# Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

Ilyas Alav, Jessica Kobylka, Miriam S Kuth, Klaas M Pos, Martin Picard, Jessica M A Blair, Vassiliy N Bavro

► To cite this version:

Ilyas Alav, Jessica Kobylka, Miriam S Kuth, Klaas M Pos, Martin Picard, et al., Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria. Chemical Reviews, 2021, 121 (9), pp.5479-5596. 10.1021/acs.chemrev.1c00055. [https://doi.org/10.1021/acs.chemrev.1c00055](https://doi.org/10.1021/acs.chemrev.1c00055). [hal-03452959](https://hal.science/hal-03452959)

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# Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

Ilyas Alav, Jessica Kobylka, ∇ Miriam S. Kuth, ∇ Klaas M. Pos, Martin Picard, Jessica M. A. Blair,* and Vassiliy N. Bavro*

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Cite This: *Chem. Rev.* 2021, 121, 5479–5596

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Supporting Information

**ABSTRACT:** Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components—the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

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## CONTENTS

1. Introduction: The Problem of Pumping across Two Membranes in Gram-Negative Bacteria 5481

2. Involvement of Tripartite Systems in Antibiotic Resistance and Drug Efflux Profiles for Principal Classes of Transporters 5484

3. Biological Functions Outside of Multidrug Resistance 5484

3.1. Virulence 5485

3.1.1. Defense against Host-Derived Molecules 5486

3.1.2. Export of Virulence Factors and Toxins 5486

3.2. Quorum Sensing 5487

3.3. Biofilm Formation 5487

3.4. Other Reported Biological Roles 5488

4. Genes and Regulation of Expression 5489

4.1. Local Regulation 5489

4.2. Global Regulation 5490

4.3. Post-transcriptional Regulation 5492

4.4. Regulation by Two-Component Systems 5492

4.5. Other Types of Regulation 5492

5. Classes of Transporter Proteins 5493

5.1. Resistance–Nodulation–Division (RND) Transporter Superfamily 5493

5.1.1. Bacterial Drug Efflux Proteins from the HAE Family of RND Transporters 5493

5.1.2. The *E. coli* HAE-1 Member Acriflavine Resistance Protein B (AcrB) 5494

5.1.3. Structural Features of AcrB 5495

5.1.4. Substrate and Inhibitor Binding to Multidrug Efflux RND Transporter 5497

5.1.5. Drug/Proton Antiport Coupling in AcrB 5499

5.1.6. Structures of Other HAE-1 RND Transporters 5500

5.1.7. HME Family 5501

5.1.8. Other Bacterial RND Transporters 5501

5.2. Major Facilitator Superfamily (MFS) Transporters 5501

5.3. Multiple Antibiotic and Toxin Extrusion (MATE) Transporters 5502

5.4. Small Multidrug Resistance (SMR) Transporters 5502

5.5. Proteobacterial Antimicrobial Compound Efflux (PACE) Transporter Family 5503

5.6. ABC Transporter Family 5503

**Special Issue:** Transporters, Porins, and Efflux Pumps

Received: January 20, 2021

Published: April 28, 2021

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<table>
  <tbody>
    <tr>
      <td>5.6.1. Type IV Family of ABC Transporters Involved in Tripartite Assemblies</td>
      <td>5503</td>
    </tr>
    <tr>
      <td>5.6.2. NisT/McjD Family of Bacteriocin Transporters</td>
      <td>5504</td>
    </tr>
    <tr>
      <td>5.6.3. Structural Features of McjD and the Standard Cycle of the Type IV ABC Family</td>
      <td>5505</td>
    </tr>
    <tr>
      <td>5.6.4. ABC Transporters with Double Functions—The Peptidase-Containing ATP-Binding Cassette Transporters (PCAT) Family</td>
      <td>5506</td>
    </tr>
    <tr>
      <td>5.6.5. Unique Features of the AMS/PCAT Transporters—N-Terminal C39 Protease and CLD Domains</td>
      <td>5506</td>
    </tr>
    <tr>
      <td>5.6.6. NBDs of PCAT Transporters</td>
      <td>5509</td>
    </tr>
    <tr>
      <td>5.6.7. Structure of AMS/PCAT Family Transporters</td>
      <td>5509</td>
    </tr>
    <tr>
      <td>5.6.8. Models of the Mechanism of Cargo Transport of the PCAT Type ATPases</td>
      <td>5510</td>
    </tr>
    <tr>
      <td>5.6.9. PCATs Associated with T1SS</td>
      <td>5512</td>
    </tr>
    <tr>
      <td>5.7. MacB/FtsX Transporter Family</td>
      <td>5513</td>
    </tr>
    <tr>
      <td>5.7.1. Biological Functions</td>
      <td>5514</td>
    </tr>
    <tr>
      <td>5.7.2. Gram-Positive Homologues of the MacB Transporters</td>
      <td>5515</td>
    </tr>
    <tr>
      <td>5.7.3. Topology and Structural Organization of MacB Family Members</td>
      <td>5516</td>
    </tr>
    <tr>
      <td>5.7.4. General Topology of MacB/FtsX Family and TM Domains</td>
      <td>5516</td>
    </tr>
    <tr>
      <td>5.7.5. Periplasmic Core Domains (PCDs)</td>
      <td>5517</td>
    </tr>
    <tr>
      <td>5.7.6. Versatility of PD Fold outside of the MacB/FtsX Family. Modularity of Assembly, Phylogenetic Connections, and Topological Linkage between Different Transporter Groups</td>
      <td>5519</td>
    </tr>
    <tr>
      <td>5.7.7. Relations of the Membrane Porter Domains of MacB/FtsX to Other ABC Transporter Families</td>
      <td>5524</td>
    </tr>
    <tr>
      <td>5.7.8. TMD and Energy Coupling</td>
      <td>5524</td>
    </tr>
    <tr>
      <td>5.7.9. Substrate Binding Site</td>
      <td>5525</td>
    </tr>
    <tr>
      <td>6. Outer Membrane Factors (OMFs): TolC and Others</td>
      <td>5526</td>
    </tr>
    <tr>
      <td>6.1. Introduction to the OMFs</td>
      <td>5526</td>
    </tr>
    <tr>
      <td>6.2. Overall Organization of OMFs</td>
      <td>5526</td>
    </tr>
    <tr>
      <td>6.3. Gates within the OMFs</td>
      <td>5528</td>
    </tr>
    <tr>
      <td>6.3.1. Gates within the OMFs: Outer Membrane Loops</td>
      <td>5528</td>
    </tr>
    <tr>
      <td>6.3.2. Gates within the OMFs: Knob-into-Holes and Bistable Switching between Opening and Closing of the α-Helical Bundle</td>
      <td>5529</td>
    </tr>
    <tr>
      <td>6.3.3. Gates within the OMFs: Primary and Secondary Periplasmic Gates</td>
      <td>5531</td>
    </tr>
    <tr>
      <td>7. Periplasmic Adaptor Proteins (PAPs)/Membrane Fusion Proteins (MFPs)</td>
      <td>5534</td>
    </tr>
    <tr>
      <td>7.1. Historical Note</td>
      <td>5534</td>
    </tr>
    <tr>
      <td>7.2. Structural Organization of PAPs</td>
      <td>5534</td>
    </tr>
    <tr>
      <td>7.3. Domain Organization of the PAPs Associated with RND and MacB-Assemblies</td>
      <td>5535</td>
    </tr>
    <tr>
      <td>7.4. PAPs Associated with MFS Transporters and T1SS</td>
      <td>5538</td>
    </tr>
    <tr>
      <td>7.5. T1SS Associated PAPs</td>
      <td>5538</td>
    </tr>
    <tr>
      <td>7.6. PAPs Associated with the Heavy Metal Efflux (HME) Family</td>
      <td>5539</td>
    </tr>
  </tbody>
</table>

<table>
  <tbody>
    <tr>
      <td>7.7. Fellowship of the Rings—The Role of PAP β-Barrel Domains in Assembly and Processivity of Tripartite Efflux Pumps</td>
      <td>5540</td>
    </tr>
    <tr>
      <td>8. Assembly and Function of the RND-Based Tripartite Complexes</td>
      <td>5542</td>
    </tr>
    <tr>
      <td>8.1. Cryo-EM Structures of Fully Assembled RND Complexes</td>
      <td>5542</td>
    </tr>
    <tr>
      <td>8.2. PAP-OMF Interface</td>
      <td>5543</td>
    </tr>
    <tr>
      <td>8.3. RND-PAP Interface: Structural Data Suggests Asymmetry of the Assembled Tripartite Assembly and Different Roles for the PAPs</td>
      <td>5545</td>
    </tr>
    <tr>
      <td>8.4. Heterooligomeric Complexes Allow Unambiguous Assignment of PAP1 and PAP2 Highlighting Their Distinct Roles during Assembly and Cycling of the Pump</td>
      <td>5548</td>
    </tr>
    <tr>
      <td>8.5. Conformational Cycling of the Protomers within the AcrB/MexB Trimer</td>
      <td>5549</td>
    </tr>
    <tr>
      <td>8.6. Toward the Unified Model of RND Pump Assembly—An Integrated Model of PAP Participation in the Efflux Cycle and Resetting of the RND Assemblies</td>
      <td>5549</td>
    </tr>
    <tr>
      <td>8.7. RND-PAP Interface and Energetic Considerations</td>
      <td>5552</td>
    </tr>
    <tr>
      <td>8.8. Tripartite Complexes of HME-RND Family</td>
      <td>5553</td>
    </tr>
    <tr>
      <td>8.9. Could the Structure of the Metal Pump CusBA Suggest a Conformation for a Pre-engagement Apo-State for the Transporter-PAP Complex?</td>
      <td>5554</td>
    </tr>
    <tr>
      <td>8.10. Additional Level of Regulation of RND Pumps May Be Provided by the Trans-membrane Modulators of the AcrZ-Family</td>
      <td>5555</td>
    </tr>
    <tr>
      <td>8.11. Functional Assays in Artificial Membrane Environments Provide Additional Insights into the Regulation of the RND Transporters and the Role of the Lipid Environments</td>
      <td>5555</td>
    </tr>
    <tr>
      <td>9. Tripartite Assembly of MacAB-TolC</td>
      <td>5557</td>
    </tr>
    <tr>
      <td>9.1. Functional Cycle of the Tripartite MacB-Pumps</td>
      <td>5560</td>
    </tr>
    <tr>
      <td>10. Tripartite Assembly of the MFS-Based Systems</td>
      <td>5561</td>
    </tr>
    <tr>
      <td>11. Assembly of Type 1 Secretion Systems</td>
      <td>5562</td>
    </tr>
    <tr>
      <td>12. Targeting Tripartite Assemblies—Pump Inhibitors and Disruptors</td>
      <td>5566</td>
    </tr>
    <tr>
      <td>12.1. Targeting the Inner Membrane Transporter</td>
      <td>5566</td>
    </tr>
    <tr>
      <td>12.2. Targeting the Periplasm Adaptor Protein</td>
      <td>5568</td>
    </tr>
    <tr>
      <td>12.3. Targeting the Outer Membrane Factor</td>
      <td>5569</td>
    </tr>
    <tr>
      <td>12.4. Other Strategies to Inhibit Efflux</td>
      <td>5569</td>
    </tr>
    <tr>
      <td>13. Conclusion</td>
      <td>5569</td>
    </tr>
    <tr>
      <td>14. Remaining Challenges and Future Directions</td>
      <td>5570</td>
    </tr>
    <tr>
      <td>Associated Content</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Supporting Information</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Author Information</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Corresponding Authors</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Authors</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Author Contributions</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Notes</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Biographies</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>Acknowledgments</td>
      <td>5571</td>
    </tr>
    <tr>
      <td>References</td>
      <td>5572</td>
    </tr>
  </tbody>
</table>
<figure><img src="image_11.png" /><figcaption>**Figure 1.** Representative structures of multidrug transporters and tripartite assemblies. Structures of members of the ATP-driven ATP-binding cassette (ABC) superfamily and the proton-motive force-dependent secondary antiporters of the major facilitator superfamily (MFS), multidrug and toxin extrusion (MATE) family, small multidrug resistance (SMR) family, and resistance nodulation-cell division (RND) superfamily are shown. A substrate for each of the transporters is indicated (colored double triangles). The single-component transporters are independent multidrug/H⁺ (or Na⁺) antiporters which transport the drugs from the cytoplasm to the periplasm. The tripartite RND superfamily member MexAB-OprM and the tripartite ABC superfamily member MacAB-TolC are postulated to sequester their substrates from the periplasm (or outer leaflet of the inner membrane) and transport them across the outer membrane. The single component multidrug efflux antiporters and the RND-type tripartite efflux pumps act in a synergistic fashion to efflux drug substrates across both inner and outer membranes. Structures of a proteobacterial antimicrobial compound efflux (PACE) member are elusive and therefore not included in the figure. ABC superfamily members and the 14-transmembrane helix MFS transporters form also tripartite pumps. The ABC superfamily member HlyBD-TolC transports hemolysin from the cytoplasm to the outside of the cell. The ABC-type tripartite MacAB-TolC complex transports macrolide antibiotics, outer membrane glycolipids, lipopeptides, and protoporphyrin and polypeptide virulence factors such as enterotoxin II. The MFS-type tripartite EmrAB-TolC system has been shown to transport several small molecular weight drugs. EmrE Protein Databank (PDB) entry: 3BSD; DinF-BH PDB: 4LZ9; MdfA PDB: 4ZOW; MexAB-OprM EM Data Bank (EMDB) entry: EMD-10395, PDB: 6TA6; EmrAB-TolC adapted from Yousefian et al.;⁹ MacAB-TolC EMDB: EMD-3652, PDB: 5NIK; MacB EMDB: EMD-3653, PDB: 5NIL. The HlyB model (based on PCAT1 (PDB: 4RY2)) and the full assembly of the HlyD-docked homology model are provided by V. Bavro. LPS, lipopolysaccharide.</figcaption></figure>

# 1. INTRODUCTION: THE PROBLEM OF PUMPING ACROSS TWO MEMBRANES IN GRAM-NEGATIVE BACTERIA

The cell envelope of Gram-negative bacteria consists of three fundamental layers: the inner or cytoplasmic membrane, the peptidoglycan cell wall, and the outer membrane. The two membrane layers are separated by an aqueous cellular compartment known as the periplasm.¹ Although the double membrane cell envelope is a sophisticated barrier that affords Gram-negative bacteria protection from various environmental insults, it also presents a biological challenge for transporting molecules out of cells. This has given rise to a plethora of transenvelope transport machinery, namely tripartite efflux systems and the related type 1 secretion systems (T1SSs).²,³ Tripartite efflux systems and the related T1SSs consist of a transmembrane inner-membrane transporter protein, a periplasmic adaptor protein (PAP) that spans the periplasm, and an outer membrane factor (OMF) protein that penetrates the outer membrane. This tripartite organization allows Gram-negative bacteria to directly transport molecules across the outer membrane to the extracellular environment. Tripartite efflux systems can be categorized into three superfamilies based on the type of the inner-membrane transporter around which they are built: with multidrug efflux pumps being formed with the participation of transporters belonging to either the ATP-

binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), or the resistance-nodulation-division (RND) superfamily, whereas the transporters associated with the related T1SS belong exclusively to the ABC superfamily (Figure 1).²,⁴,⁵ The T1SSs secrete diverse proteins, many of which are involved in host pathogenesis and virulence.⁶ Tripartite efflux systems play a major role in the multidrug resistance of Gram-negative bacteria, including ESKAPE pathogens such as *Acinetobacter baumannii*, *Enterobacter* spp., *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa*, which have contributed significantly to the increasing rate of antimicrobial resistance (AMR)-related infections.⁷ AMR is a major global public health crisis that undermines not only human health, but also animal health, food security, and development. The severity of this problem has led the World Health Organization to adopt the Global Action Plan on AMR that consists of five strategic objectives to combat AMR, one of which is to stimulate the development of new medicines.⁸ Therefore, to develop new therapeutics to target tripartite efflux systems and T1SSs, we must better understand their physiology and roles in Gram-negative bacteria.

