrogen bonds with both D405 and D406. In the extrusion state the K948 forms hydrogen bonds with N949 and T985 passing the proton through the transmembrane domain.
Figure 6.
Figure 6.
The closed/open states of CmeB and the gates of CmeC. (a) Ribbon diagram of the CmeB homotrimer viewed in the membrane plane (adapted from PDB ID 5T0O). Each subunit of CmeB is labeled with a different color. (b) The closed “resting” state protomer of CmeB (slate) superimposed onto the open binding state protomer (light green). Subdomains DN, DC, PN1, PN2, PC1 and PC2 are labeled on the front of the protomers. In the “resting” state the periplasmic cleft between PC1 and PC2 is closed in each protomer. The “binding” state of the CmeB protomer creates a channel through the opening of the periplasmic cleft and is exposed to solvent. (c) Ribbon diagram of the CmeC trimer viewed in the outer membrane plane (adapted from PDB ID 4MT4). Each subunit of CmeC is labeled with a different color. (d) Top view of the CmeC trimer looking down from the extracellular space. The three R104 residues (colored sticks) from each protomer are found to interact and form the first gate of the channel. (e) Periplasmic view from the top of the CmeC trimer showing the charged and polar residues Q412, D413, E416 and N420 form each protomer forming the second gate to block the channel.
Figure 7.
Figure 7.
Drug and lipid binding sites of the AdeB pump. (a) Ribbon diagram of the cryo-EM structure of the trimeric AdeB multidrug efflux pump viewed in the inner membrane plane. Each protomer of AdeB is labeled with a different color (dark red, green, light blue). (b) The F-loop that forms part of the proximal multidrug binding site with residues (dark red sticks) that are important (dark red) for drug binding in this F-loop. The G-loop (green) with residues (green sticks) important for delivering drug molecules to the distal multidrug binding site. (c) The PE lipid (yellow sticks) binding site at the interior surface of the central cavity of an AdeB protomer. The residues (grey sticks) of the AdeB protomer that are involved in binding the lipid. (d) The PE lipid binding site at the interface between two AdeB protomers (green and light blue) with the residues (grey sticks) involved in binding the lipid.
Figure 8.
Figure 8.
Heavy metal binding sites of CusA and ZneA. (a) The ribbon diagram of the Cu+ ion (dark orange sphere) bound CusA trimer pump (adapted from PDB ID 3KSS) viewed parallel to the inner membrane plane. Each protomer of CusA is labeled with a different color (ruby, purple and green). (b) Ribbon diagram of the Zn2+ ion (slate sphere) bound structure of ZneA (adapted from PDB ID 4K0J), viewed parallel to the inner membrane plane with colored protomers (orange, sand and dark magenta). (c) Metal extrusion pathway of CusA from the periplasm. The Cu+ ion (dark orange sphere) is coordinated by the three methionine binding site M573, M623 and M672 (black sticks) The methionine pair M271-M755 is where the metal ion could then be released for extrusion through CusB. (d) Metal extrusion pathway of CusA from the cytoplasm. Metal ions are passed through the transmembrane via the residues (black sticks) that form the methionine pair channel. (e) The Zn2+ ion binding site within the ZneA periplasmic cleft with a Zn2+ ion (slate sphere) coordinated by acidic residues (black sticks).
Figure 9.
Figure 9.
Components of the CusCFBA efflux system. (a) Model of the CusCBA tripartite efflux system. The system contains of the inner membrane pump CusA protomers (shades of blue), the membrane fusion adaptor CusB subunits (shades of red), and the outer membrane channel CusC monomers (shades of green) adapted from PDB ID 4DNT and PDB ID 4K7R. (b) Extracellular top view of the tripartite CusC monomers (shades of green) by a ribbon diagram (adapted from PDB ID 4K7R). (c) Ribbon structure of the Cu+ ion (dark orange sphere) bound CusF metallochaperone (adapted from PDB ID 3E6Z). The binding of the Cu+ ion is coordinated by the residues H36, M47 and M49 (black sticks). (d) The ribbon diagram of the CusBA subunit with each subunit of CusBA containing one CusA molecule (blue) and two CusB molecules (red and light pink) adapted from PDB ID 4DNT. (e) Ribbon diagram of single flexible CusB subunit coordinating two Cu+ ions (dark orange sphere) adapted from PDB ID 3OW7.
Figure 10.
Figure 10.
PAβN binds to AcrB (a) Trimeric architecture of AcrB (adapted from PDB ID 1T9Y). AcrB crystallized with three-fold symmetry with PAβN bound at two sites, a periplasmic site (cyan spheres) and the central cavity (wheat spheres). (b) Magnified view of PAβN bound at the periplasmic site. Residues F664, S715, R717 and P718 (yellow sticks) contribute electrostatic interactions to stabilize PAβN (cyan sticks).