In the sections below, we will discuss the genes, function, and structural organization of tripartite efflux systems and T1SSs. This is a vast topic, which is also reflected in the volume of this systematic review, and to facilitate the reader,
<table><caption>Table 1. List of Characterized Tripartite Systems in Gram-Negative Bacteria and Their Substrate Profiles<sup>a</sup></caption><thead><tr><th>Microorganism</th><th>Efflux pump family</th><th>Efflux system</th><th>Substrates</th><th>refs</th></tr></thead><tbody><tr><td rowspan="6">Acinetobacter baumannii</td><td rowspan="6">RND</td><td>AdeABC</td><td>AG, BL, CHL, CHX, ML, FA, FQ, TET, TGC, TMP</td><td>11, 19, 20</td></tr><tr><td>AdeFGH</td><td>CHL, FQ, SUL, TET, TGC, TMP</td><td>21</td></tr><tr><td>AdeIJK</td><td>BL, CHL, CHX, ERY, FA, FQ, NOV, TET, TGC, TMP, TRI</td><td>11</td></tr><tr><td>AbuO</td><td>AG, BL, TGC</td><td>22</td></tr><tr><td>ArpAB</td><td>AG</td><td>23</td></tr><tr><td>ABC</td><td>MacAB-TolC</td><td>ML</td><td>24</td></tr><tr><td>Acinetobacter Genomic DNA Group 3</td><td>RND</td><td>AdeDE</td><td>CHL, FQ, TET</td><td>25, 26</td></tr><tr><td rowspan="3">Achromobacter spp.</td><td rowspan="3">RND</td><td>AdeXYZ</td><td>CHL, FQ, BL, TET</td><td>25</td></tr><tr><td>AxyXY-OprZ</td><td>AG, FQ</td><td>27</td></tr><tr><td>NccABC</td><td>Cd<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup></td><td>28</td></tr><tr><td>Aeromonas hydrophila</td><td>RND</td><td>AheABC</td><td>RIF</td><td>29</td></tr><tr><td>Aggregatibacter actinomycetemcomitans</td><td>ABC</td><td>LtxDB-TolC</td><td>LTX</td><td>30</td></tr><tr><td>Bacteroides fragilis</td><td>RND</td><td>BmeABC3</td><td>BL, FQ</td><td>31</td></tr><tr><td>Borrelia burgdorferi</td><td>RND</td><td>BesABC</td><td>ML</td><td>32</td></tr><tr><td>Bordetella pertussis</td><td>ABC</td><td>CyaBDE</td><td>ACT</td><td>33</td></tr><tr><td>Bradyrhizobium japonicum</td><td>RND</td><td>BdeAB-?</td><td>AG</td><td>34</td></tr><tr><td rowspan="2">Brucella suis</td><td rowspan="2">RND</td><td>BepDE-BepC</td><td>ACR, AMP, DOC, EB, FQ, NOV, PMB, SDS, TET</td><td>35</td></tr><tr><td>BepFG-BepC</td><td>DOC, NAL, SDS</td><td>35</td></tr><tr><td rowspan="3">Burkholderia cenocepacia</td><td rowspan="3">RND</td><td>CeoAB-OpcM</td><td>CHL, FQ</td><td>36</td></tr><tr><td>BCAL2820-BCAL2822</td><td>ATM, AG, CHL, FQ</td><td>37</td></tr><tr><td>AmrAB-OprA</td><td>AG, ML</td><td>38</td></tr><tr><td rowspan="3">Burkholderia pseudomallei</td><td rowspan="3">RND</td><td>BpeAB-OprB</td><td>AG, CHL, ML, TET</td><td>38, 39</td></tr><tr><td>BpeEF-OprC</td><td>CHL, TET, TMP</td><td>40</td></tr><tr><td>AmrAB-OprA</td><td>AG, ML, TET</td><td>41</td></tr><tr><td rowspan="2">Campylobacter jejuni</td><td rowspan="2">RND</td><td>CmeABC</td><td>CHL, CTX, ML, FA, FQ, TRI</td><td>42, 43</td></tr><tr><td>CmeDEF</td><td>CTX, FQ, TRI</td><td>44</td></tr><tr><td>Caulobacter crescentus</td><td>RND</td><td>NczABC</td><td>Co<sup>2+</sup>, Cd<sup>2+</sup>, Ni<sup>2+</sup>, Zn<sup>2+</sup></td><td>45</td></tr><tr><td>Cupriavidus metallidurans</td><td>RND</td><td>ZneCAB</td><td>Zn<sup>2+</sup></td><td>46</td></tr><tr><td>Dickeya zeae</td><td>RND</td><td>DesABC</td><td>ZMN</td><td>47</td></tr><tr><td>Enterobacteriaceae<sup>b</sup></td><td>RND</td><td>AcrAB-TolC</td><td>ACR, BAC, BL, CHL, EB, ERY, FA, FQ, NOV, OXN, TET, TGC, TRI, RIF, R6G</td><td>10, 48–50, 52</td></tr><tr><td rowspan="10">Escherichia coli</td><td rowspan="10">RND</td><td>OqxAB-TolC<sup>c</sup></td><td>CHL, FQ, ML, NIT, TGC</td><td>53</td></tr><tr><td>AcrAD-TolC</td><td>AG, BL</td><td>54, 55</td></tr><tr><td>AcrEF-TolC</td><td>ACR, BL, CHL, FQs, EB, ERY, R6G, TET, TGC</td><td>10, 56, 57</td></tr><tr><td>MdtEF-TolC</td><td>BL, BAC, ML, OXN</td><td>10, 56–58</td></tr><tr><td>CusABC</td><td>Ag<sup>+</sup>, Cu<sup>+</sup></td><td>59</td></tr><tr><td>ABC</td><td>MacAB-TolC</td><td>ML</td><td>60</td></tr><tr><td>HlyBD-TolC</td><td>AH</td><td>61</td></tr><tr><td>YhbFGSR-</td><td>EB, TET</td><td>62</td></tr><tr><td>TolC</td><td>MJ25</td><td>63</td></tr><tr><td>Yojl-?-TolC</td><td>MFS</td><td>EmrAB-TolC</td><td>FQ, NOV, TRI</td><td>10, 56</td></tr><tr><td rowspan="2">Helicobacter pylori</td><td rowspan="2">RND</td><td>EmrKY-TolC</td><td>TET</td><td>64</td></tr><tr><td>AaeAB-?</td><td>ACR, PUR</td><td>56</td></tr><tr><td rowspan="2">Klebsiella aerogenes</td><td rowspan="2">RND</td><td>AaeAB-?</td><td>pHBA</td><td>65</td></tr><tr><td>HefABC</td><td>AG, BL, CHL, EB, ERY, FQ, NOV, TET</td><td>66</td></tr><tr><td rowspan="7">Klebsiella pneumoniae</td><td rowspan="7">RND</td><td>HefDEF</td><td>Cd<sup>2+</sup>, Ni<sup>2+</sup>, Zn<sup>2+</sup></td><td>67</td></tr><tr><td>EefABC</td><td>CHL, BL, ERY, FQ</td><td>68</td></tr><tr><td>AcrAB</td><td>BAC, BL, ML, FQ, TET, NOV</td><td>69, 70</td></tr><tr><td>EefABC</td><td>BAC, BL, FQ, ERY, TET, NOV</td><td>70</td></tr><tr><td>KexEF</td><td>BL, ERY, TET, NOV</td><td>70</td></tr><tr><td>KexD</td><td>ML</td><td>71</td></tr><tr><td>KexVWX</td><td>BL, NOV</td><td>72</td></tr><tr><td>MFS</td><td>KpnGH-TolC</td><td>BL, AG, ML</td><td>73</td></tr><tr><td>Legionella pneumophila</td><td>RND</td><td>LpeAB</td><td>ML</td><td>74</td></tr><tr><td>Moraxella catarrhalis</td><td>RND</td><td>AcrAB-OprM</td><td>CTX, ML</td><td>75</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>Microorganism</th><th>Efflux pump family</th><th>Efflux system</th><th>Substrates</th><th>refs</th></tr></thead><tbody><tr><td rowspan="3">Neisseria gonorrhoeae</td><td>RND</td><td>MtrCDE</td><td>BL, BS, FQ, ML, TET</td><td>76, 77</td></tr><tr><td>ABC</td><td>MacAB-MtrE</td><td>ML</td><td>78</td></tr><tr><td>MFS</td><td>FarAB-MtrE</td><td>FA</td><td>79</td></tr><tr><td>Porphyromonas gingivalis</td><td>RND</td><td>XepCAB</td><td>RIF</td><td>80</td></tr><tr><td rowspan="11">Pseudomonas aeruginosa</td><td rowspan="11">RND</td><td>MexAB-OprM</td><td>AG, BL, CHL, COL, EB, FQ, ML, SDS, SUL, TET, TMP, TRI</td><td>12, 81</td></tr><tr><td>MexCD-OprJ</td><td>BL, CHL, CHX, COL, FQ, ML, QAC, TET, TGC, TMP, TRI</td><td>81</td></tr><tr><td>MexEF-OprN</td><td>CHL, FQ, TET, TMP, TRI</td><td>82</td></tr><tr><td>MexGHI-OpmD</td><td>ACR, EB, FQ, TET</td><td>83</td></tr><tr><td>MexJK-OprM/ OpmH</td><td>ERY, TET, TRI</td><td>84</td></tr><tr><td>MexMN-OprM</td><td>CHL</td><td>85</td></tr><tr><td>MexPQ-OpmE</td><td>ML, FQ</td><td>85</td></tr><tr><td>MexVW-OprM</td><td>ACR, CHL, EB, ERY, FQ, TET</td><td>86</td></tr><tr><td>MexXY-OprA/ OprM</td><td>AG, BL, CHL, FQ, ML, TET, TGC</td><td>81</td></tr><tr><td>MuxABC-OpmB</td><td>ATM, ERY, FQ, NOV, TET</td><td>87</td></tr><tr><td>TriABC-OpmH</td><td>TRI</td><td>88</td></tr><tr><td rowspan="7">Pseudomonas putida</td><td rowspan="7">RND</td><td>ArpABC</td><td>BL, CHL, ERY, TET</td><td>89</td></tr><tr><td>MepABC</td><td>BL, ERY, TET</td><td>90</td></tr><tr><td>ParXY/TtgC</td><td>AG, FQ</td><td>91</td></tr><tr><td>SrpABC</td><td>OS</td><td>92</td></tr><tr><td>TtgABC</td><td>BL, CHL, ERY, OS, TET</td><td>93</td></tr><tr><td>TtgDEF</td><td>OS</td><td>94</td></tr><tr><td>TtgGHI</td><td>OS</td><td>95</td></tr><tr><td>Pseudomonas fluorescens</td><td>RND</td><td>EmhABC</td><td>CHL</td><td>96</td></tr><tr><td>Pseudomonas syringae</td><td>RND</td><td>PseABC</td><td>ACR, ERY, TET</td><td>97</td></tr><tr><td rowspan="2">Riemerella anatipestifer</td><td rowspan="2">RND</td><td>RaeABC</td><td>AG, SDS</td><td>98</td></tr><tr><td>RaeEF-RopN</td><td>AG, SDS</td><td>99</td></tr><tr><td rowspan="6">Salmonella enterica</td><td rowspan="6">RND</td><td>AcrAD-TolC</td><td>ATM, BL, NOV, SDS</td><td>100</td></tr><tr><td>AcrEF-TolC</td><td>ACR, BAC, BL, CHL, EB, FUS, FQs, NOV, R6G, SDS, TET, TRI</td><td>101</td></tr><tr><td>MdtABC-TolC</td><td>NOV</td><td>101</td></tr><tr><td>MdsABC</td><td>Au<sup>3+</sup>, BAC, BL, CHL, NOV</td><td>102</td></tr><tr><td>SilABC<sup>c</sup></td><td>Ag<sup>+</sup></td><td>103</td></tr><tr><td>ABC</td><td>MacAB-TolC</td><td>ML</td><td>101</td></tr><tr><td>MFS</td><td>EmrAB-TolC</td><td>NAL, NOV, TRI</td><td>101</td></tr><tr><td rowspan="5">Serratia marcescens</td><td rowspan="5">RND</td><td>SdeAB</td><td>CHL, FQ, QAC</td><td>104</td></tr><tr><td>SdeGH</td><td>FQ</td><td>105</td></tr><tr><td>SdePQ</td><td>FQ</td><td>105</td></tr><tr><td>SdeXY-HasF</td><td>BAC, CHL, CHX, FQ, ML, QAC, TET, TGC, TRI</td><td>106, 107</td></tr><tr><td>ABC</td><td>SmeAB-TolC</td><td>ACR, CHL, ERY, NAL, RIF, SDS, TET</td><td>108</td></tr><tr><td rowspan="2">Sinorhizobium meliloti</td><td rowspan="2">RND</td><td>SmeCD-TolC</td><td>CHL, NAL</td><td>108</td></tr><tr><td>SmeEF-TolC</td><td>ERY, TET</td><td>108</td></tr><tr><td rowspan="10">Stenotrophomonas maltophilia</td><td rowspan="10">RND</td><td>SmeABC</td><td>AG, BL, FQ</td><td>109</td></tr><tr><td>SmeDEF</td><td>CHL, FQ, ML, TET, TRI</td><td>110</td></tr><tr><td>SmeGH</td><td>BL, CHL, FQ, ML, TET</td><td>109, 111</td></tr><tr><td>SmeIJK</td><td>AG, TET</td><td>109</td></tr><tr><td>SmeOP-TolC</td><td>AG, FQ, ML, TET</td><td>112</td></tr><tr><td>SmeVWX</td><td>CHL, FQ, TET</td><td>113</td></tr><tr><td>SmeYZ</td><td>AG, TET</td><td>114</td></tr><tr><td>MFS</td><td>EmrCAB</td><td>CCCP, NAL</td><td>115</td></tr><tr><td rowspan="5">Vibrio cholerae</td><td rowspan="5">RND</td><td>MacABC</td><td>AG, ML, PMB</td><td>116</td></tr><tr><td>FuaABC</td><td>FUA</td><td>117</td></tr><tr><td>SmaAB</td><td>AMK</td><td>118</td></tr><tr><td>SmaCDEF</td><td>LEV</td><td>118</td></tr><tr><td>VexAB- TolC</td><td>AMP, ERY, NOV, PEN, PMB, SDS</td><td>119, 120</td></tr><tr><td>VexCD-TolC</td><td>BS, DTG, ERY</td><td>119</td></tr><tr><td>VexEF- TolC</td><td>BAC, DOC, EB, ERY, NOR, NOV, SDS, TET, TMP</td><td>119</td></tr><tr><td>VexGH</td><td>DTG, NOV</td><td>121</td></tr><tr><td>VexIJK</td><td>BS, DTG</td><td>120</td></tr><tr><td>MFS</td><td>VceCAB</td><td>CCCP, DOC, NAL</td><td>122</td></tr></tbody></table>
<table><caption>Table 1. continued</caption><thead><tr><th>Microorganism</th><th>Efflux pump family</th><th>Efflux system</th><th>Substrates</th><th>refs</th></tr></thead><tbody><tr><td rowspan="7">Vibrio parahaemolyticus</td><td rowspan="7">RND</td><td>VmeAB-VpoC</td><td>ACR, BL, BS, EB, FQ, ML, NOV, R6G, SDS, TET, TMP</td><td>123</td></tr><tr><td>VmeCD-VpoC</td><td>BAC, BL, BS, ML, NOV, R6G, SDS</td><td>124</td></tr><tr><td>VmeEF-VpoC</td><td>BC, EB, NOV, R6G, SDS</td><td>124</td></tr><tr><td>VmeGHI-VpoC</td><td>SDS</td><td>124</td></tr><tr><td>VmeTUV-VpoC</td><td>ACR, BL, BAC, BC, CHX, EB, R6G, SDS</td><td>124</td></tr><tr><td>VmeYZ-VpoC</td><td>BC, NOV, SDS</td><td>124</td></tr></tbody></table>

<sup>a</sup>ABC, ATP-binding cassette; ACR, acriflavine; ACT, adenylate cyclase toxin; AG, aminoglycosides; AH, α-hemolysin; AMK, amikacin; AMP, ampicillin; ATM, aztreonam; BAC, benzalkonium chloride; BL, β-lactams; BS, bile salts; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CHL, chloramphenicol; CHX, chlorhexidine; COL, colistin; CTX, cefotaxime; DOC, deoxycholate; DTG, detergents; ERY, erythromycin; EB, ethidium bromide; FA, fatty acids; FUS, fusidic acid; FQ, fluoroquinolones; FUA, fusaric acid; LEV, levofloxacin; LTX, leukotoxin; MFS, major facilitator superfamily; MJ25, microcin J25; ML, macrolides; NAL, nalidixic acid; NIT, nitrofurantoin; NOR, norfloxacin; NOV, novobiocin; OS, organic solvents; OXN, oxazolidinones; PEN, penicillin; pHBA, p-hydroxybenzoic acid; PMB, polymyxin B; PUR, puromycin; QAC, quaternary ammonium compounds; R6G, rhodamine 6G; RIF, rifampicin; RND, resistance-nodulation-division; SDS, sodium dodecyl sulfate; SUL, sulfonamides; TET, tetracycline; TGC, tigecycline; TMP, trimethoprim; TRI, triclosan; ZMN, zeamines. <sup>b</sup>A large family of Gram-negative bacteria that includes several important pathogenic species such as Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, and Shigella flexneri. <sup>c</sup>Pumps that are encoded in plasmids.

we would like to highlight the structure of this manuscript. It is organized into four major divisions, which cover first the biological roles of the tripartite pumps including their contribution to antimicrobial resistance, as well as their genetic organization and regulation (sections 2–4). The second group of sections (5–7) deals with the molecular organization of the individual components of the tripartite complexes, with particular effort being put into highlighting the modularity and similarities in the structural organization of the different transporter groups, notably the ABC and RND families (sections 5.7.6–5.7.7), as well as the unexpected correlations in the organization of the PAP proteins (section 7.7) that may be of interest to structural biologist beyond the immediate field of efflux pumps. The third group of chapters (8–11) focuses on the current knowledge of the mechanisms of assembly of the respective tripartite pumps but also aims to provide a synthesis of the data in the form of a synthetic new model of assembly and functional cycling of the RND-based tripartite pumps (section 8.6), which spotlights the new roles for the PAPs highlighted by the latest research in the field. Insights from this model allow us to underline the communality of the assembly between the RND-, MacB-, MFS-, and T1SS-based systems (sections 9–11). The review concludes with an overview of the current strategies for disruption of the pump function (section 12).

## 2. INVOLVEMENT OF TRIPARTITE SYSTEMS IN ANTIBIOTIC RESISTANCE AND DRUG EFFLUX PROFILES FOR PRINCIPAL CLASSES OF TRANSPORTERS

Numerous tripartite efflux complexes have been characterized and defined across Gram-negative bacteria (Table 1). The overwhelming majority of tripartite efflux pumps belong to the RND family, although a few pumps in the ABC and MFS families have also been described. Tripartite efflux complexes often recognize and export a range of physicochemically diverse substrates, consisting of some lipophilic (quinolines, macrolides, and glycylcyclines) as well as cationic (amino-glycosides), anionic (β-lactams), and neutral (chloramphenicol) compounds. For instance, AcrAB-TolC is the predominant efflux system in members of Enterobacteriaceae and has a remarkably wide substrate profile, consisting of multiple different classes of antibiotics (chloramphenicols, tetracyclines,

quinolones, macrolides, and β-lactams).¹⁰ Other examples include MexAB-OprM of P. aeruginosa and AdeABC of A. baumannii, which can also export multiple different classes of antibiotics.¹¹,¹² Tripartite efflux pumps and their regulatory systems have been shown to be involved in clinically relevant antibiotic resistance. For example, overexpression of the MtrCDE efflux pump due to mutations in mtrR have contributed to penicillin resistance in Neisseria gonorrhoeae, which has contributed to the inefficacy of older β-lactams in the treatment of gonorrhoeae.¹³ There is now evidence that the efficacy of azithromycin, which is a current first-line treatment option for gonorrhoeae, is also under threat due to overexpression of the MtrCDE pump in clinical isolates.¹⁴ In another example, overexpression of AcrAB-TolC and MdfA in clinical Escherichia coli UTI isolates has been correlated with fluoroquinolone resistance,¹⁵ severely limiting treatment options. In addition to clinically relevant antibiotics, several tripartite efflux pumps also export commonly used biocides. The AcrAB-TolC pump of E. coli can export benzalkonium chloride, chlorhexidine, and triclosan, and P. aeruginosa possesses multiple efflux pumps capable of exporting triclosan.¹⁶ This has serious implications for infection control in a wide range of healthcare settings by jeopardizing the efficacy of important biocides and selecting for cross-resistance to clinically relevant antibiotics.¹⁷ Tripartite systems also include T1SSs, such as HlyBD-TolC from E. coli and LipBCD from S. marcescens, which are involved in the export of virulence factors.¹⁸

## 3. BIOLOGICAL FUNCTIONS OUTSIDE OF MULTIDRUG RESISTANCE

Efflux-mediated antibiotic resistance was first discovered in the 1970s by Levy and McMurry,¹²⁵ who showed active efflux of tetracycline from bacterial cells possessing the pBR322 plasmid. Since then, efflux-mediated resistance has been identified for nearly all classes of antibiotics. Although efflux pumps are associated with antimicrobial resistance, they likely existed long before the advent of modern medicine. This is evident from the highly conserved nature of efflux pump genes across bacterial genomes. A recent review by Teelucksingh et al.¹²⁶ found that the majority of E. coli efflux pumps are highly conserved and make up 1% of the core genome. This indicates that despite the significant diversity across the E. coli strains,
<table><caption>Table 2. List of Tripartite Systems That When Deleted or Inactivated Result in Attenuated Virulence in Their Cell/Host Model of Infection<sup>a</sup></caption><thead><tr><th>Microorganism</th><th>Efflux pump family</th><th>Efflux system</th><th>Cell/host infection model</th><th>ref(s)</th></tr></thead><tbody><tr><td rowspan="4">S. Typhimurium</td><td rowspan="4">RND</td><td>AcrAB-TolC</td><td>Human epithelial cells, murine macrophages, Galleria mellonella, mouse, chicken</td><td>101, 158–160</td></tr><tr><td>MdtABC</td><td>Mouse</td><td>101</td></tr><tr><td>MdsABC</td><td>Mouse</td><td>101</td></tr><tr><td>ABC</td><td>MacAB-TolC</td><td>Mouse</td><td>101, 138, 139</td></tr><tr><td>K. pneumoniae</td><td>RND</td><td>AcrAB-TolC</td><td>Cattle and bovine enterocytes</td><td>156</td></tr><tr><td rowspan="3">E. coli</td><td>RND</td><td>AcrAB-TolC</td><td>Mouse</td><td>51</td></tr><tr><td>ABC</td><td>MacAB-TolC</td><td>Galleria mellonella and murine mammary glands</td><td>161</td></tr><tr><td>RND</td><td>MdtB</td><td>Mouse spleen</td><td>161</td></tr><tr><td rowspan="2">A. baumannii</td><td rowspan="2">RND</td><td>MdtEF</td><td>Human macrophages</td><td>162</td></tr><tr><td>AdeABC</td><td>Galleria mellonella</td><td>163</td></tr><tr><td>E. cloacae</td><td>RND</td><td>AdeIJK</td><td>Caenorhabditis elegans</td><td>164</td></tr><tr><td>F. tularensis</td><td>RND</td><td>AcrAB-TolC</td><td>Mouse</td><td>165</td></tr><tr><td>N. gonorrhoeae</td><td>RND</td><td>AcrAB</td><td>Human neutrophils, mouse</td><td>166</td></tr><tr><td>P. aeruginosa</td><td>RND</td><td>MtrCDE</td><td>Human neutrophils, mouse</td><td>146, 167</td></tr><tr><td rowspan="2">S. maltophilia</td><td rowspan="2">RND</td><td>MexAB-OprM</td><td>Mouse, canine epithelial cells</td><td>168</td></tr><tr><td>MexGHI-OpmD</td><td>Rat</td><td>83</td></tr><tr><td>B. pseudomallei</td><td>RND</td><td>SmeYZ</td><td>Mouse</td><td>114</td></tr><tr><td>B. burgdorferi</td><td>RND</td><td>BpeAB-OprB</td><td>Human epithelial cells and macrophages</td><td>169</td></tr><tr><td>C. jejuni</td><td>RND</td><td>BesABC</td><td>Mouse</td><td>32</td></tr><tr><td rowspan="2">S. flexneri</td><td rowspan="2">MFS</td><td>CmeABC</td><td>Acanthamoeba polyphaga, Chicken</td><td>170</td></tr><tr><td>EmrKY</td><td>Human macrophages</td><td>143</td></tr><tr><td>L. pneumophila</td><td>ABC</td><td>LssBD-TolC</td><td>Ameoeba and human macrophages</td><td>144</td></tr><tr><td>V. cholerae</td><td>RND</td><td>VexAB-TolC, VexCD-TolC, VexIJK</td><td>Mouse</td><td>157</td></tr><tr><td rowspan="3">Riemerella anatipestifer</td><td rowspan="3">RND</td><td>RaeEF-RopN</td><td>Duck</td><td>120</td></tr><tr><td>MdtABC</td><td>Apple rootstock</td><td>99</td></tr><tr><td>MdtUVW</td><td></td><td>147</td></tr><tr><td>E. amylovora</td><td>RND</td><td>AcrAB</td><td></td><td>171</td></tr><tr><td>R. solanacearum</td><td>RND</td><td>AcrAB</td><td>Tomato plant</td><td>148</td></tr></tbody></table>

efflux pumps have remained relatively stable. Phylogenetic analysis of RND-type transporters by Zwama et al.<sup>127</sup> revealed that AcrB from *Haemophilus influenzae* is evolutionarily ancient compared to AcrB from *Escherichia coli*. Despite its evolutionary age, AcrB of *H. influenzae* was shown to export the same range of antibiotics as AcrB of *E. coli*. Based on their findings, they speculated that multidrug recognition by RND-type transporters is an ancient trait rather than an evolutionarily acquired ability. A different phylogenetic analysis found that the MdtB and MdtC inner membrane RND transporters are conserved throughout Proteobacteria and that their existence was due to a single gene duplication event before the split of Proteobacteria into the α-, β-, and γ-classes. This also suggests that efflux pump genes have remained relatively stable throughout bacterial evolution.<sup>128</sup> Similarly, ABC transporters constitute one of the largest transporter superfamilies. In *E. coli*, there are approximately 80 distinct ABC transporters, which represent 5% of the genome.<sup>129</sup> Many ABC transporters are linked with multidrug resistance; however, it remains unclear how multidrug recognition was acquired. Studies on the resistome of ancient bacteria in archeological samples have also identified multiple different efflux pump genes.<sup>130</sup> Accordingly, if efflux pumps are so conserved and were present before the age of modern medicine, what are their physiological roles in bacteria?

Compared to our knowledge on the role of efflux pumps in antibiotic resistance, our understanding of the biological roles of efflux pumps is lagging. However, studies have suggested increasingly diverse roles for efflux pumps in bacterial physiology.

Genes encoding tripartite efflux systems are ubiquitous among Gram-negative bacteria, including pathogenic and nonpathogenic species, indicating that efflux pumps are evolutionarily ancient with fundamental physiological roles in bacteria.<sup>131</sup> Studies have demonstrated increasingly diverse roles for efflux pumps in bacterial biology, including virulence, biofilm formation, and quorum sensing (QS). The role of efflux pumps in detoxification and pH homeostasis has been recently reviewed by Teelucksingh et al.<sup>126</sup> and therefore will not be discussed here. In this section, the role of tripartite efflux systems in virulence, QS, and biofilm formation will be discussed.

### 3.1. Virulence

Tripartite efflux systems have been shown in numerous studies to play an essential role in the ability of Gram-negative bacteria to colonize and disseminate during host infection. These studies have demonstrated that the inactivation or deletion of tripartite efflux components has detrimental effects on virulence (Table 2). Tripartite efflux pump genes are

<footnote><sup>a</sup>ABC, ATP-binding cassette; MFS, major facilitator superfamily; RND, resistance-nodulation-division.</footnote>
conserved regardless of the host species, highlighting an
important role for virulence.

**3.1.1. Defense against Host-Derived Molecules.** The host environment can be challenging for bacteria due to the presence of various host antimicrobial compounds, such as bile salts, antimicrobial peptides (AMP), and fatty acids.<sup>132</sup> This especially holds for the mammalian gut, which is enriched with bile salts that confer protection to the gut mucosa against bacteria.<sup>133</sup> Therefore, tripartite efflux systems are likely to be involved in the extrusion of host innate defense compounds, thereby enabling the survival of bacteria within the host. This is best exemplified by the tripartite efflux system AcrAB-TolC of Enterobacteriaceae, which can actively efflux and confer resistance to bile salts, AMP such as LL-37 and defensin HBD-1, and fatty acids.<sup>134–136</sup> A recent metabolomics study strongly suggests that oxidized fatty acids are the native substrates for AcrB in both *Salmonella* and *E. coli*.<sup>137</sup> Additionally, the ABC pump MacAB-TolC has been shown to protect S. Typhimurium from oxidative stress. Reactive oxygen species (ROS), such as hydrogen peroxide, are produced by macrophages as a defense mechanism against intracellular pathogens. Bogomolnaya et al.<sup>138</sup> demonstrated that macAB mutants of S. Typhimurium were unable to grow in cultured macrophages that produce ROS but grew normally in ROS-deficient macrophages. Furthermore, macAB mutants were unable to confer resistance to hydrogen peroxide upon exogenous administration *in vitro*, indicating a role for MacAB in ROS detoxification. Recently, the same group also showed that the linearized enterobactin trimer dihydroxybenzoylserine, an enterobactin metabolite, protects S. Typhimurium against peroxide-mediated killing and is a natural substrate of the MacAB pump.<sup>139</sup> This indicates that the MacAB efflux system plays an important role in the survival of S. Typhimurium in the host environment during infection by conferring protection against ROS-mediated oxidative stress. As a pathogen with only one host, *N. gonorrhoeae* is particularly well adapted to infecting humans. The MtrCDE efflux system of *N. gonorrhoeae* has been reported to confer resistance to neutrophil-derived AMP and proteins, indicating that the MtrCDE pump contributes to the defense of *N. gonorrhoeae* against immune cells. The local regulator of the MtrCDE pump, MtrR, has also been shown to bind bile as a physiological inducer, resulting in the depression of *mtrCDE* expression to confer protection from host-derived and clinically relevant antimicrobials.<sup>140</sup> In a different study, the RND transporter MtrD was shown to export the hormone progesterone and the cationic antimicrobial peptide polymyxin B.<sup>141</sup> The MFS tripartite pump FarAB-MtrE of *N. gonorrhoeae* can extrude long-chained antibacterial fatty acids, such as linoleic acid, oleic acid, and palmitic acid, thereby enhancing the *in vivo* survival of *N. gonorrhoeae*.<sup>79</sup> In the related species *N. meningitidis*, the MtrCDE pump has also been reported to confer resistance to human cationic AMP.<sup>142</sup> Other efflux systems, including VexAB and VexCD of *V. cholerae*, CmeABC of *C. jejuni*, and AcrAB of *S. flexneri*, have also been reported to confer resistance bile salts and AMP.<sup>120,143,144</sup> In *E. coli*, the AcrAB-TolC and EmrAB-TolC efflux systems can export mammalian steroid hormones, including estradiol, progesterone, and hydrocortisone.<sup>145</sup> Steroid hormones have been reported to inhibit growth of *N. gonorrhoeae* and *N. meningitidis*; hence, some efflux systems may protect bacteria from such compounds<sup>146</sup> Recently, the AdeIJK efflux system of *A. baumannii* was shown to export and confer resistance to host antimicrobial fatty acids, such as

arachidonic and docosahexaenoic acid.²⁰ The plant pathogen *E. amylovora* also requires tripartite efflux systems MdtABC and MdtUVW to cause infection in apple rootstock. Intriguingly, both efflux systems are also involved in resistance to some antimicrobial plant flavonoids, which are produced and commonly used by plants as a defense mechanism against invading pathogens.¹⁴⁷ The plant pathogen *Ralstonia solana-cearum*, the causative agent of bacterial wilt, also requires the RND efflux genes *acrAB* to cause infection. Compared to wild-type, the *acrAB*-deleted mutant strain was significantly less virulent on tomato plants. Furthermore, various plant antimicrobial compounds were found to induce *acrAB* expression, suggesting a protective role for AcrAB against plant antimicrobials.¹⁴⁸

**3.1.2. Export of Virulence Factors and Toxins.** Tripartite efflux systems can also contribute to the infection process by extruding virulence factors and toxins that cause damage to the host. Siderophores are virulence factors which are secreted by bacteria to acquire iron and other required metals from their environment. Freely available iron is scarce within the host since it is mostly bound to heme and other circulating proteins; hence, bacteria use siderophores to scavenge for iron to use for intracellular processes.<sup>149</sup> Several tripartite efflux pumps have been found to secrete siderophores to allow iron acquisition. The VexGH-TolC efflux system of *V. cholerae* can export the siderophore vibriobactin to acquire iron and maintain cellular homeostasis.<sup>150</sup> In *E. coli*, the AcrAB-TolC, AcrAD-TolC, and MdtABC tripartite efflux systems are required for the secretion of enterobactin, a siderophore with the highest known affinity for iron.<sup>151</sup> Pyoverdines are another group of siderophores synthesized and secreted by fluorescent Pseudomonads. In *P. aeruginosa*, pyoverdine is exported by an ABC family tripartite efflux system, PvdRT-OpmQ. Furthermore, it can export pyoverdine that has already transported iron into the periplasm and any other pyoverdine-metal complex, suggesting a role for PvdRT-OpmQ in maintaining iron and metal homeostasis.<sup>152</sup> *S. marcescens* employs the T1SS HasDEF/TolC to secrete the heme-binding protein HasA, which can bind here heme and also acquire heme from host hemoglobin.<sup>153</sup> Toxins are another virulence factor which are exported by tripartite efflux systems. The ABC family pump MacAB-TolC has been reported to be involved in the secretion of enterotoxin II from the periplasm into the extracellular environment in *E. coli*.<sup>154</sup> MacAB-TolC is also involved in the export of protoporphyrin, the immediate heme precursor. It has been proposed that MacAB-TolC may play a role in maintaining protoporphyrin levels to avoid excess accumulation, which can be toxic to bacteria since it can degrade to ROS in the presence of oxygen.<sup>155</sup> The *V. cholerae* RND transporter VexH of the VexGH-TolC<sub>Vc</sub> efflux system has been reported to be involved in the production of the cholera toxin, although its role in secretion has not been determined.<sup>121</sup>

Type I secretion systems (T1SSs) are another class of tripartite complexes involved in toxin secretion. T1SSs play a critical role in the virulence of these pathogens by helping to disseminate toxins to host cells. HlyBD-TolC is a T1SS found in pathogenic *E. coli* strains involved in the secretion of the α-hemolysin (HlyA) toxin. Hemolysins plays a major role in the virulence of pathogenic *E. coli* by forming pores on the surface of host cells, such as erythrocytes, thereby causing lysis. This allows bacterial cells to penetrate mucosal barriers and infect effector immune cells to prevent clearance of infection. Another important T1SS in *E. coli* is CvaAB-TolC, which is
responsible for exporting colicin V (CvaC), a pore-forming toxin.⁶ As an important human pathogen, *B. pertussis* employs the T1SS CyABDE to export the adenylate cyclase toxin (CyaA) to colonize host cells.³³ In *S. enterica*, the SiiCDF T1SS is involved in the secretion of the large nonfimbrial adhesin SiiE. Deletion of either *siiE* or *siiF* results in attenuated colonization in cattle and invasion of bovine enterocytes.¹⁵⁶ LssBD-TolC is a T1SS from *L. pneumophila*, which has been reported to be important for the secretion of the RtxA toxin.¹⁵⁷

## 3.2. Quorum Sensing

Quorum sensing (QS) is a process of intercellular communication, wherein bacteria synthesize, secrete, and respond to extracellular signaling molecules known as autoinducers (AI) to modulate gene expression according to cell population density. Therefore, QS allows bacteria to only express genes involved in energetically costly behaviors once the conditions are deemed necessary, for instance during host infection. Bacteria use the concentration of AI in their environment to monitor changes in cell density and modulate the expression of quorum-specific genes. QS has been reported to regulate a wide range of bacterial processes and behaviors, such as antibiotic production, biofilm formation, motility, sporulation, and virulence.¹⁷² In Gram-negative bacteria, acylated homoserine lactones (AHL) are predominantly used as AI, whereas Gram-positive bacteria use processed oligopeptides. To exert their effect, AHL must be secreted from the cytosol of Gram-negative bacteria to the external environment.¹⁷³

Tripartite efflux systems have been reported to be involved in the export of AHL in some Gram-negative bacteria, thereby contributing to QS. The MexAB-OprM efflux system of *P. aeruginosa* actively exports a class of AHL known as 3-oxo-acyl-homoserine lactones (HSL) of different acyl chain lengths.¹⁷⁴ Furthermore, MexAB-OprM also influences the accessibility of noncognate HSLs to LasR, the intracellular receptor for HSLs in *P. aeruginosa*.¹⁷⁵ This suggests a role for MexAB-OprM in regulating the QS response. A recent study reported that in MexAB-OprM overproducing *P. aeruginosa* mutant strains, the observed impairment in the QS response was due to the impaired synthesis of alkyl quinolone QS signals, likely due to the decreased availability of a precursor molecule, such as octanoate.¹⁷⁶ The MexEF-OprN efflux system has been shown to export the precursor of the AI *Pseudomonas* quinolone signal (PQS), which is important for the virulence of *P. aeruginosa*.¹⁷⁷ By exporting an AI precursor, MexEF-OprN may play a role in modulating *P. aeruginosa* virulence by limiting the intracellular availability of AI precursors. In the phytopathogen *P. syringae*, MexEF-OprN was also reported to act as a negative determinant of AHL production and accumulation, suggesting that it may also export AI precursors in other Pseudomonads to regulate AHL production.¹⁷⁸ Interestingly, overexpression of the MexCD-OprJ efflux system in *P. aeruginosa* has been reported to reduce the QS response. Additionally, the MexXD-OprJ pump extrudes kynurenine, a precursor of alkyl-quinolone signals, such as PQS.¹⁷⁹ Similar to MexEF-OprN, the MexCD-OprJ pump may also play a role in regulating QS by modulating the intracellular levels of PQS precursors and thus PQS production. Although MexEF-OprN and MexCD-OprJ are inducible efflux systems, they seem to be involved in the QS response. This can be puzzling within the context of an antibiotic-free host environment, which can be considered noninducible. However, RND efflux pumps are induced not

just by antibiotics but also by host-derived compounds, such as bile and fatty acids,¹⁸⁰ and possibly the host immune response.¹⁴⁴ In *B. pseudomallei*, the BpeAB-OprB efflux system is involved in the active efflux of several different types of AHL. Besides, inactivation of *bpeAB-oprB* in *B. pseudomallei* results in complete inhibition of QS and diminished virulence, indicating a critical role for this efflux system in the physiology of *B. pseudomallei*.¹⁸¹ In *E. coli*, the AcrAB-TolC efflux system may also be involved in the efflux of AI. A study by Yang et al.¹⁸² suggested that AcrB-TolC efflux may facilitate more efficient efflux of AI, although the identity of the AI was not determined. These studies suggest a role for tripartite efflux systems in QS; however, further research is required to elucidate their role in regulating QS.

## 3.3. Biofilm Formation

A biofilm is an aggregation of bacteria that is enclosed in a self-produced matrix of extracellular polymeric substances and commonly attached to a surface and/or to each other. Bacteria within a biofilm display an altered phenotype concerning growth rate and gene transcription to reflect the biofilm lifestyle, which differs significantly from planktonic bacterial cells. The biofilm matrix is a highly diverse environment, consisting of polysaccharides, lipids, proteins, nucleic acids, and even inorganic materials, such as mineral crystals and clay particles. The matrix plays a critical role as a structural scaffold to maintain the integrity of the biofilm. Importantly, biofilms are characterized by heterogeneity, such as oxygen, nutrient, pH, and QS gradients, as well as social interactions, consisting of microbial communication, cooperation, and competition.¹⁸³ Bacteria form biofilms in response to a wide range of environmental and mechanical signals, which can trigger a host of processes, including QS, to promote biofilm formation.¹⁸⁴

Studies suggest several roles for tripartite efflux systems in biofilm formation, including export of harmful metabolic intermediates, export of extracellular polymeric substances, export of AI to modulate biofilm formation, and export of protein factors to enable adhesion.¹⁸⁵ Inactivation of genes encoding tripartite efflux systems can result in impaired biofilm formation. Kvist et al.¹⁸⁶ demonstrated that inactivation of efflux pumps in *E. coli* caused impaired biofilm formation. Specifically, the *aaeX*-deleted *E. coli* mutant exhibited significantly lower biofilm formation.¹⁸⁶ The *aaeX* gene encodes a membrane component of the MFS efflux system AaeAB, which has been previously reported to be involved in the efflux of toxic metabolic intermediates.⁶⁵ The *emrY* gene, encoding a component of the MFS efflux system EmrKY-TolC, has been reported to be important for the growth of *E. coli* biofilms.¹⁸⁷ Matsumura et al.¹⁸⁸ reported that the inactivation of all genes encoding for the components of tripartite efflux systems in *E. coli* resulted in significantly reduced biofilm formation. In particular, *acrD*-, *acrE*-, *emrK*-, and *mdtE*-deleted mutants displayed extremely lower biofilm formation. In agreement with this study, Baugh et al.¹⁸⁹ reported that *acrB*-, *acrD*-, *acrEF*-, *mdtABC*-, *mdsABC*-, *emrAB*-, and *macAB*-deleted *S. Typhimurium* mutants also exhibited significantly reduced biofilm formation. These studies suggest that tripartite efflux systems contribute to biofilm formation. In *E. coli*, inactivation of *emrK* or *emrY* has been found to significantly impair survival of cells in response to physical and chemical stresses, including UV radiation, mitomycin C, and hydrogen peroxide.¹⁹⁰ Therefore, the EmrKY-TolC system may protect
<figure><img src="image_12.png" /><figcaption>**Figure 2.** Diverse genetic organization of tripartite efflux systems across a representative sample of Gram-negative bacteria. ABC, ABC-binding cassette; MFS, major facilitator superfamily; RND, resistance-nodulation-division.</figcaption></figure>

cells by exporting toxic metabolic intermediates induced by environmental stresses. A different study found that the EmrB and MdtB tripartite efflux components contribute to extreme acid survival in *E. coli*.¹⁹¹ The MdtEF-TolC efflux system has been shown to protect *E. coli* against nitrosative damage in anaerobic conditions. Specifically, the *mdtEF*-deleted mutant was found to grow significantly more slowly during anaerobic respiration of nitrate.¹⁹² This suggests MdtEF-TolC plays an important role in detoxifying cells in anaerobic conditions by exporting harmful nitrosyl indole derivatives. Several *E. coli* cellular metabolites have been found to induce the expression of the *acrAB* operon, possibly by inactivating *acrR* and/or upregulating *marA* and *soxS* expression. Thus, it is possible that the *AcrAB-TolC* system exports toxic metabolites and/or signaling molecules to maintain homeostasis of gene expression.¹⁹³ T1SSs have also been reported to be involved in biofilm formation. The T1SS LapEBC from *P. fluorescens* is involved in the secretion of LapA, a biofilm-promoting adhesin, into the extracellular environment to enable bacterial cells to adhere to surfaces. Additionally, mutant strains lacking *lapA*, *lapB*, or *lapE* have been shown to display impaired biofilm formation and maturation.¹⁹⁴

How might tripartite efflux systems contribute to biofilm formation? As described above, the biofilm environment is heterogeneous, with the biofilm core being more anoxic, acidic, and nutrient-deprived than the biofilm surface.¹⁸³ This can exert pressure and stress on cells within the biofilm core; hence, tripartite efflux systems may contribute to their survival. There is some evidence that expression of some efflux pumps is induced by stress within the biofilm core. De Kievit et al.¹⁹⁵ showed that expression of *mexAB-oprM* and *mexCD-oprJ* in *P. aeruginosa* was the highest in the bottom of the biofilm, near the solid substratum. These efflux systems may also extrude QS molecules to regulate QS, thereby also regulating the expression of biofilm-specific genes. Some tripartite efflux systems like *AcrAB-TolC* and *AaeAB* may play roles in exporting harmful metabolic intermediates to prevent toxic accumulation within biofilm cells. Owing to their role in biofilm formation, tripartite efflux systems could be attractive drug targets to inhibit biofilm formation in clinical settings.