Figure 11.
Figure 11.
D13–9001 binds to the hydrophobic trap. (a) D13–9001 binds to AcrB (PDB ID 3W9H). The hydrophobic trap is lined with Phe residues while hydrophilic residues contribute important electrostatic interactions. F136, F178, F610, F615 and F628, Q176, S180, N274 and R620 (yellow sticks) stabilize D13–9001 (cyan sticks) at this site. (b) D13–9001 binds to MexB (PDB ID 3W9J). The hydrophobic trap of MexB is similar to that of AcrB. Residues F136, F178, F573, F610, F615, F628, K151, D274 and R620 (yellow sticks) create favorable electrostatic interactions with D13–9001 (cyan sticks).
Figure 12.
Figure 12.
The structure of B. multivorans HpnN. (a) HpnN structural organization. A monomer of HpnN consists of 12 transmembrane domains (TMs 1–12; blue) and four periplasmic domains: PD1 (red), PD2 (yellow), PD3 (cyan) and PD4 (magenta). (b) Important residues in HpnN. Important conserved residues have been identified in HpnN through a combination of structural and mutational studies. Residues F270, V332, V339 and L826 (cyan spheres) form the tunnel entrance and are important for hopanoid recognition in the inner membrane. Residue L48 (yellow spheres) constricts the tunnel and controls the flow of hopanoids through the pump. Residues F117, F541 and W661 (Wheat spheres) are conserved residues that form the exit site of the tunnel. Residues D344, T818 and T819 (red spheres) participate in the proton transport chain (PTC) to fuel the pump. (c) Dimeric forms of HpnN. Form I (left) and Form II (right) differ by a >5 Å swinging motion of the periplasmic domains. This motion constricts the tunnel at L48 and is believed to regulate hopanoid shuttling by HpnN. All figures adapted from PDB ID 5KHN (Form I) and ID 5KHN (Form II).
Figure 13.
Figure 13.
The structure of M. smegmatis MmpL3. (a) Ribbon diagram depicting the crystal structure of MmpL3. Colors are consistent for all panels. Dark blue represents the 12 α-helices that constitute the transmembrane region of the protein as well as two small α-helices (TM1a and TM7a) that are located on the inner leaflet of the inner membrane. The TM helices are mostly embedded within the inner membrane with TM2 and TM8 extending into the periplasm. PD1 and PD2 are tethered to the membrane through flexible loops, allowing significant mobility and interaction between the two periplasmic domains. PD1 is light blue along with the helix α5 (green). PD2 is green with the helix α1 (light blue). Other features include the start of the cytoplasmic c-terminal domain (red), DDM detergent molecule (green and red) and PE located in the cavity formed by PD1 and PD2 (orange). (b) magnified view of the PE binding pocket. The side chains of MmpL3 that can stabilize PE binding are shown in purple. (c) Cartoon schematic identifying each secondary structural component. (d) Magnified view of important side chains for proton translocation (orange) TMs are labeled in yellow. All figures adapted from PDB ID 6OR2.
Figure 14.
Figure 14.
Structures of MmpL3-associated proteins. (a) Crystal structure of LpqN (adapted from PDB ID 6E5D). The central ribbon is depicted in green with α-helices in blue and loop regions in pink. (b) and (c) Overlay of substrate bound LpqN with the apo-form shown in (a). Arrows point to areas of significant shift upon substrate binding, within the central ribbon (compare light green (bound) to dark green (unbound)) and the proximal α-helix (compare light blue (bound) to dark blue (unbound)). L6T (b) is shown as orange (carbon atoms) and red (oxygen atoms) and T6D (c) is shown as light grey (carbon atoms) and red (oxygen atoms). L6T-bound LpqN ane T6D-bound LpqN structures adapted from PDB ID 6E5F and PDB ID 6MNA, respectively. (d) Crystal structure of TftA (adapted from PDB ID 6T84). The central antiparallel β-sheet region (tan) is surrounded by α-helices (light blue) and flexible loops (grey).
Figure 15.
Figure 15.
MmpL3 as a drug target. (a) Mutations within MmpL3 are able to confer resistance to a variety of antibiotics. Red spheres represent residues whose side chains can directly affect the putative binding site of SQ109 and other MmpL3 inhibitors. Tan spheres represent residues that are outside of the inhibitor binding pocket (adapted from PDB ID 6OR2). (b) SQ109 directly interrupts the proton translocation chain in MmpL3. α-helices are depicted in green while β-strands are blue. SQ109 is shown in tan (carbon atoms) and blue (nitrogen atoms) (adapted from PDB ID 6AJH). A magnified view of the SQ109 binding pocket shows how the critical Asp-Tyr pairs are disrupted when SQ109 is present. Side chains are shown in grey (carbon atoms) and red (oxygen atoms). Parts of TM5 and TM6 are removed for clarity.
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