### 3.4. Other Reported Biological Roles

The multifunctional OMF protein TolC is also involved in colicin E1 import¹⁹⁶ and is employed by bacteriophage TLS as a cell surface receptor to enter *E. coli* cells.¹⁹⁷ Similarly, in *Salmonella Typhimurium*, the TolC appears to be essential for
the infection by the Chi-phage.¹⁹⁸ Likewise, TolC from V. cholerae is also utilized by the VP3 phage to gain entry into cells.¹⁹⁹ The AcrEF-TolC complex from E. coli has been posited to play a role in the maintenance of normal cell division. Lau and Zgurskaya²⁰⁰ reported that AcrEF-deficient cells exhibited defective chromosome condensation and segregation during cell division. Furthermore, the absence of AcrEF was also found to result in cell filamentation. The SmeIJK efflux system of S. maltophilia has been reported to contribute to cell envelope integrity and the envelope stress response. Compared to wild-type, the smeIJK-deleted mutant strain was found to exhibit increased sensitivity to membrane damaging agents and elevated RpoE-mediated envelope stress response. Additionally, sublethal concentrations of membrane damaging agents were reported to induce smeIJK expression in a RpoE-dependent manner.²⁰¹ In A. baumannii, the RND transporter AdeJ has been reported to play a role in surface-associated motility.¹⁶⁴ Similarly, the AcrD RND transporter from S. enterica has also been shown to play a role in swarming motility.²⁰² There is also some evidence that tripartite efflux pumps play a role in lipid modulation, which could potentially effect resistance to membrane targeting antibiotics. In P. fluorescens, the RND pump EmhABC has been found to be involved in the efflux of fatty acids. Furthermore, changes in the growth temperature altered the composition of fatty acids in the membrane and significantly increased expression of emhABC, with increased extracellular fatty acids. This suggests that EmhABC is involved in the efflux of fatty acids that are replaced due to membrane damage or phospholipid turnover.²⁰³ The AdeIJK RND pump of A. baumannii has also been implicated in membrane modulation. Jiang et al. found that the AdeIJK pump can export the *ω*-6 fatty acid arachidonic acid and the *ω*-3 fatty acid decosahexaenoic acid. Furthermore, lipid analyses showed that adeJ deletion resulted in significant changes in endogenous lipid concentrations.²⁰ The role of efflux pumps in lipid modulation could act as a defense mechanism against membrane targeting antibiotics, which has been previously shown in *Staphylococcus auereus* as a daptomycin resistance mechanism.²⁰⁴,²⁰⁵

In nitrogen-fixing symbiotic bacteria, tripartite efflux systems have been reported to play various roles in nitrogen-fixation and nodulation. In *B. japonicum*, the RND efflux system BdeAB is crucial for symbiotic nitrogen-fixation of the soybean plant. The *bdeAB*-deleted *B. japonicum* mutant strain has been shown to exhibit strongly diminished nitrogen fixation, which was evident by discolored plant leaves, a typical sign of nitrogen starvation.³⁴ In *S. meliloti*, the RND system SmeAB-TolC plays a role in nodulation competitiveness by protecting cells from antimicrobial compounds produced by the host plant.¹⁰⁸ In the filamentous cyanobacterium *Anabaena* sp. PCC 7120, the tripartite ABC systems DevBCA-HgdD and HgdBCD have been shown to play critical roles for envelope formation in heterocysts, which are specialized nitrogen-fixing cells.²⁰⁶,²⁰⁷ These studies suggest an increasingly important and diverse role of tripartite efflux systems in bacterial physiology.

## 4. GENES AND REGULATION OF EXPRESSION

Genes encoding tripartite efflux systems are generally organized as operons, wherein the inner membrane transporter and the PAP genes are located within the same operon (Figure 2). The OMF gene can be encoded within the same operon, such as *oprN* of P. aeruginosa,⁸² or elsewhere in the genome, such as *tolC* of E. coli and S. enterica.²⁰⁸ The majority of tripartite efflux

systems also have a gene encoding for a local regulator protein within the operon, such as *acrR* and *mexR*, which typically act to repress efflux gene expression.²⁰⁹,²¹⁰ However, a few local regulators can activate efflux gene expression, such as *mexT* and *vexR*, which activate expression of the *mexEF-oprN* and the *vexAB* efflux genes, respectively.⁸²,²¹¹ Some tripartite efflux systems, such as MacABC of S. maltophilia and AdeABC of A. baumannii, are regulated by two-component systems (TCS), which are encoded divergently upstream of the efflux pump operon.¹¹⁶,²¹² Certain tripartite efflux systems also have more than one type of PAP or inner membrane transporter encoded within their operon. For example, the *triABC* system of P. aeruginosa comprises two PAP genes *triA* and *triB*,⁸⁸ and the *mdtABC* system of S. enterica possesses two inner membrane transporter genes *mdtB* and *mdtC*.²¹³ T1SSs are also usually organized as operons, with the cognate toxin gene located within the same operon as the efflux pump genes. For example, the *hlyA* gene encoding the *α*-hemolysin toxin is found upstream of the *hlyB* and *hlyD* genes.²¹⁴ As an extreme example, the genes encoding the AcrAD-TolC efflux system are all located in different genetic loci within the genome.¹⁰⁰ Therefore, there is significant diversity in the genetic organization of tripartite efflux systems (Figure 2).

Efflux pump overexpression frequently contributes to clinically relevant levels of antibiotic and biocide resistance. Therefore, it is critical to understand how tripartite efflux pumps are regulated to elucidate the mechanisms of over-expression, which could be used to identify novel drug targets to prevent or reverse efflux pump overexpression.²¹⁵ The regulation of tripartite efflux systems can vary between species and even within a species depending on environmental and physiological factors. Normally, efflux pumps are expressed at basal levels through tight regulation of highly complex and interconnected systems. Therefore, the deletion or inhibition of one efflux pump can affect the expression of other pumps. For example, deletion of *acrAB* in S. Typhimurium results in significant upregulation of many other genes encoding for tripartite efflux systems, including *acrD*, *acrE*, *mdsB*, *mdtB*, *macA*, and *emrA*.²¹⁶ Hence, the complexity of efflux pump regulatory networks can often cloud resistance phenotypes.

### 4.1. Local Regulation

Genes encoding for components of tripartite efflux systems are regulated by local and global transcriptional regulators that act to maintain baseline expression (Figure 3). These regulators respond to a wide range of environmental signals, including antibiotic exposure and iron limitation, to increase the expression of efflux pump genes.²¹⁵ The majority of the genes encoding tripartite efflux systems are arranged in an operon, which also contains a divergently oriented gene encoding for a local TetR family regulator (TFTR). TFTRs generally consist of a N-terminal DNA binding domain, which recognizes and binds to a palindromic DNA sequence located in the intergenic region between the regulator and the regulated gene, and a larger C-terminal domain, which is responsible for ligand binding.²¹⁷ Functioning as a dimer, TFTRs act to locally repress the expression of the efflux genes.²¹⁸ Examples of local TFTRs include AcrR, which represses expression of *acrAB* in several different members of Enterobacteriaceae;²⁰⁹ MtrR, which represses expression of *mtrCDE* in N. gonorrhoeae;²¹⁹ and CmeR, which represses the expression of *cmeABC* in C. jejuni.²²⁰ Binding of ligands to the C-terminal domain of TFTRs induces conformational changes
<figure><img src="image_13.png" /><figcaption>**Figure 3.** Schematic diagram of the different mechanisms of tripartite efflux pump regulation. (A) Local regulation usually involves a TetR family transcriptional regulator, such as AcrR, that acts to locally repress efflux gene expression and maintain basal levels of expression. Global regulation generally involves AraC/XylS family transcriptional regulators, such as MarA, RamA, and SoxS, that activate efflux pump gene expression. These global regulators themselves are locally regulated by their own TetR family transcriptional regulators, such as MarR, RamR, and SoxR. The presence of antibiotics or other external stressors can release these repressors to allow activation of efflux gene expression. Other types of regulators, such as the quorum sensing regulator SdiA, can also activate efflux gene expression. Two component systems (TCSs) consist of a sensor histidine kinase (EvgS) that detects external stimuli, such as drugs, and phosphor-ylates a response regulator (EvgA), which becomes activated to trigger efflux pump gene expression. Post-transcriptional regulation includes RNA-binding proteins, such as CrsA, that stabilize efflux gene mRNA to promote efficient efflux protein translation. (B) In V. cholerae, the TetR family transcriptional regulator VexR activates expression of the vexA and vexB efflux pump genes.</figcaption></figure>

in their structure, thus preventing their binding to target DNA.<sup>217</sup> For instance, rhodamine 6G, a substrate of the AcrAB-TolC pump, can bind to AcrR and prevent its binding to target DNA, thereby resulting in derepression and expression of *acrAB*.<sup>209</sup> Mutations conferring loss-of-function of local repressors can result in overexpression of tripartite efflux systems. For example, mutations in *acrR* can lead to overexpression of AcrAB and have been documented in the fluoroquinolone resistance of clinical *E. coli*<sup>221</sup> and *K. pneumoniae*<sup>222</sup> isolates. Likewise, in clinical isolates of *N. gonorrhoeae*, mutations in *mtrR* have been reported to contribute to decreased azithromycin<sup>223</sup> and cephalosporin susceptibility.<sup>224</sup>

In contrast to trans-acting mutations, cis-acting mutations often have a far greater impact on efflux gene expression. Cis-acting mutations mainly consist of changes in the promoter region of efflux pump genes, whereas trans-acting mutations abrogate repressor activity. For example, a 13-bp inverted-repeat sequence between the divergent *mtrR* and *mtrC* genes in *N. gonorrhoeae*, corresponding to the promoter regions of *mtR* and *mtrC*, has been shown to significantly increase *mtrC* expression and MtrC protein levels, resulting in increased resistance to antiubiotics, detergents, and bile salts.<sup>76</sup> It has also been found that a 1-bp deletion in a 13-bp inverted repeat within the *mtrR* promoter region is prevalent in 80% of *N. gonorrhoeae* isolates with increased resistance to antimicrobials, bile salts, detergents, and host-derived compounds. These cis-acting mutations impair transcription of *mtR* and promote transcription of *mtrCDE*, even more so than loss of MtrR.<sup>14</sup>

However, negative regulation of tripartite efflux systems is now known to be diverse. Contrasting the conventional negative regulation of the *acrAB* operon, studies have shown that multiple regulators can function as a network to regulate the same efflux pump gene and that non-TFTRs can also regulate efflux pump genes. For example, the *mexAB-oprM* operon in *P. aeruginosa*, which encodes for the MexAB-OprM efflux system, is repressed by the divergently encoded MarR-family protein MexR. Yet, *mexAB-oprM* expression is also repressed by the TFTRs NalC and NalD, which are encoded elsewhere in the *P. aeruginosa* genome.<sup>225,226</sup> Recently, two additional repressors of the *mexAB-oprM* operon, MdrR1 and MdrR2, have been characterized.<sup>227</sup> Together, these regulators work as a network to maintain the constitutive basal levels of MexAB-OprM efflux pump expression. Furthermore, not all TFTRs function to repress efflux pump genes. For instance, the RND pump VexAB of *V. cholerae* is organized in an operon with a divergently encoded TFTR VexR, much like AcrAB. However, VexR does not repress *vexRAB* but rather is necessary for the activation of *vexRAB* expression in response to noxious substances (Figure 3).<sup>211</sup> Notably, some TFTRs are now understood to be much more than simply regulators of efflux pump genes. For example, in addition to repressing the *cmeABC* operon, the TFTR CmeR of *C. jejuni* has been found to regulate the expression of many other genes encoding transport proteins and enzymes involved in capsular polysaccharide biosynthesis. Furthermore, loss-of-function mutations in *cmeR* were shown to diminish the ability of *C. jejuni* to colonize chickens.<sup>228</sup> In *N. gonorrhoeae*, the TFTR MtrR has been shown to modulate the expression of at least 69 genes, which encode for proteins involved in metabolism, general stress, and heat shock response.<sup>229</sup> Therefore, it has become increasingly evident that TFTRs possess further physiological roles within bacteria.

## 4.2. Global Regulation

Global regulation of tripartite efflux systems is mediated chiefly by the AraC/XylS family of transcription factors. These global regulators are involved in the transcriptional regulation of many processes inside cells, including induction of efflux pump gene expression. In Enterobacteriaceae, characterized AraC/XylS transcription factors include MarA, SoxS, and Rob, all of which can activate expression of the *acrAB* operon in response to harmful substances or stressors (Figure 3).<sup>230</sup> In *K. pneumoniae*, in addition to *acrAB*, RamA has also been recently shown to upregulate the expression of *oqxAB*.<sup>231</sup> These regulators recognize and bind to a degenerate approximately
20 bp sequence, which is located in the promoter sequence of *acrAB* and many other genes.²³² The majority of AraC/XylS transcription factors are encoded alongside their local repressor. For example, MarA, SoxS, and RamA are autoregulated by their local repressors, *marR*, *soxR*, and *ramR*, respectively (Figure 3).²³³ AraC/XylS family transcriptional regulators involved in efflux pump induction also exist in members of other families, including MtrA of *N. gonorrhoeae* which induces expression of *mtrCDE*.²³⁴ Repression of global transcriptional regulators is relieved through binding of ligands to the local repressor proteins, which prevents their binding to DNA. For instance, binding of ligands, such as ethidium bromide, rhodamine 6G, and crystal violet, to RamR reduces its binding affinity to target DNA, thus resulting in increased expression of *ramA*.²³⁵ However, the AraC/XylS family is not the only family of transcription factors involved in the regulation of efflux genes. SdiA is a LuxR-type transcription factor involved in the regulation of cell division through quorum sensing. In addition, SdiA has also been shown to be involved in the positive regulation of AcrAB protein levels (Figure 3).²³⁶ The T1SS HlyBD of *E. coli* is regulated by the transcription antitermination protein RfaH, which enhances *hlyCABD* transcript elongation. Additionally, RfaH has been found to increase the efficacy of the HlyA toxin protein.²³⁷ In *S. enterica*, RfaH also regulates the *siiCDEF* operon encoding for the SiiCDEF T1SS and the large nonfimbrial adhesin SiiE.²³⁸

The increased expression of global transcriptional regulators leads to the induction of efflux pump gene expression. Therefore, mutations that result in derepression of global transcriptional regulators can trigger the overexpression of tripartite efflux pumps. For example, reduced tigecycline susceptibility in clinical *K. pneumoniae* isolates has been associated with loss-of-function mutations in *ramR*, resulting in increased *ramA* and *acrAB* expression.²³⁹ Mutations in the promoter sequence of global transcriptional regulators can also lead to overexpression by preventing binding of local repressor proteins. Baucheron et al.²⁴⁰ demonstrated that mutations in the promoter sequence of *ramA* can prevent binding of RamR. Furthermore, they showed that the MDR phenotype of a clinical *E. coli* isolate was due to a 2-bp deletion in the *ramA* promoter sequence, which compromised RamR-mediated repression and increased *acrAB* and *tolC* expression. Interestingly, a study that investigated the differential impact of *ramRA* mutations in clinical MDR S. Typhimurium isolates found that mutations in the promoter region of *ramA* had a greater impact in increasing expression of *ramA* than loss-of-function mutations in *ramR*.²⁴¹ There are also reports of novel mutations that trigger overexpression of global transcriptional regulators. For instance, a study found that in clinical MDR *E. coli* isolates, overexpression of the SoxS protein was not due to mutations in *soxR* or the promoter region of *soxS* but instead a novel alanine to serine substitution (A12S) mutation in *soxS*. Furthermore, overexpression of *acrB* was reported with increased resistance to fluoroquinolones, chloramphenicol, and tetracycline.²⁴² Considering that the position of the substitution is not within the DNA binding region of SoxS, the mechanism by which this mutation results in *soxS* overexpression remains to be elucidated. Lastly, overexpression of global transcriptional regulators has been reported to increase mutation rate and frequency. A recent study reported that *ramR* mutants, which overproduce RamA, and thus AcrB, exposed to ciprofloxacin selected for quinolone-resistant gyrA

mutants. Furthermore, inactivation of *ramA* was found to select for *soxR* mutants, which overproduce SoxS, suggesting a compensatory role for global transcriptional regulators.²⁴³

Although antibiotics are known to induce expression of efflux pumps, worryingly, nonantibiotic drugs have also been shown to trigger efflux pump overexpression. One study found that upon exposure to the antidepressant fluoxetine, *E. coli* K-12 developed significantly increased resistance to ampicillin. chloramphenicol, and tetracycline. This was shown to be due to ROS-mediated mutations in the efflux pump regulators *marR* and *rob*, resulting in increased AcrAB-TolC and MdtEF-TolC expression.²⁴⁴ Several commonly used biocides have also been documented to trigger efflux pump overexpression. Exposure to subinhibitory concentrations of benzalkonium chloride has been reported to promote antibiotic resistance in *P. aeruginosa* due to mutations in *nfxB*, resulting in overexpression of MexAB-OprM and MexCD-OprJ efflux pumps thereby increasing benzalkonium chloride and ciprofloxacin resistance.²⁴⁵ In agreement with this, a recent study also reported the overexpression of the MexCD-OprJ efflux pump in *P. aeruginosa* upon exposure to benzalkonium chlordide, resulting in increased ciprofloxacin and tetracycline resistance.²⁴⁶ Several other studies have also reported similar findings in *P. aeruginosa*, pointing to a link between biocide exposure and cross-resistance to clinically relevant antibiotics.²⁴⁷,²⁴⁸ Similarly, triclosan exposure has also been reported to promote significantly high levels of cross-resistance to other antibiotics, including ciprofloxacin, tetracycline, and erythromycin, in *P. aeruginosa*. The triclosan resistant mutants were found to harbor mutations in *nfxB*, the local repressor gene of the * mexCD-oprJ* operon, which resulted in the overexpression of the MexCD-OprJ plasmids carrying antibiotic resistance genes.²⁴⁹ Worryingly, the presence of biocides in the environment can also select for MDR. Amsalu et al.²⁵⁰ found that 19% of *P. aeruginosa* isolates collected from diverse ecological niches exhibited MexAB-OprM overexpression due to mutations in the efflux pump regulators MexR, NaIc, or NaID, resulting in MDR.

Owing to their role as global transcriptional regulators, MarA, SoxS, Rob, and RamA have been shown in several studies to have wider roles in bacterial physiology. For example, MarA, SoxS, Rob, and RamA have been reported to repress flagellar gene expression and motility in S. Typhimurium. Specifically, each of these global regulators was found to directly interact with the promoter of *flhDC*, the master regulator of motility in *Salmonella*.²⁵¹ In *E. coli*, MarA has been demonstrated to regulate genes involved in DNA repair and lipid trafficking. Identified MarA targets include the *xseA* gene and the *mlaFEDCB* operon, which encode an endonuclease subunit and a lipid trafficking ABC transporter complex, respectively.²⁵² Lastly, the elucidation of the RamA regulon in *K. pneumoniae* found that RamA binds directly to the promoters of *lpxC*, *lpxL-2*, and *lpxO* genes, which are involved in lipid A biosynthesis. This suggests that RamA plays a role in the modification of the lipid A moiety of lipopolysaccharide (LPS). Furthermore, overexpression of RamA was found to reduce *K. pneumoniae* adhesion and uptake into macrophages, thus conferring protection against the innate host response.²⁵³ Together, these results signify that in addition to their role in MDR, regulators of tripartite efflux systems also play a wider physiological role in bacteria.
### 4.3. Post-transcriptional Regulation

In contrast to transcriptional regulation, less is known about the regulation of tripartite efflux systems at a post-transcriptional and translational level. However, recent studies have demonstrated that post-transcriptional regulation is likely to play an important role in fine-tuning efflux pump expression. One mechanism of post-transcriptional regulation is through the action of RNA-binding proteins, which are seen as increasingly important modulators of protein function.²⁵⁴ The RNA-binding protein CsrA (Carbon Storage Regulator A) acts as a global regulator of a wide range of genes. Ricci et al.²⁵⁵ reported that CsrA binds to and stabilizes *acrAB* mRNA, presumably through preventing the formation of repressive RNA structures that hinder ribosome binding (Figure 3). Furthermore, deletion or mutagenesis of *crsA* was shown to decrease the production of AcrAB. Therefore, CsrA may be involved in facilitating the efficient translation of AcrAB. Another study reported that Hfq, an RNA-binding protein and RNA chaperone, may also be involved in the post-transcriptional regulation of AcrB. Deletion of *hfq* in *E. coli* does not affect the transcription of the *acrAB* operon; however, it does diminish the levels of AcrB protein production and increase susceptibility to antibiotics.²⁵⁶ In a different study, SdsR, a noncoding small RNA expressed at the stationary phase, was shown to repress the expression of *tolC* by binding to the region upstream of its ribosomal binding site. Furthermore, overexpression of SdsR was found to result in the loss of *tolC* mRNA, a reduction in TolC protein production, and increased susceptibility to some AcrAB-TolC substrates, including novobiocin and crystal violet.²⁵⁷ The role of SdsR-mediated *tolC* repression has also been reported in S. Typhimurium.²⁵⁸

### 4.4. Regulation by Two-Component Systems

Bacteria can adjust their physiological behavior in response to environmental changes through two-component systems (TCSs). In essence, TCSs mediate signal transduction by enabling bacteria to sense external stimuli, such as pH, temperature, nutrient levels, and antibiotics, and convey this signal to the intracellular environment to facilitate changes in gene expression.²⁵⁹ TCSs are present in all domains of life and typically consist of a membrane-bound sensor histidine kinase (HK) and its cognate response regulator (RR). Upon sensing external stimuli, the sensor HK catalyzes the autophosphorylation of a conserved histidine residue in its C-terminal domain using ATP. The phosphoryl group is then transferred to a conserved aspartate residue on the receiver domain of the RR. This triggers a conformational change in the effector domain of the RR, resulting in a cellular response. The majority of the characterized RR belongs to the DNA binding superfamily and thus is assumed to function as transcription factors to induce changes in gene expression. TCSs regulate many physiological processes, such as motility, cell division, and nutrient uptake, but also antibiotic resistance and virulence.²⁶⁰

In addition to transcriptional regulation, many of the tripartite efflux systems (Table 1) are also regulated by TCSs. For example, the AdeABC efflux system of *A. baumannii* is regulated by the TCS AdeRS, encoded by the divergently encoded *adeRS* operon upstream of *adeABC*. The AdeS protein is a sensor HK that detects environmental stimuli, and the AdeR protein is a DNA-binding RR that induces changes in gene expression.²¹² AdeR activates *adeABC* expression by binding to a direct-repeat motif in the intergenic region

between *adeR* and *adeA*.²⁶¹ Importantly, mutations in *adeRS* can result in overexpression of the AdeABC pump. This has been reported in clinical MDR *A. baumannii* isolates due to various point mutations in both AdeR and AdeS.²⁶² As a TCS, AdeRS also regulates genes involved in several other processes, including biofilm formation, motility, and virulence.¹⁶³ The tripartite ABC efflux pump MacAB-TolC is regulated by the pleiotropic TCS PhoPQ, which governs virulence, adaptation to magnesium limiting environments, resistance to antimicrobial peptides, and various other processes. Nishino et al.¹⁰¹ demonstrated that in *S. enterica*, phosphorylated PhoP binds to a PhoP box upstream of the *macAB* operon thereby directly repressing *macAB* expression. However, a recent study found that PhoP-inducing signals, such as low magnesium, generally act to increase *macAB* transcription in *Salmonella*. The difference in the role of PhoPQ in *macAB* regulation may arise from differences in experimental conditions, PhoP turnover, and changes in rRNA levels in response to low magnesium. Whereas MacAB-TolC remains functional in the majority of S. Typhimurium lineages, the S. Typhimurium ST313 lineage has been reported to exhibit a pattern of evolutionary convergence toward an impaired MacAB-TolC efflux system. This possibly represents a unique adaptation to allow systemic infections of permissive hosts who are unable to clear infection.²⁶³ Interestingly, the MacAB-TolC homologue in *S. maltophilia*, MacABC, has been reported to be intrinsically expressed as inactivation of the TCS genes *macRS* was found to not affect *macABC* expression.¹¹⁶ Some TCS are capable of regulating more than one tripartite efflux complex. For example, EvgAS in *E. coli* has been demonstrated to regulate the expression of *acrAB*, *emrKY*, *mdtEF*, and *tolC* (Figure 3),²⁶⁴ which encode AcrAB-TolC, EmrKY-TolC, and MdtEF-TolC pumps, respectively. Likewise, the TCS BaeSR in *E. coli* and S. Typhimurium can detect heavy metals and in response activate the expression of both *mdtABC* and *acrD*, which encode MdtABC and the AcrD RND transporter of AcrAD-TolC efflux systems, respectively.²¹³,²⁶⁵ In *A. baumannii*, BaeSR can detect antimicrobial compounds, such as tigecycline and tannic acid, and in response is believed to regulate the expression of AdeABC, AdeIJK, and MacAB-TolC tripartite efflux systems.²⁶⁶ Owing to their role in antibiotic resistance and virulence, TCSs could be attractive drug targets for the development of inhibitors.

### 4.5. Other Types of Regulation

Aside from the canonical pathways of efflux pump regulation, several other types of regulatory mechanisms have been posited. Another possible mechanism of efflux pump regulation is through the action of bacterial secondary messenger molecules, such as ppGpp (guanosine pentaphosphate), which is involved in the stringent response. A recent study found that the deletion of the *A1S_0579* gene, which is involved in the synthesis of ppGpp, increased antibiotic susceptibility in *A. baumannii*. To elucidate the mechanism of increased antibiotic susceptibility, expression levels of efflux pump genes were determined. Interestingly, the expression of the genes *adeB* and *adeIJK*, which encode components of tripartite efflux systems, was significantly reduced (at least 10-fold reduction).²⁶⁷ This suggests a link between secondary messenger molecules and efflux pump regulation; however, further research is required to elucidate the molecular mechanisms. A recent study found that post-translational modification is another determinant of efflux pump activity. In
<figure><img src="image_14.png" /><figcaption>**Figure 4.** Overview of the principal components of the tripartite assemblies. The primary and tertiary structures of the main group of proteins involved in the formation of tripartite efflux complexes and type 1 secretion systems. The coloring of the key structural features is the same on the primary and the 3D structure. (A) Outer membrane factor (OMF) family on the example of the *Pseudomonas aeruginosa* OprM channel monomer, based on the PDB ID 1WP1.²⁶⁹ (B) Periplasmic adaptor protein (PAP), also known as the Membrane Fusion Protein (MFP) family, on the example of the *Pseudomonas aeruginosa* MexA associating with the RND transporter MexB. Two conformers are presented based on the PDB ID 6TA6;²⁷⁰ see text in section 7.2 for details. (C) A representative RND-family transporter—MexB, from *P. aeruginosa*. Trimeric assembly based on PDB ID 6T7S.²⁷⁰ PN and PC—periplasmic or porter domain N-terminal and C-terminal subdomains; DN and DC—docking/funnel domain N- and C-terminal subdomains, respectively. See section 5.1 and Figures 5 and 6 for details. (D) A representative structure of the Major Facilitator Superfamily (MFS) transporter with 14-transmembrane helices based on the POT-family member PepTSo from *Shewanella oneidensis*. PBD ID 4UVM,²⁷¹ monomer presented. See Section Y for details. (E) A dimeric structure of the MacB transporter from *E. coli* based on PDB ID 5NIK.²⁷² (F) Representative structure of the ABC transporters involved in the formation of the type 1 Secretion system (T1SS), on the example of Hemolysin alpha exporter HlyB. Dimer structure based on a homology model derived from the peptidase containing ABC transporter PCAT1 structure (PBD ID 4RY2).²⁷³ For details see section 5.6. NBD, nucleotide binding domain; CLD, C39 like domain.</figcaption></figure>

C. jejuni, the CmeABC RND pump is subjected to N-linked glycosylation, which was shown to enhance protein thermostability, stabilize protein complexes, and promote protein-protein interaction, thereby facilitating efflux activity.²⁶⁸

## 5. CLASSES OF TRANSPORTER PROTEINS

The multidrug efflux transporters of Gram-negative bacteria are diverse and are categorized into one of six families: the Resistance-Nodulation-Division (RND) superfamily, the Major Facilitator Superfamily (MFS), the Multidrug and Toxic Compound Extrusion (MATE) family, the Small Multidrug Resistance (SMR) family, the Proteobacterial Antimicrobial Compound Efflux (PACE) transporter family, and the ATP-Binding Cassette (ABC) superfamily. The representative known structures of the transporters belonging to these families, along with their representative partner proteins, which participate in tripartite complex formation are shown in Figure 4. The individual components, structure, and assembly of each of these systems are described as comprehensively as possible below.

### 5.1. Resistance-Nodulation-Division (RND) Transporter Superfamily

Representatives of the RND superfamily can be found in all domains of life. Members are characterized by conserved transmembrane structures comprising 12, sometimes 13 or 14, transmembrane helices (TMs) arranged in two pseudosymmetric bundles. The transmembrane domains (TMDs) of these secondary active transport proteins are involved in the binding and release of H⁺ (or Na⁺). Between TM1 and TM2 and between TM7 and TM8, soluble loops of lesser conservation constitute domains involved in various biological functions.²⁷⁴ From the known structural and functional analysis of these proteins, the soluble domains often display large conformational changes as a result of ligand/substrate binding and release. The energy required for the entire transport process is transduced via the TMD upon consumption of the proton (or sodium-ion) motive force (PMF or SMF). Based on these functions and their phylogeny, RND transporters are assigned to distinct families.²⁷⁵,²⁷⁶

#### 5.1.1. Bacterial Drug Efflux Proteins from the HAE Family of RND Transporters. The hydrophobe/amphiphile
<figure><img src="image_15.png" /><figcaption>**Figure 5.** Structure of asymmetric AcrB comprising three protomers in the loose (L, blue), tight (T, yellow), and open (O, red) conformation. (A) Side view of trimeric AcrB along the membrane plane. Indicated are the transmembrane, periplasmic porter, and funnel domains (TMD, PD, and FD). (B) Top view on the porter domain. In the L protomer (blue), the PN1, PN2, PC1, and PC2 subdomains are indicated. The PC1 and PC2 subdomains constitute a cleft as part of the access pocket (AP, dashed oval). The T protomer contains also an open PC1/PC2 cleft but is less voluminous compared to the one in the L protomer. Between the PN2 and PC1 subdomains, the deep binding pocket (DBP, dashed oval) is indicated. The switch loop (green) separates the AP and the DBP. In the O protomer (red), the PC1/PC2 cleft, the AP, and the DBP are closed. From the closed DBP, a tunnel is present which exits at the funnel domain. In the asymmetric LTO AcrB porter domain representations (top view) on the left, binding of drugs to the access pocket is shown. Rifampicin (sphere representation in pink) binds in the L protomer (highlighted in blue) at the proximal side of the access pocket, and erythromycin (sphere representation in pink) binds to most distal part of the access pocket, underneath the switch loop (green).<sup>289</sup> On the right, the T protomer is highlighted (in yellow), and bound minocycline (sphere representation in pink) is located in the distal groove region of the deep binding pocket,<sup>288,290</sup> whereas puromycin (sphere representation in cyan) binds to the more proximal cave region of the deep binding pocket underneath the switch loop (in green).<sup>305</sup> (C) Top view of the funnel domain (FD). The N-terminal FN and the C-terminal FC subdomains are indicated. The AcrB trimer is stabilized by a loop protruding from the FN subdomain connected to the FD subdomain of the neighboring protomer emerging from subdomain DN to DC of the adjacent protomer. The distal FD remains largely unaffected by the conformational cycling and remains structurally unchanged during the LTO cycle. (D) Top view (from the periplasm) of the transmembrane domain (TMD). The TMD displays 12 transmembrane helices (TMHs). The proton relay network consists of residues D407, D408, and K940 associated with R971 and T978 located in the center of the TM-domains. Adjacent protomers interact via TM1 and TM8. The large central cavity is depicted by a circle. This image was constructed with PDB 4DX5 (in complex with minocycline)<sup>290</sup> for the structure images in complex with drugs. Coordinates for rifampicin (PDB 3AOB), erythromycin (PDB 3AOC), minocycline (PDB 4DX5), and puromycin (PDB 5NC5) were superposed to the 4DX5 structure.</figcaption></figure>

efflux exporter (HAE) family is subdivided into three subfamilies.<sup>275</sup> The HAE-1 family is from Gram-negative bacterial origin and includes the secondary active drug efflux RND transporters which are only active *in vivo* in conjunction with PAPs and outer membrane factors/channels (OMFs). The HAE-2 family members are lipid exporters of Gram-positive bacteria, and recently, structures of the monomeric mycolic acid RND transporter MmpL3 from *Mycobacterium smegmatis* were solved in apo-form and in complex with anti-TB drugs.<sup>277</sup> Members of the HAE-3 family include archaeal and bacterial exporters of hopanoids.<sup>275</sup> A crystal structure of the homodimeric hopanoid exporter HpnN from *Burkholderia multivorans* was elucidated.<sup>278</sup>

Structures of apo and substrate bound *E. coli* HAE-1 member AcrB,<sup>279–294</sup> *P. aeruginosa* MexB,<sup>291,295,296</sup> apo and substrate bound *N. gonorrhoeae* MtrD,<sup>297,298</sup> *C. jejuni* CmeB,<sup>299</sup> *A. baumannii* AdeB,<sup>300</sup> AcrB from *S. Typhimurium*,<sup>301</sup> and TriC from *P. aeruginosa*<sup>302</sup> could be solved by crystallography or recently by single particle cryo-EM. HAE-1 family RND transporters are embedded in the inner membrane as homotrimers and rarely also as heterotrimers, such as MdtBC.<sup>303</sup> The RND pumps form larger two-membrane

spanning tripartite complexes in conjunction with a PAP and an OMF; the latter two form channel structures to create a long drug exit tunnel toward the outside of the Gram-negative cell.<sup>270,287,304–307</sup>

### 5.1.2. The *E. coli* HAE-1 Member Acriflavine Resistance Protein B (AcrB).
Acriflavine resistance protein B (AcrB) from *E. coli* was identified in 1978 to be a factor in resistance against the topical antiseptic acriflavine.<sup>308</sup> Later, AcrB was shown to act as an efflux pump of bile salts, and its gene is upregulated under several stress conditions.<sup>180,309</sup> Early reports indicated the large substrate spectrum of this efflux pump,<sup>309,310</sup> exporting a broad range of structurally diverse molecules comprising diverse classes of antibiotics, including β-lactams, macrolides, quinolones, rifamycins, aminocoumarins, tetracyclines, and oxazolidinones, as well as anticancer drugs, dyes, bile salts, detergents, and solvents<sup>5</sup> (Table 1). The substrates of the pump commonly harbor hydrophobic moieties. Bianionic β-lactams are apparently poor substrates for AcrB, and the more hydrophilic aminoglycosides are not substrates for the pump. The latter compounds are preferably extruded by the homologous AcrAD-TolC efflux pump in *E. coli*.<sup>55,311,312</sup>
<figure><img src="image_16.png" /><figcaption>**Figure 6.** Topology of AcrB protomer. The structure of AcrB is a representation of an architecture that is shared across the HAE and HME families of RND transporters. The protomer of AcrB presents a pseudo-2-fold symmetry, associated with a gene duplication of the ancestral gene, giving two semi-identical N-terminal and C-terminal portions of the protein, each including a 6 TM-helical component with a large periplasmic loop spliced between the TM1/TM2. These loops form the porter or periplasmic domain (PD) and a docking domain. Porter domains display highly modular organization, presenting a mixed α-β sandwich of a general (β-α-β-β-α-β) configuration dubbed the PD-module (lower inset). This is repeated through the porter subdomains PN1/PN2 (belonging to the N-terminal loop) and PC1/PC2 (C-terminal loop), respectively. Subdomains PN1/PC2 and PN2/PC1 form discrete units coupled by a shared β-sheet. The docking or funnel domain comprises two subdomains, DN (or FN) and DC (or FC) respectively, which are topologically derived from the basic PD-fold of the PN/PC domains, by a flippage of the β2-β3 hairpin out of the central β-sheet (upper inset). The long β-hairpins of the DN and DC domains connect neighboring protomers by forming interprotomer β-sheets.</figcaption></figure>

The membrane topology of the AcrB transmembrane domain was initially accurately predicted to comprise 12 TMHs and a periplasmic region composed of two large loops.<sup>313</sup> Periplasmic domain swapping between AcrB and AcrD<sup>314</sup> or exchange of the periplasmic domains of MexB and MexY<sup>315</sup> clearly indicated that the substrate specificity of the RND pumps was determined inside the periplasmic domains. Furthermore, Tikhonova et al.<sup>316</sup> showed with AcrB and MexB fusions that the specificity of interaction is encoded in the periplasmic T60-V612 region of these transporters. Soon after initial diffraction studies were published,<sup>317</sup> the first crystal structure of the symmetric trimeric AcrB was solved at a resolution of 3.5 Å in 2002.<sup>279</sup> The three protomers of the trimer are arranged in 3-fold symmetry perpendicular to the membrane plane, all adopting the same distinct conformation (later defined as the loose (L) or access conformation) and thus constituting a LLL trimer.

In 2006 and 2007, three independent studies reported an asymmetric trimeric setup of AcrB<sup>288,318,319</sup> (Figure 5). The use of designed ankyrin-repeat proteins (DARPins) as crystallization chaperones resulted in a higher resolution structure (2.5 Å) of the otherwise congruent asymmetric AcrB trimer.<sup>319</sup> In 2012, under optimized conditions, a high-resolution structure at 1.9 Å was obtained allowing detailed insight on substrate binding.<sup>290</sup> Moreover, the presence of water molecules in the TMD suggested the presence of water channels to the H<sup>+</sup>-titratable residues (D407, D408).<sup>292</sup> The “asymmetric” crystal structures revealed three distinct con-

formations assigned to consecutive states of the transport cycle, generally denoted as access, binding, and extrusion states<sup>288</sup> or loose (L), tight (T), and open (O) states,<sup>318</sup> with the latter designation in analogy to the conformations established for the functional rotation (binding change) mechanism of the F₁F₀ ATP-synthase<sup>320</sup> (Figure 5). The functional rotation mechanism hypothesis was supported by structure-based site-directed disulfide cross-linking studies. Experiments confirmed not only the presence of asymmetric AcrB *in vivo* but also the necessity of conformational cycling in substrate extrusion.<sup>321,322</sup> Studies with a genetic fusion construct encoding a covalently linked AcrB trimer provided further evidence for interdependent conformer cycling by demonstrating that the inactivation of a single protomer abolishes the function of the entire AcrAB-TolC system.<sup>323</sup>

The first structures in complex with the AcrB substrates minocycline and doxorubicin were obtained by Murakami et al.<sup>288</sup> at 2.8 Å resolution. The use of brominated minocycline was of crucial importance, to assign the different densities to minocycline, as the anomalous signal of bromine yields unequivocal proof of its presence in the region defined as the deep binding pocket (DBP) (Figure 5B). The use of brominated substrates was later also used to identify a novel binding site for carboxylated drugs, by the use of brominated fusidic acid.<sup>324</sup>

**5.1.3. Structural Features of AcrB.** The three protomers within the AcrB trimer<sup>279</sup> each comprise a transmembrane domain (50 Å height) characterized by two parallel pseudo-2-
<figure><img src="image_17.png" /></figure>

**Figure 7.** Structural features of AcrB. The asymmetric AcrB trimer comprises three protomers adopting different conformations (L, loose—blue; T, tight—yellow; O, open—red). (a) AcrB can be structurally subdivided into a funnel (FD), a porter (PD), and a transmembrane domain (TMD). The TMD consists of 12 transmembrane helices (TMs) per protomer (shown are the L (blue) and T (yellow) protomers; the O protomer is omitted for clarity). The TMD of the three protomers encloses a central lipid-filled cavity. Likewise, the FD of the AcrB trimer forms a funnel region, which is exposed to the channel structures of AcrA and TolC and facilitates the export of drugs across the OM. (b) Drug entrance sites are highlighted on the L protomer (blue). The vestibules are interprotomeric entrance channels (shown here is the vestibule between the O (red) and L protomers) toward the central cavity. The indicated TM7/8 groove in the L protomer is the entrance toward channel 1 (CH1, shown in panel c). This groove is also present in the T protomer. The cleft between the PC1 and PC2 subdomains, apparent in both the L and T protomers (indicated only for the L protomer), is the entry side for channel 2 (CH2, shown in panel c). (c) The asymmetric AcrB trimer comprises substrate entry channels in the L and T conformations. The entrance of channel 1 (CH1) starts above the TM8/TM9 groove and guides substrates from the outer leaflet of the inner membrane to the AP and further to the DBP. The cleft pathway via channel 2 (CH2) likewise leads to the AP. Compounds sequestered in the central cavity via the vestibules between the protomer interfaces can access channel 3 (CH3) for transport directly to the DBP. All entry channels are closing during the T to O transition. Concomitantly, an exit channel is created in the O protomer that connects the closed DBP to the FD.

fold symmetric repeats of 6 TM helices (Figure 5A, D). Two central helices, TM4 from repeat 1 and TM10 from repeat 2, are surrounded by the other TMs. TM1 and TM7 are located more at the periphery of the TMD of each protomer (Figure 5D). Structures and mutational studies were conducted on charged residues within the TMD, i.e. residues D407 and D408 in TM4, K940 in TM10, and R971 in TM11,²⁷⁹,³²⁵⁻³²⁸ and indicated the importance of the respective charges at these positions. Both TMD repeats are connected between TM6 and TM7 by a horizontal amphipathic helix (Iα), which is located in the cytoplasm presumably near the membrane (Figure 5A). Between the protomers, there is a small contact interface located between TM1 and TM8.³²⁹ The TMDs of the protomers encircle a lipid filled central cavity²⁷⁹,³³⁰ (Figure 5D). The extensive periplasmic part with a maximum diameter of 100 Å is formed by the periplasmic loops protruding into the periplasm between TM1 and TM2 as well as TM7 and TM8 (Figure 5A). This part is subdivided into the membrane-proximal porter domain (40 Å length) and the distal funnel domain (30 Å) (Figure 5A, B, C). The porter domain comprises four subdomains (Figure 5B; Figure 6). The periplasmic N-terminal PN1 and PN2 subdomains are protruding from TM1 and TM2; subdomains PC1 and PC2 are the C-terminal segments of the TM7/TM8 loop. The subdomain structures display two α-helices at the external side together with four- (PN2 and PC2) or five- (PN1 and PC1) stranded antiparallel β-sheets at the interior. The cores of the PN1/PN2 and PC1/PC2 domains are organized around a similar basic architecture, which we refer to as the porter domain or PD module, presenting a mixed α-β sandwich of a general (β-α-β-β-α-β) configuration (Figure 6). This is a very versatile module, that is shared across a number of transporters both within and outside the RND family, as well

as with a number of other, predominantly periplasmic proteins, as will be discussed in detail in the MacB section below.

Subdomains PN1/PC2 and PN2/PC1 form discrete units coupled by a shared β-sheet. A top view of the porter domain (Figure 5B; Figure 6) exhibits an arrangement of the PN1 subdomain located in the trimer's inner surface and the more peripheral exposed PN2, PC1, and PC2 subdomains. A cleft between the PC1 and PC2 subdomains in the L and T conformation forms a solvent exposed protomer substrate entry site. The funnel domain (also called the docking domain) comprises two subdomains, FN (DN) of the N-terminal periplasmic loop and FC (DC) of the C-terminal periplasmic loop (Figure 5C; Figure 6). The funnel domain is a large water exposed cavity and is involved in the collection of exported drugs from the O protomer (Figure 7A). The membrane-located and proximal central cavity and the distal periplasmic funnel are separated by the central pore helices of the PN1 subdomains at the center of the trimer porter domain (Figure 6B; Figure 7A). The protomers are noncovalently connected by long interconnecting loops emerging from the FN to the FC subdomains protruding in the neighboring protomers²⁷⁹ (Figure 6A) and have been found essential for the trimeric assembly.³³¹,³³² The asymmetric structure of AcrB comprising the L, T, and O protomers revealed the presence of a substrate tunnel or channels (referred to as CH1 to CH4)³¹⁸,³¹⁹,³³³,³³⁴ (Figure 7). The concept of tunnels leading from the surface of the protein to the active/substrate binding site of proteins was known for soluble proteins; however, the peristaltic pump concept³¹⁸ was rather different, as it suggested that drugs are not diffusing through these tunnels (Figure 7C) but guided along the path by conformational change, therefore enforcing directionality (to the outside). The initial hypothesis anticipated an ordered binding process (assumed to be valid for all drugs recognized by this pump, e.g., β-lactams,
tetracyclines, fluoroquinolones, macrolides, detergents), comprising initial binding to the access pocket, followed by binding to a deep binding pocket and finally extrusion via an exit tunnel. In the L protomer, the AP is open and the DBP is closed. Substrates can access the AP from the periplasm via the open PC1/PC2 cleft (Figure 5B). Furthermore, a large groove is formed by TM7-9, where the substrate dodecylmaltoside is observed in several AcrB structures.<sup>290,319</sup> However, the T protomer appears to have multiple channels leading to both the AP (reduced in volume compared to the L protomer access pocket) and the DBP (Figure 7C). Channel 1 (CH1) starts at the TM7/8 groove (also referred to as the TM8/9 groove) and leads to the AP, past the switch loop, to the DBP.<sup>290,319,335</sup> The channel 2 (CH2) entrance is at the PC1/PC2 cleft and also leads to both binding pockets.<sup>318,319,333</sup> Channel 3 starts from the lipid filled central cavity and leads directly to the DBP (and not to the AP).<sup>333</sup> Finally, a fourth channel (CH4, not shown) was postulated starting from the TM1/2 groove also directly toward the DBP.<sup>334</sup> Interestingly, despite the overall substrate polyspecificity of the AcrB efflux pump, the channels appear to have restricted access to certain drugs, although it is not yet clearly defined for every channel and channels might have overlapping specificities.<sup>333</sup> CH1 is possibly involved in the transport detergents and smaller drugs, such as β-lactams. CH2 has a wide entrance and leads directly from the periplasm to the AP and is most likely involved in the transport of high molecular weight drugs such as macrolides. CH3 has been shown to be involved in planar aromatic cations, such as ethidium or rhodamine 6G.<sup>333</sup> CH4 is proposed to be involved in the transport of carboxylated drugs, such as fusidic acid and β-lactams.<sup>334</sup> Further studies have to be conducted to analyze the precise substrate preferences and overlap between the different channels.

**5.1.4. Substrate and Inhibitor Binding to Multidrug Efflux RND Transporter.** Structural analysis has been conducted for several RND transporters, among them structures revealing the binding of substrates (drugs) to various areas within the porter domain and to the TMD grooves between TM1 and TM2 and between TM7 and TM8. Here, we describe the substrate and inhibitor binding to the multidrug efflux RND transporters from the hydrophobic/amphipathic exporter-1 (HAE-1) family. For an excellent review of antibiotic/inhibitor complex structures of this family and those of the related heavy metal ion exporter family (HME), the reader is referred to the work of the group of Edward Yu and his current review in the same issue of this journal.<sup>336</sup> The L, T, and O states are structurally different both in the TMD and the porter domain, but the differences are especially apparent at the structural level of the porter domains (Figure 5B). Of note, crystallographic analysis of the symmetric (LLL) state<sup>279–287,337</sup> suggests that this trimeric conformation might be the resting state of the transporter. Some of the structures presented in the articles referenced above also present drugs bound to the L protomer TMD/periplasm interface region and the PC1/PC2 cleft region in this symmetric state.

For the asymmetric AcrB structures,<sup>288–294</sup> binding in the trimeric HAE-1 pumps occurs in the L and T states. In the compact O state, the only volume inside the periplasmic porter domain is the exit tunnel (Figure 7C). The drug binding access pocket (AP) is located between the β-sheets of the PC1 and PC2 subdomains in the L state. AcrB co-structures with high molecular weight compounds such as rifampicin, erythromycin<sup>289</sup> (Figure 5B), and dimeric doxorubicin<sup>290</sup> indicate different binding preferences within the AP. In accordance with the multisite-drug oscillation hypothesis,<sup>338</sup> drug binding occurs at various subsites with the AP. While rifampicin and dimeric doxorubicin bind in the approximate center of the AP, the interaction surface of erythromycin is shifted deeper into the AP of the protein (Figure 5B).

During conformational change from the L to the T conformation, the DBP opens and is accessible for drug binding. The AP and DBP are separated by a glycine rich (G614, G616, G619, G621) switch loop emerging from subdomain PC1. Consisting of only 11 amino acids, the switch loop is highly flexible. Structures revealed its function as an adapter for the binding of various drugs, modulating the shape and interaction surface of both binding pockets without changing the overall AcrB structure.<sup>289</sup> The switch loop is therefore directly involved in substrate binding, adopting diverse conformations. The important role of the switch loop for the drug transport mechanism was further emphasized in biochemical assays, exhibiting dramatic effects of single side chain substitutions (especially substitutions of glycine to proline) on the resistance phenotype.<sup>289,290,339,340</sup> On the other hand, a complete inactive AcrB variant, where the glycine to proline substitutions apparently rigidify the loop, can be rescued by additional substitution of F615 and F617 to alanine.<sup>340</sup> Moreover, binding of erythromycin to its binding site as observed in the wild-type protein, appears to be unaffected in an AcrB variant devoid of the switch loop (by deletion).<sup>293</sup>

The opening of the DBP during the L to T conversion is mediated by an outward movement of the PN2/PC1 subdomains, while the PN1/PC2 subdomain unit remains almost rigid.<sup>292</sup> Simultaneously, the AP is contracting and narrowing the available binding sites. It is anticipated in this step that substrates can slide underneath the switch loop from the AP into the DBP. Substrates are observed at diverse positions of the DBP.<sup>290,305,341,342</sup> Figure 8 illustrates the positions of the substrate molecules minocycline, doxorubicin,

<figure><img src="image_18.png" /><figcaption>**Figure 8.** Drug binding to the DBP of the AcrB T conformer. Superimposition of the coordinates of minocycline (MIN, carbon = green; PDB: 4DX5), doxorubicin (DOX, carbon = violet; PDB: 4DX7), rhodamine 6G (R6G, carbon = pink; PDB: 5ENS), and puromycin (PUY, carbon = orange; PDB: 5NC5) in the DBP. Substrates are shown as sticks, the PN2/PC1 subdomains are represented as a cartoon, colored in yellow. The switch-loop is highlighted in green.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Table 3. Assignment of Potentially Interacting Residues with Substrates Found in Crystal Structures<sup>a</sup></figcaption></figure>

<sup>a</sup>All residues with atoms found in a radius of 3.5 Å around one of the co-crystallized ligands were assigned according to their localization to the outer AP (cleft), the AP-DBP interface, and the DBP cave and groove regions. Potential interactions with a substrate are indicated in green. The numbers after the ligand/substrate give the sum of potential interacting residues per substrate. Rifampicin and the doxorubicin dimer are found exclusively interacting with residues of the AP and the interface region between the AP and DBP, while erythromycin also interacts with parts of the DBP cave and groove. Puromycin, binding underneath the switch loop, makes contact to all five indicated sections. Rhodamine 6G, minocycline, and doxorubicin bind to the DBP, primarily (but not only) to the groove region. The inhibitors, ABIPP and MBX3132 as a consequence of their orthogonal orientation, are interacting with both groove and cave regions of the DBP. Adapted with permission from Kobylka et al.<sup>342</sup> Copyright 2019, Kobylka, Kuth, Müller, Geertsma & Pos under Creative Commons Attribution 4.0 International Public License: https://creativecommons.org/licenses/by/4.0/legalcode.

rhodamine-6G, and puromycin in the DBP of AcrB. Binding by conserved Phe residues (F136, F178, F615, F617, and F628) is especially apparent for many of the substrates in the DBP. The phenyl rings of rhodamine 6G favor an interaction via π-π stacking in the DBP cave<sup>341,343</sup> near the hydrophobic trap, located in the center of the DBP (Figure 5. The planar tetracycline minocycline on the other hand is bound in the more distal narrow groove (DBP groove) in the upper part of the pocket<sup>288,290</sup> (Figure 5B, Figure 8). The AP in the L protomer binds high molecular weight drugs such as macrolides or ansamycins<sup>289</sup> (Figure 5B). Direct transport competition of macrolides with another high molecular mass compound, the fluorophore DiBAC<sub>4</sub>-(3), substantiated the drug size-preference by the AP,<sup>344</sup> whereas low molecular weight drugs such as tetracyclines, which are observed to preferentially bind to the DBP, do not interfere with DiBAC<sub>4</sub>-(3) transport. Low molecular weight compounds rather affect the transport of the low molecular mass fluorophore Nile Red.<sup>344,345</sup> While planar aromatic cations are preferably transported via CH3, all substrates have to traverse the DBP region during the T to O transition which results in transport toward the funnel domain via the exit gate (Figure 5C).

Analysis of the published AcrB/substrate co-structures indicates which and how many residues are located around the individual substrates within a radius of 3.5 Å, a distance which makes them likely to be involved in substrate binding. Table 3 shows an organization of the residues into the outer and the inner AP, the AP-DBP interface at the switch loop, and the DBP groove and cave as well as an assignment of the (potentially) interacting substrates.

The assignment of substrates binding to the upper DBP groove region or the lower DBP cave region or binding to both regions of the DBP (i.e., mixed binders) is based on docking and MD simulations.<sup>343,346</sup> The groove region is formed by converging β-sheets of the PN2 and PC1 subdomains in the upper part of the DBP, while the cave (limited downward by the TMD) provides a much larger cavity. In contrast to substrate binding, AcrB inhibitors of the pyridopyrimidine (ABI-PP) and the pyranopyridine class (e.g., MBX3132) bind to a much larger area of the DBP.<sup>291,341</sup> Both inhibitors were

shown to significantly potentiate the activity of known antimicrobials without exhibiting membrane disruption effects or antibacterial activity.<sup>347,348</sup> The co-structures display extensive interactions of the inhibitors with both groove and cave regions. From this, the mechanism of action was suggested to be competition with substrates to bind to the DBP and/or conformational arrest of the T protomer.<sup>291,341</sup> The aromatic moieties of the inhibitors engage in intensive π-π stacking interactions with specifically side chains F178 and F628. The pyranopyridine core structures of other MBX derivatives (MBX2319, MBX2931, and MBX3135) showed thereby essentially the same binding properties as MBX3132.<sup>341</sup> Binding and stabilization of the inhibitor includes water-mediated protein-ligand interactions, and computational structural analysis indicated that the binding affinities of the inhibitors were significantly exceeding those of AcrB substrates.<sup>341,349</sup> Both inhibitors ABIPP and MBX3132 are of similar size as DBP binding drugs; however, the inhibitors contact 2-3 additional side chains (Table 3) apparently stabilizing the inhibitor/protein interaction. Substrate (drug) binding appears to be less reliant on specific interactions as van der Waals interactions are preferred over hydrogen bonding. Cocrystallized rifampicin, erythromycin, and doxorubicin dimer are in contact with 13-20 amino acid side chains within the AP, and residues such as M575 and F666 are contacted by any of these drugs (Table 3). Smaller drugs such as minocycline, doxorubicin monomer, rhodamine 6G, and puromycin display less contact sites in the DBP. The additional hydrogen bonding contacts with the inhibitors might stabilize the overall geometry of inhibitor binding and intensify the van der Waals interactions in other regions of inhibitor contact.

Some progress has also been made in understanding the residues involved in binding of natural substrates to RND transporters. For example, in MtrD critical residues including F136, F176, I605, F610, F612, and F623 have been highlighted to be essential for bacterial resistance to cationic antimicrobial peptides including polymyxin B.<sup>141</sup> Additionally, the W136 and R176 residues in MtrR have also been shown to be important for binding of the bile salt chenodeoxycholate.<sup>140</sup>
<figure><img src="image_20.png" /><figcaption>**Figure 9.** Proton binding or release induces conformational change of the transmembrane domain structural repeats. (A) Substrate/proton antiport catalyzed by AcrB can be described by two coupled mechanisms. A conformational switch from L (blue) to T (yellow) is mediated by substrate binding in the DBP of the porter domain. The rearrangement of subdomain unit PN2/PC1 leads to a downward movement of linked TM2. Consequently, the TMR1–TMR2 interface rearranges and opens a periplasmic proton entry site. Protonation of residues D407 and/or D408 results in separation of the TMR1–TMR2 interaction network. A conformational change to the disengaged state results in a switch to the O-conformation (red). TM2 moves upward and the upper part of TM8 undergoes a random-coil-to-helix transition, while binding pockets in the porter domain collapse and the exit channel becomes accessible. The proton binding site opens to the cytoplasmic side. Protons are released in the cytoplasm, down the electrochemical gradient. Charged D407 and/or D408 switch the TM domain back to the engaged state. This conformational change translates back to the porter domain. Proton entry and exit are presumably mediated through a network of water molecules. (B) Close view of the proton relay comprising D407, D408, K940, and R971. These residues undergo the most dramatic conformational change during the T to O transition. In the L (blue) and T (yellow) conformations, water molecules mediate electrostatic contact between D407/D408/K940 with R971. In the O state (red), the transmembrane domain is (almost) dehydrated, greatly affecting the pKₐ values of the titratable residues. (C) Insight into the structural details of the interaction of D407 in the O conformation; the H-bonding interaction of the carboxyl group of D407 with the carbonyl group of G403 is only possible if D407 is protonated. A central water molecule connects (by hydrogen bonding) the interaction partners on TM4 (D407), TM10 (K940), and TM11 (T978).</figcaption></figure>

**5.1.5. Drug/Proton Antiport Coupling in AcrB.** For all characterized RND efflux pumps thus far, energy coupling is mediated via the proton-motive force (in one described case, sodium-ion motive force).¹¹⁹ In the TMDs of all RND transporters, at least one acidic residue appears to stay central to proton binding and release.²³³ Proton binding or release is coupled to changes in the electrostatics at the interface of the N- and C-terminal transmembrane repeats (TMRs) leading to conformational change and transduction of that movement toward the soluble parts of the transporters involved in substrate or ligand binding. For AcrB, this process has been

studied by many laboratories in more detail compared to other RND transporters. Substitution of the central acidic residue or residues has been conducted most of the time for all studied RND proteins and was shown to be essential for activity. For AcrB, proton binding and release is facilitated by residues D407, D408 (on TM4), and K940 (TM10), the proton relay triad, located in the TMD core of each protomer at the interface of the TMRs described below (Figure 9). A further critical residue is R971 on TM11, possibly involved in the coordination of a network of water molecules connecting R971 with D407 and D408 and, in this way, affecting the pKₐ values
of the carboxylates.²⁹² Substitution of one of these highly conserved residues abolishes substrate extrusion altogether.²⁷⁹,³²⁵⁻³²⁸ Two TMRs consisting of transmembrane helix 1 (TM1) and TM3-TM6 (transmembrane repeat 1, TMR1) as well as TM7 and TM9-TM12 (TMR2) are arranged in parallel pseudosymmetric fashion.²⁹² During conformational cycling, the TMR1 and TMR2 repeats act as rigid bodies and remain internally largely unchanged. However, relative movements between the TMRs are a rocking motion during L to T transition and a shear motion when the TMD changes from T to O. The cytoplasmic horizontal helix Iα (Figure 6A), connecting TMR1 and TMR2, shows a rigid motion in relation to TMR2. Flanking helices TM2 and TM8 are presumably the coupling elements mediating crosstalk between the TMD and the porter domain of AcrB²⁹² (Figure 9); TM2 is thereby linked to the PN2/PC1 subdomain unit, and TM8 is connected to the PN1/PC2 tandem (Figure 6B). A downward movement of TM2 relative to TMR1 occurs during the conformational change from L to T. This is most likely due to the binding of substrate to the DBP, which stabilizes the separation of the PN2/PC1 subdomain unit. This process is reversed during the T to O transition. The latter transition is catalyzed by the protonation of the TMD causing conformational change between the two TMRs. As a result, TM8 switches from a disengaged state from TMR2 in the L and T state, to an engaged state in the O conformation. A short sequence between TM8 and the PC2 subdomain, the hoisting loop,³⁵⁰ thereby undergoes a random-coil-to-helix transition, and TM8 becomes a compact elongated α-helix.²⁹² The hoisting loop is an important factor in conformational coupling of TMD and the periplasmic porter domain. Mutagenesis studies revealed that it is not actively involved in energy transduction, but its high flexibility is crucial for conformational cycling.³⁵⁰ High resolution structures of the L-, T-, and O-states, both of wild-type AcrB and D407N, D408N, K940A, and R971A variants, reveal different interaction networks in this energy conversion machinery.²⁹⁰,²⁹² In the L- and T-conformations, TMR2 K940 forms an ionic interaction with D407 and D408 in TMR1. An additional hydrogen bond between D407 and T978 in the second repeat further contributes to the stabilization of the TMR1–TMR2 complex. Residue R971 in TMR2 forms yet another interaction with TMR1 via E414 and N415. With change to the O-conformation, the proton relay residues twist and reconfigure as a result of the rigid body motion of TMR 1 and TMR2. In the disengaged state, connections of TMR1 and TMR2 are replaced by intra-TM repeat interactions.²⁹² As such, the TMRs switch between an engaged and a disengaged state during cycling (Figure 9). One of the most discussed questions on the proton transport is the number of protons transported per LTO cycle. Since there are two titratable residues (D407 and D408), the anticipated number is either one or two. There is good biochemical, structural, and molecular dynamic evidence that D408 is protonated and deprotonated during the entire cycle.²⁹²,³²⁸,³⁵¹⁻³⁵³ For D407, molecular dynamics studies show that it remains deprotonated (charged) in the O state,³⁵¹,³⁵² whereas other structural and molecular dynamics studies suggest a protonation of D407 (and D408) in the O state.²⁹² In particular, the D407 residue in the wild-type AcrB structure (at 1.9 Å resolution) is in hydrogen bond distance with the carbonyl oxygen of G403 of TM4, implicated to be responsible for the stabilization of the observed kink in TM4 in the O state (Figure 9C). A hydrogen bond as observed in the

structure will only be feasible if the D407 is protonated. It is very clear that further analysis, including determination of proton per cycle stoichiometry in *in vitro* reconstituted systems and the protonation state of K940 during the cycle, have to be done to obtain more insights onto this basic question.

### 5.1.6. Structures of Other HAE-1 RND Transporters.

Besides AcrB, HAE-1 RND transporter structures have been solved via X-ray crystallography or single-particle cryo-EM. For MexB from *P. aeruginosa*, crystal costructures showed dodecylmaltoside (DDM),²⁹⁵ a pyridopyrimidine inhibitor (D13-9001, also referred to as ABI-PP,²⁹¹ lauryl maltose neopentyl glycol (LMNG), CYMAL6 neopentyl glycol, or CYMAL-7 neopentyl glycol²⁹⁶ bound to the DBP of the T protomer. For MtrD of *N. gonorrhoeae*, an X-ray structure of a symmetric LLL trimer²⁹⁷ showed a possible resting state of this pump. Recently, a cryo-EM structure of the asymmetric LTO trimer in complex with hydrolyzed, decarboxylated ampicillin or erythromycin in the DBP of the T protomer was solved.²⁹⁸ In the case of erythromycin binding, it clearly revealed that a high molecular weight, despite its size, can be present in the DBP cave region. Further structures of HAE-1 family transporters included those of AdeB of *A. baumannii*³⁰⁰ and CmeB from *C. jejuni*.²⁹⁹ The AdeB trimer, with its structure solved by single particle cryo-EM, showed an OOO conformation, where every protomer lacks a distinct drug binding site. This OOO conformation was previously suggested to be sterically precluded in the case of the AcrB trimer transport cycle.²⁹² For CmeB, two different trimer conformations were observed from structures obtained by X-ray crystallography. One state appeared to be again an approximate OOO state, i.e. with every protomer in the trimer in the extrusion state revealing an exit channel toward the funnel domain. In the other trimeric state, three different conformations were observed, one binding state, similar to the T state of AcrB, and one extrusion or O state, and the third protomer was in an intermediate state, unlike the L, T, and O states, which was designated a “resting” state. A similar protomer conformation was found for the HME-family Cu⁺-RND pump, CusA, in the apo-state³⁵⁴ of the crystal structure. The functional implication for this observation was tested with CmeB-containing proteoliposomes and different labeling of the protomers in order to observe single-molecule FRET signals upon conformational change of the protomers relative to each other. Interestingly, the results were not in accordance with a concerted cycling of the protomers in the trimer but rather supported the independent (i.e., not dependent on the conformation of the neighboring protomer) conformational change of each of the protomers during transport. Of note, the study was done with the isolated RND component (CmeB). Concerted cycling, however, might be highly supported by the PAPs (like in this case CmeA) and the outer membrane channel (CmeC) to generate a concerted cycling *in vivo*. Recently, single-particle cryo-EM structures of AcrB wild-type and of a clinically relevant resistance mutant G288D³⁵⁵ from S. Typhimurium were solved using purified samples in styrene maleic acid (SMA) copolymer.³⁰¹ The AcrB trimer structure revealed a similar state as observed for the *E. coli* LLL state;²⁷⁹ however, slight differences between the protomers were observed, in accordance with previously predicted flexibility within the porter domain of *E. coli* AcrB by molecular dynamics simulations.³⁵⁶ For the G288D variant, it was observed that the residues around D288 show a notable shift, which may represent the observed increase in bulk waters
and large change in electrostatics.355 All-atom molecular dynamics simulations indicated the increase in volume in the area close to D288 compared to the wild-type (G288) and increase in the number of water molecules present in that region.

**5.1.7. HME Family.** The members of the Gram-negative heavy metal-ion exporter (HME) family catalyze the efflux of heavy metal ions from the periplasm across the outer membrane albeit that *in vitro* studies show that these proteins are also able to transport heavy metal ions across the inner membrane. These family members can be further categorized into several subfamilies (HME1-5).357 The tripartite Zn²⁺-exporter ZneCAB is involved in the extrusion of specifically Zn²⁺ ions, and the structures of the RND-component ZneA were solved in different conformations of the anticipated transport cycle, with Zn²⁺ bound to a proximal or distal binding site in the periplasmic domain.358 Reconstitution of ZneA in liposomes and Zn²⁺ transport experiments suggests transport of Zn²⁺ from the cytoplasm or periplasm, in an electrogenic fashion. The CusCBA complex catalyzes the efflux of Cu⁺ or Ag⁺. Structures of CusA alone and in complex with the PAP CusB have been solved.354,359 The TMD and the periplasmic part of the RND transporter CusA contains five methionine clusters which are proposed to coordinate metal ion transport through the protein. The uptake of Cu⁺ or Ag⁺ from the periplasm involves also the nonessential metal-ion chaperone CusF, from which Cu⁺ is transported via the PAP CusB to the Cu⁺-binding site in the periplasmic part of CusA.360

**5.1.8. Other Bacterial RND Transporters.** Nodulation factor exporter (NFE) family members are supposed to act as a single component of nodulation factors as NolGHI, coding for a single gene,361 was identified in the Gram-negative root nodule bacterium *Rhizobium meliloti*.362 An NFE transporter of the plant pathogen *Pseudomonas syringae*, involved in the export of the lipodepsipeptides syringopeptin and syringomy-cin, and YerP from the Gram-positive bacterium *Bacillus subtilis*, involved in the secretion of lipodepsipeptide surfactin, are further examples of NFE family members.274 Members of the SecDF family are of bacterial or archaeal origin and have been shown to assist in protein translocation over the cytoplasmic membrane mediated by the SecYEG translocon. *Thermus thermophilus* SecD and SecF display a heterodimeric 2 × 6 TM setup with a typical monomeric RND motif.363–366 SecDF captures the SecYEG transported precursor protein on the periplasmic side with its soluble periplasmic domain and can independently finalize the complete translocation at the expense of the PMF, even in the absence of SecA-mediated ATP-hydrolysis (see section below).

## 5.2. Major Facilitator Superfamily (MFS) Transporters

The diverse major facilitator superfamily (MFS) is thought to be the largest group of membrane transporters found in all organisms.367–371 Members are uniporters, symporters, and antiporters comprising approximately 400 to 600 amino acids and 12 or 14 transmembrane helices (TMHs). Many MFS-members are involved in the uptake of nutrients, such as sugars and amino acids. This occurs in symport with H⁺ and depends on the substrate and stoichiometry, at the expense of the electrochemical gradient across the inner membrane. The general architecture of the prototypical MFS transporters (Figure 10) consists of a polypeptide chain that contains 2 lobes of 6 (or 7) transmembrane α-helices, which gives rise to

<figure><img src="image_21.png" /><figcaption>**Figure 10.** General topology of MFS transporters based on a representative of the structure of *E. coli* EmrD (PDB ID: 2GFP).380 The topological diagram highlights the inverted structural repeats, forming the N- and C-terminal lobes of the transporter, suggesting multiple gene duplication events.</figcaption></figure>

a 12-TM or 14-TM “clam-like” assembly with a central symmetry between the two respective N- and C-terminal lobes,372 although 24 TM-spanners exist, such as NarK from *Paracoccus pantotophus*,373 which can be demonstrated to have evolved from the fusion of two homologous, but distinct 12-TM MFS permeases. The substrate pathway is thought to be formed between the two lobes of the permeases using an alternating access mechanism with a single substrate binding site.374 The high-resolution structures of the glycerol-3-phosphate:P antiporter GlpT374 and the lactose:H⁺ symporter LacY375,376 confirmed the existence of a pseudo-2-fold symmetry, which was predicted based on the sequence similarity of the two 6 TM-lobes of the prototypical MFS transporters, suggested to have arisen from a 3 TM helical element, known as MFS fold via two successive gene-duplication events,377,378 although this evolutionary scenario has been challenged more recently.379

In MFS transporters four motifs, A–D, have been identified. Motifs A and B can be found in antiporters as well as symporters. In the multidrug efflux antiporter MdfA from *E. coli*, the sequence of motif A is 73GPLSDRIGRRP83 (mapping to the TM2–TM3 junction; numbers relate to the position in the *EcMdfA* sequence)381 and has been proposed to stabilize the outward facing state,382 and that of motif B is 108FTLLRFLQG116 (TM4), with R112 as a pivotal residue involved in the alternative access mechanism of drug/H⁺ antiport (Figure 11). Motif-C (154PLGLPVGA162; TMS) is unique for antiporters including MdfA381,383 and MdtM384 and because of this is called the antiporter motif.385 Preceding motif C is TM5, which shows a large deviation when superposing the inward and outward open structures of

<figure><img src="image_22.png" /><figcaption>**Figure 11.** Alternating access mechanism by MFS transporters. The transport cycle of MFS proteins seems to involve ordered binding and release of proton and substrate; however, drug/proton stoichiometry varies between family members.</figcaption></figure>
MdfA.³⁸³ Motif-D (²⁶EFSTYIGNNDMIQPGM⁴⁰; TM1) is not very common in MFS transporters but conserved in MdfA orthologs. It includes the important titratable residues E26 and D34 involved in proton binding and release (see below).

The X-ray structures of the GlpT³⁷⁴ and Lacy³⁷⁵ also provided a mechanistic model for the operation of the MFS transporters which has become known as the “rocker-switch” mechanism. In this model, the clamlike transporter opens up only to one side of the membrane at a time, preventing formation of a continuous aqueous conduit across it. In the case of LacY (symporter) this cycle involves binding of lactose and protons on the periplasmic side, causing closure of the “clam” to the periplasm but opening toward the cytoplasm releasing the cargo. The cycle for efflux pumps is suggested to be inverted. In terms of energy coupling, the MFS transporters catalyze uniport and symport of solutes using cation gradients (predominantly protons but occasionally Na⁺) and, in the case of exporters/efflux pumps, the antiport of solutes against protons,³⁶⁹ often in conjunction with the negative-inside membrane potential.³⁸⁶ Most MFS transporters exhibit a very distinct substrate specificity; however, MFS transporters involved in multidrug resistance, i.e. having a polyspecific substrate spectrum, have also been identified.³⁸⁵ Drug/H⁺ antiporters (DHA) from the DHA-1 (12 TM segments) and DHA-2 (14 TM segments) families are most commonly found in bacteria. MFS transporters of the DHA-1 family are arranged in 2 domains of 6 TMH bundles related in a pseudo-2-fold symmetry.³⁷⁴,³⁸⁸ The well-studied DHA-1 family protein *E. coli* MdfA transports a broad range of zwitterionic and uncharged drugs from the cytoplasmic side to the periplasm.³⁸⁹ Crystallographic structures indicate a central cavity at the interface of the two TM bundles containing exposed side chains involved in both drug and proton binding. In MdfA, drug binds to the central cavity, formed by TM1, TM4, TM7, and TM10. For MdfA, an inward open³⁸¹ and an outward-open conformation was recently reported.³⁸³ Transport is mediated in an alternating access mechanism³⁸¹,³⁹⁰,³⁹¹ (Figure 11).

Two conserved acidic residues, E26 and D34, have been shown in MdfA to be involved in proton binding and release, and the negative charge on D34 is not essential for substrate binding as it could be replaced with asparagine without having an effect on *in vitro* substrate binding.³⁹² MdfA is broadly flexible in recognizing electroneutral or monovalent cationic drugs and catalyzes the efflux of these drugs in exchange with a single proton, and there appears to be an indirect competition mechanism between protons and substrates. However, MdfA was shown to also transport divalent cations where the charges were separated by a long linker. It is anticipated that the export of these kinds of divalent drug molecules is in need of two consecutive transport cycles, thereby consuming 2 protons.³⁹³

In Gram-negative bacteria, this large superfamily comprises not only single component transporters but also tripartite efflux machineries. Tripartite multidrug pumps of the MFS family are encoded by chromosomal or plasmid genes, and usually PAP and MFS are coded on a single operon (e.g., *emrAB* in *E. coli*, *salAB* in *Rhyzobium leguminosarum*, or *emrKY* in *Shigella flexneri*), while the OMF is borrowed from another operon. However, in some cases all three pump components are encoded on one operon, such as the *emrAB-emrC* from *Chromobacterium violaceum*.³⁹⁴ As an example, a tripartite system is formed by the *E. coli* DHA-2 family MFS transporter *E. coli* EmrB, which interacts with the PAP EmrA, connecting it

to the OMF TolC.³⁹⁵ While this is one of the first multidrug efflux systems to be identified,³⁹⁶ direct structural information about the complete assembly has not been available until very recently.⁹ The EmrAB-TolC system provides resistance to a number of antibiotics, from thiolactomycin (TLM) and cerulenin³⁹⁷ to nalidixic acid and nitroxolone and of hydrophobic proton uncouplers, including carbonyl-cyanide *m*-chlorophenylhydrazone (CCCP).³⁹⁶,³⁹⁸ EmrB contains 14 transmembrane helices, which sets it apart from other related MFS exporters such as MdfA and EmrD, which feature 12 TM helices. On TM1 and TM7, candidate aspartic acid candidates for H⁺ and/or drug binding are present. To our knowledge, no mutational analysis has been conducted on EmrB or EmrA, e.g. to determine residues involved in H⁺ or drug binding. Other identified tripartite MFS transporters are the VceABC system from *V. cholerae*³⁹⁹ and the FarAB-MtrE system from gonococci,⁷⁹ with the latter involved in fatty-acid resistance. The expression of the *farAB* genes is under the control of the same regulator protein (MtrR) as the genes encoding the RND-tripartite MtrCDE system, and for resistance, the FarAB system shares the OMF (MtrE) with the MtrCD system.

## 5.3. Multiple Antibiotic and Toxin Extrusion (MATE) Transporters

Based on phylogenetic analyses, bacterial MATE transporters can be divided into the NorM and DinF subfamilies of 12TM drug/Na⁺ or drug/H⁺ antiporters⁴⁰⁰ (for xenobiotic-transporting eukaryotic MATE transporters the reader is referred to Miyauchi et al.⁴⁰¹). MATE family members transport polyaromatic and cationic compounds, such as rhodamine 6G, ethidium bromide, and tetraphenylphosphonium (TPP).²³³,⁴⁰⁰ Structures of Na⁺-dependent NorM subfamily transporters have been solved by X-ray crystallography at 3.6 Å resolution in cation- and substrate-bound states.⁴⁰²,⁴⁰³ X-ray structures (3.2 Å resolution) of the *Bacillus halodurans* DinF (DinF-BH) transporter of the DinF subfamily were solved in complex with rhodamine 6G bound to the central cavity via mostly hydrophobic interactions.⁴⁰⁴ From the same subfamily, structures of pfMATE from *Pyrococcus furiosus* in two distinct apo-form conformations, and in complexes with a derivative of norfloxacin and three thioether-macrocyclic peptides, were solved at 2.1–3.0 Å resolution.⁴⁰⁵ Despite MATE and MFS transporters having different topologies in their pseudosymmetrical 6 TM helix bundles, an alternating access mechanism with ordered binding and release of proton and substrate similar to that for MFS transporters has been proposed.⁴⁰⁶

## 5.4. Small Multidrug Resistance (SMR) Transporters

Members of the small multidrug resistance (SMR) transporter family are the smallest membrane transporters identified thus far.⁴⁰⁷ SMR transporters form parallel or antiparallel homo- or heterodimers comprising 4 TMs to transport drugs in dependence of the PMF.⁴⁰⁸,⁴⁰⁹ EmrE is the most studied SMR transporter and contains an interprotomeric hydrophobic drug binding pocket including a highly conserved Glu residue that is essential for transport. In *A. baumannii*, a homologue of EmrE, AbeS⁴¹⁰ selects for the size and planarity of the conjugated aromatic moieties on drugs. Moderate changes in the property of side chains in the substrate binding site resulted in large phenotypic differences.⁴¹¹ SMR family members recognize and transport cationic lipophilic molecules such as ethidium, tetraphenylphosphonium (TPP), or acriflavine, which are extruded in an alternating access mechanism.⁴¹² An apo-structure of EmrE and in complex
with various substrates was created by molecular dynamic (MD) simulations⁴¹³ on the basis of a low-resolution EmrE X-ray structure in complex with TPP.⁴¹⁴

## 5.5. Proteobacterial Antimicrobial Compound Efflux (PACE) Transporter Family

A transcriptomic study when *A. baumannii* was challenged with the disinfectant chlorhexidine, resulted in the discovery of AceI, which was the founding member of the proteobacterial antimicrobial compound efflux (PACE) transporter family.⁴¹⁵ PACE family members are small membrane proteins comprising approximately 150 amino acids. Topology prediction revealed putative 4 TMs in a setup similar to members of the SMR family.⁴¹⁶ AceI homologues were shown to confer resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. For the AceI homologue from *Vibrio parahaemolyticus* resistance to proflavine and acriflavine was shown to be mediated by an active efflux mechanism. Recently, it has been reported that AceI of *A. baumannii* exists in a monomer-dimer equilibrium that is regulated by pH. The binding of chlorhexidine and an increase in pH was found to facilitate the functional form of the protein.⁴¹⁷

## 5.6. ABC Transporter Family

The ATP-binding cassette (ABC) transporters are an extremely diverse superfamily of transporters with equally diverse functions within the cell that are widespread across both pro- and eukaryotic organisms. Classified as the TC# 3.A.1 by the transporter classification database, TCDB (http://www.tcdb.org),⁴¹⁸ the ABC superfamily forms the largest functional superfamily of primary active transporters known, and in *Escherichia coli* alone 79 ABC proteins have been identified, making the ABC transporters the largest paralogous family of proteins in its genome comprising 5% of its total,⁴¹⁹ forming 69 independent functional systems, with some 57 being ABC transporters.¹²⁹,⁴²⁰ Several different classification systems have been proposed over the years to address this diversity within the family, see refs 421–423, taking into account both functional and topological data. Despite their considerable diversity across the family, in general organization, the ABC transporters are built around a common basic blueprint and in a first approximation can be seen as composed of a dimeric transmembrane porter section, typically in a 6 + 6 transmembrane helix configuration, and a pair of cytoplasmic nucleotide binding domains (NBDs), which provide energy coupling for the transport process by ATP-hydrolysis and could be found either as a part of a single polypeptide chain or as separate subunits.⁴²⁴ While the NBDs of the ABC transporters are relatively conserved across the superfamily and contain highly conserved nucleotide binding motifs, known as Walker A (GXXXXGS/T) and Walker B (hhhhD, or h₄B where “h” is a hydrophobic residue),⁴²⁵ as well as the ABC signature sequence LSGGQ (aka the C-motif), that defines the family⁴¹⁹,⁴²² (see section 5.6.6 for detail and graphic), the transmembrane porter domains are rather diverse⁴²⁶ (see section 5.7.7 for details). Extensive bioinformatic analysis⁴²⁷ has suggested that the porter domains of ATP-binding cassette transport systems are polyphyletic and have evolved three times independently, forming three families which the authors designated ABC1, ABC2, and ABC3, upon which the monophyletic ATPases have been superimposed for energy-coupling purposes. It has been suggested that ABC1 transporters have originated from intragenic triplication of a

primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins, while the ABC2 transporters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, which is nonhomologous to the ABC1 proteins.⁴²⁷ ABC3 clade of transporters on the other hand is suggested to have arisen via duplication of a primordial four-TMS-encoding genetic element, yielding either 8- or 10-TMS proteins.

Recent reclassification of the ABC transporters has established some order in this overwhelming diversity of ever-expanding structural and topological data, resulting in 7 main family “types”,⁴²³,⁴²⁸ and to avoid confusion the discourse below follows this latest classification. Of these newly defined seven classes, three types are importers, which are of limited interest to the topic of interest here, while the others include the following families: “type IV”, possessing 6 + 6 TM-helices membrane topology, includes both exporters (e.g., Sav1866 and MsbA floppase) and importers (e.g., YbtPQ; Rv1819c); “type V”, in prokaryotes a 6 + 6 TM topology of exporters/floppases including the Wzm-WztN O-antigen transport system;⁴²⁹ and the “extractor”, ABC transporters that remove compounds from the outer leaflet of the membrane, that includes members with a 6 + 6 TM membrane topology such as LptB₂FG,⁴³⁰ which possess specialized β-jellyroll domains forming the type VI group, as well as the 4 + 4 TM members of the MacB/FtsX family constituting the newly defined type VII group.⁴²³ The known structures of the latter group include MacB itself,²⁴,²⁷,²⁹,⁴³¹ as well as the heterodimeric LolCDE lipoprotein sorting system (LoLC, PDB ID SNAA;⁴³² LoLE, PDB ID 5UDF⁴³²) and the FtsX family members (PDB ID 4N8N; 6HEE, 6HFX).⁴³³,⁴³⁴

In addition, the maintenance of phospholipid asymmetry (Mla) MlaFEDB complex,⁴³⁵,⁴³⁶ the structure of which has recently become available (PDB ID 7CGN; 7CH0; 7CGE; 6XBD),⁴³⁷⁻⁴³⁹ revealed unexpected structural organization of the MlaE transmembrane domain, which might merit its isolation into a new, separate topological group.

As the ABC transporters are subject to a number of reviews (see refs 423 and 440), including in the current special issue of *Chemical Reviews* itself,⁴⁴¹ to avoid redundancy, below we will focus on aspects of the organization of the ABC transporters that are specific to their function within the context of the tripartite assemblies. Indeed, despite the diversity of folds and architectures described above, only two groups of prokaryotic ABC transporters participate in tripartite assemblies—namely the transporters associated with protein secretion and type I secretion systems (belonging to the type IV [exporter] group) and the MacB/FtsX-like transporters (belonging to the newly defined type VII),⁴²⁸,⁴⁴² with only the latter of them conclusively associated with efflux-function, despite some recent circumstantial evidence.⁴⁴³

### 5.6.1. Type IV Family of ABC Transporters Involved in Tripartite Assemblies.
As mentioned above the ABC transporters present a great structural variety, which corresponds to an equally vast diversity of transported cargoes, which vary from xenobiotics and dyes, to lipid and glyco-lipid molecules; lipoproteins and outer-membrane components to S-layer proteins; as well as an array of virulence factors, including large adhesins, toxins, and smaller antibacterial peptides.⁴²²,⁴²⁸ The latter two functions, that allow secretion of proteinaceous cargoes bypassing the Sec-pathway, are served by two related families of type IV ABC transporters, the first of
<figure><img src="image_23.png" /><figcaption>**Figure 12.** General view of the type IV ABC transporter topology and features of the typical type IV ABC transporter structure. Schematic representation of the topology of the isolated monomer of ABC transporters belonging to the type IV group (panel A) and the dimeric formation (panel B), highlighting the cross-protomer engagement of the nucleotide binding domains (NBDs) by the coupling helices 1 and 2 (CH1 and CH2, respectively). The optional N-terminal C39 or C39-like domain (CLD), which is present in a subset of the family, is shown as a "packman". The TMD is presented as a rainbow from N- to C-terminal, and the EH represents the elbow helix. The second protomer of HlyB is presented in gray. Approximate membrane boundaries are represented by the blue rectangle. (C) View of a microcin-transport associated McjD based on the PDB ID 4PL0 (two side views 90° apart) highlighting the principal structural elements. Coloring as in panels A and B. (D) PCAT1 in complex with its transported peptide CtA, based on the PDB ID 6VZ. The C39-domain is colored wheat.</figcaption></figure>

which, often called the NisT/McjD family after its representative members,⁴⁴⁴,⁴⁴⁵ is solely dedicated to cargo transport and exclusively deals with smaller cargoes without changing them during transport, while the second family covers bifunctional transporters, which provide maturation as well as transport function, proteolytically processing their substrates, which are hence known as the peptidase-containing ATP-binding transporters (PCAT) or ABC transporter maturation secretion (AMS) family.⁴⁴⁶,⁴⁴⁷ These AMS/PCAT family transporters, apart from small antimicrobial peptides, are capable of transporting very large cargoes such as toxins and adhesins and participate in formation of type 1 secretion systems (T1SSs) in Gram-negative bacteria.⁴⁴⁸,⁴⁴⁹ A detailed description of these tripartite systems alongside a graphic overview is provided in section 10. In the following paragraphs, we will discuss both these transporter families in turn, focusing on the transporters from Gram-negative organisms, which contribute to tripartite assemblies. For comprehensive reviews of the ABC transporters associated with the antimicrobial transport in Gram-positive (Firmicutes) bacteria please consult refs 444 and 445

**5.6.2. NisT/McjD Family of Bacteriocin Transporters.**
In their interspecies warfare, bacteria have developed a number

of adaptive strategies, including the production of narrow-spectrum antimicrobial peptides, which they deploy against their competitors, which are generally termed "bacteriocins" (a term that also includes the heavily modified lantibiotics, which do not always perform bactericidal function), and which in Gram-positive organisms exert their action by disrupting the membrane potential.⁴⁵⁰,⁴⁵¹ When applicable to Gram-negative bacteria these antimicrobial peptides are designated "microcins".⁴⁴⁷,⁴⁵² A large class of these are the so-called ribosomally synthesized and post-translationally modified peptides (RiPPs),⁴⁵³,⁴⁵⁴ and they are primarily secreted by dedicated ABC transporter systems, bypassing the Sec-pathway. Microcins are gene-encoded antibacterial peptides, with molecular weight below 10 kDa,⁴⁵⁵ and could be both modified and nonmodified, with the latter group encompassing a great variety of lasso peptides, siderophore peptides, nucleotide peptides, and linear azole(in)e-containing peptides.⁴⁵² While the peptide antibiotics produced by Gram-positive organisms mainly act as ionophores, the microcins, which are secreted by the Gram-negative bacteria attack a diverse range of cellular targets including DNA gyrase (microcin B17),⁴⁵⁶ protein synthesis (microcin C7),⁴⁵⁷ and dissipation of the membrane potential (colicin V).⁴⁵⁰
**5.6.3. Structural Features of McJD and the Standard Cycle of the Type IV ABC Family.** The best studied member of the family is the enterobacterial McjD transporter,⁴⁵⁸,⁴⁵⁹ which is involved in the transport of the eight-residue macrolactam-containing lasso-peptide microcin J25 (aka MccJ25).⁴⁶⁰ MccJ25 has a narrow spectrum and is principally active against *Salmonella* and *E. coli* and provides the cells expressing it self-immunity.⁴⁶¹ McjD is related to the prototypical NisT ABC transporter from the Gram-positive *Lactococcus lactis* which is involved in the transport of the commercially important lantibiotic nisin,⁴⁴⁴ with both of them giving the name to the wider family. In *E. coli*, MccJ25 is secreted by a dedicated ABC transporter McjD; albeit, additional ABC transporters of the same group may be involved, including YojL.⁶³,⁴⁶² Both McjD and YojL require the action of the OMF TolC for the export of MccJ25, although it is not clear whether any of them participate in bona fide tripartite systems or there is a soluble intermediate of transport.⁶³,⁴⁶²

The structure of the McjD (PDB ID 4PL0)⁴⁵⁹ is representative of the type IV family of ABC transporters, sharing close topological and structural similarity to other canonical type IV transporters, such as Sav1866,⁴⁶³ MsbA,⁴⁶⁴ and Pgp/ABCB1⁴⁶⁵ (Figure 12). The structure of the biological unit is homodimeric (overall dimension of ~125 × 55 × 50 Å), with each protomer composed of an N-terminal TMD composed of 6 TM-helices and a C-terminal NBD. The TM-helices are connected by extracellular and intracellular loops, with the intracellular loops between TM2 and TM3 and between TM4 and TM forming the “coupling helices” CH1 and CH2, respectively. Coupling helices interact with the NBDs transducing the conformational changes induced by ATP-binding and hydrolysis to conformational changes of the TMDs. In a configuration that is typical for the type IV ABC transporters,⁴⁶³,⁴⁶⁵ the CH1 contacts predominantly the NBD of its cognate protomer, while the CH2 interacts exclusively with the NBD from the opposing protomer, in a swapped-protomer manner⁴⁵⁹ (see topological schematic Figure 12).

The membrane protein structural biology revolution has dramatically increased our understanding of the mechanistic operation of ABC transporters, largely displacing the earlier models derived from kinetic and biochemical data, e.g. the “alternating catalytic sites”⁴⁶⁶ and the connected “two-cylinder engine” model.⁴⁶⁷ The “alternating access” allosteric model, first postulated for major facilitator transport proteins,³⁹⁰ has become a paradigm for ABC transporter action being used for rationalization of the available crystal structures of Sav1866, with the notable distinction being the suggestion that ATP-binding and hydrolysis rather than substrate acquisition are the main drivers of the conformational state transition.⁴⁶³ As its name suggests, the functional cycle of the transporter is based on the alternating inward- and outward-facing TMD conformations of the protomers. This rather straightforward model has undertaken a number of iterations and increased in complexity over the years as more diverse ABC transporters came to light. Some notable variations include the “Switch Model”, which proposes that hydrolysis in each protomer is sequential and that as the sites are freed of nucleotide, the protomers lose contact.⁴⁶⁸⁻⁴⁷⁰ In this model, the ATP binding and ATP-hydrolysis, respectively, induce formation and dissociation of an NBD dimer, as seen in a number of crystal structures, e.g., Sav1866⁴⁶³,⁴⁷¹ and MsbA;⁴⁶⁴,⁴⁷² furthermore, a

derived model of the P-glycoprotein (Pgp, aka ABCB1) binding has been based on this approach.⁴⁶⁵

A second major group of models, known as “Constant Contact”,⁴⁷³ has evolved from the alternating catalytic site ideas,⁴⁶⁶,⁴⁷⁴ which were derived mostly from observations of the cooperativity between the two nucleotide binding sites in the P-glycoprotein. In that model, there is an alternating ATP-hydrolysis in each NBD, with one site opening at the point of ATP-hydrolysis, while the second site remains closed with ATP bound and occluded, with the process repeating itself in alternating cycles without the NBDs losing contact fully. This model has gained support from a number of structures of heterodimeric transporters, e.g., the TM287/TM288 from *T. maritima*,⁴⁷⁵,⁴⁷⁶ BmrCD,⁴⁷⁷ and TmrAB.⁴⁷⁸,⁴⁷⁹ This may not be restricted to heterodimeric transporters with degenerate sites; however, e.g., experiments by Siarheyeva et al.⁴⁸⁰ identified an asymmetric and catalytically active state of the Pgp/ABCB1 that did not show reduced binding of drug substrate, suggesting that ATP-binding does not drive transport, contradicting the “switch model”. It thus seems plausible that the mechanistic cycles of different ABC transporters may align closer to either one or the other model, or indeed by a tuned-up, blended version of each, giving rise to a considerable variety of allosteric modulation, and these possibilities are further explored in dedicated reviews (see refs 423, 473, and 481

The crystal structure of McjD reveals the transporter in an outward “occluded TMD” conformation, presenting a substrate-binding cavity, which is sealed both from the periplasm and the cytoplasmic ends. This cavity, at ~5900 Å³ is sufficiently large to accommodate the full microcin allowing for a single-step translocation, with several residues being specifically important for cargo recognition, notably F86, N134, N302,⁴⁵⁹ and TM3/TM6 lining the walls of the cavity. In comparison, the structure of the MsbA in an occluded, ligand-bound state⁴⁶⁴ presents a conformation of its TM3 and TM6, which bend toward the center of the TMD eliminating the corresponding cavity.

As the McjD has been co-crystallized with the ATP analogue AMP-PNP and MgCl₂, both of its NBDs were found in a dimerized, ATP-bound state, with the P-loop and ABC-signature motifs involved in binding of the nucleotides, and correspondingly the TMD is also closed up.⁴⁵⁹ The TMD-dimer interface is formed by TM2 and TM5/TM6 from one subunit, the binding of which to the equivalent TM5/TM6 and TM2 from the opposite subunit buries a surface area of ~7,100 Å² per protomer. The structure of McjD suggested a model of its conformational cycling,⁴⁵⁹ where the inward facing open (IF₀) apo-conformation allows binding of the transported substrate to the transporter, which facilitates the transition into an inward-closed conformation (IF_c) which becomes ATP-binding capable. The binding of the ATP to the NBDs at this point was proposed to bring the transporter into a hypothetical substrate-engaged occluded conformation (Occ), and it is supposed to transit into a nucleotide bound outward-facing open (OF) conformation. Upon release of the substrate, the transporter was suggested to adopt an the outward occluded (Occ) conformation yet again, however this time in a substrate free state. The structure solved (PDB ID 4PL0)⁴⁵⁹ was associated with this post-transport occluded stated, and subsequent ATP-hydrolysis and release of the bound ADP and Pi eventually resets the transporter to the initial (IF₀) configuration completing the cycle. Later the structure of the
<figure><img src="image_24.png" /><figcaption>**Figure 13.** C39 and CLD domains in AMS/PCAT transporters. (A) Structure of the catalytically active C39 domain from PCAT1 (based on PDB ID 6V9Z).⁵⁰⁵ The main elements of the secondary structure, as well as the key catalytic residues are highlighted. (B) The structure of the C39-like domain (CLD) of the HlyB transporter in identical orientation based on PBD ID 3ZUA.⁵⁰⁶ While the general architecture is preserved, the active site cysteine residue is substituted by a tyrosine.</figcaption></figure>

eukaryotic P-gp (ABCB1)⁴⁶⁵ has been used to further refine this cycle.

Further research, adding pulsed electron-electron double resonance (PELDOR) and single molecule Förster-resonance energy transfer (sm-FRET) measurements to probe the conformational changes and the dynamics of McJd along its transport cycle,⁴⁶¹,⁴⁸² has led to the suggestion that irrespective of the presence or absence of nucleotides, in the absence of MccJ25, McJd remains in an occluded conformation and that it requires both MccJ25 and ATP to open, presenting a mechanism for the substrate transport and selectivity that was dubbed the “occluded-mechanism with transient opening”. Such a mechanism would make it distinct from the other type IV ABC transporters discussed above, which are capable of adopting an outward open conformation in the absence of substrate.

**5.6.4. ABC Transporters with Double Functions—The Peptidase-Containing ATP-Binding Cassette Transporters (PCAT) Family.** These peptidase-containing ATP-binding cassette transporters (or PCATs for short) are also sometimes designated in the ABC transporter maturation and secretion (AMS) family and also belong to the wider family of type IV exporters, which possess a 6 + 6 TM-helix topology⁴²³,⁴⁴⁴ and are present in both Gram-positive and Gram-negative bacteria. The transporters involved in protein secretion and maturation are unique among ABC transporters, due to the presence of extra domains required for substrate recruitment and processing. These domains typically have a proteolytic function and are a result of a gene fusion with peptidases belonging to the cysteine protease superfamily, classified as family C39, bacteriocin-processing peptidase, with their protease activity being dependent upon the catalytic cysteine, histidine, or aspartate residues that are conserved among papain-like cysteine proteases⁴⁸³ and are hence designated either as PEP or C39 domains. Crystal structures of C39 domains, e.g.,

ComA,⁴⁸⁴ have confirmed their association with the Cys-protease family. The substrates of the PCAT transporters contain an N-terminal “leader sequence” that typically ends in a double Gly-motif (variations G-G, G-A, or G-S), and mutational analysis, based primarily on the quorum-sensor pathway-associated transporter ComA, identified a consensus leader-peptide recognition sequence of Leu(-12)-(X)₃-Glu(-8)-Leu(-7) located upstream from that double Gly-motif.⁴⁸⁴

In Gram-positive bacteria, PCATs function as both maturation proteases and exporters for peptides involved in quorum-sensing⁴⁸⁵ or antimicrobial peptides, including lantibiotic and nonlantibiotic bacteriocins, e.g., colicin V and similar class II bacteriocins, and various classes of RiPPs, which are extensively post-translationally modified over their C-terminal core peptide.⁴⁵³,⁴⁸⁶⁻⁴⁸⁸ The biosynthetic clusters of bacteriocins typically contain a dedicated PCAT exporter that directs the processing and secretion of their respective cargoes prior to membrane translocation at the conserved double Gly-leader sequence described above,⁴⁴⁶,⁴⁸⁹ removing a roughly 2.5 kDa N-terminal fragment, which is necessary for cargo secretion and activation of their antibacterial function.⁴⁹⁰

In Gram-negative organisms, PCATs, by pairing with respective PAPs and OMFs, form type 1 secretion systems (T1SSs) capable of exporting a range of virulence factors including ligases, scavenging molecules, and proteases,¹⁵⁷,⁴⁹¹,⁴⁹² as well as gigantic adhesins with a mass over 1 MDa,⁶,⁴⁹³ e.g. in the export of hemolysin A the PCAT transporter HlyB pairs with HlyCD.²¹⁴,⁴⁹⁴

**5.6.5. Unique Features of the AMS/PCAT Transporters—N-Terminal C39 Protease and CLD Domains.** Many of the T1SS transporters, as well as the PCAT transporters involved in microcin/bacteriocin secretion possess unique N-terminal C39-protease domains,³,⁴⁴⁷ e.g., the *E. coli* MchF (UniProtKB: Q9EXN5), involved in the secretion of microcin H47 ref 495; RaxB from *Xanthomonas oryzae*
<figure><img src="image_25.png" /><figcaption>**Figure 14.** PCAT-cargo interactions mediated by their C39 and CLD domains. (A) Side view of the PCAT1-CtA transporter-cargo complex based on the PDB ID 6 V9Z (Kieuvongngam et al.⁵⁰⁵). (B) Zoomed-in view of the C39 domain highlighting the interaction with the secretion signal of the CtA. (C) Structure of the equivalent CLD domain in HlyB, with CtA superposed into the substrate binding groove. (D) View of the CtA-threading into the substrate binding cavity of PCAT reveals the large substrate access cavern delineated by TM4 and TM6. (E) Position of the cargo targeting motif of the CtA on the surface of the C39 domain, indicating the location of the conserved hydrophobic residues which provide the docking and the GG-motif where the proteolytic cleavage occurs.</figcaption></figure>

(UniProtKB: Q5GWX4).⁴⁹⁶ These C39 domains are partic-
ularly associated with Gram-negative T1SSs involved in the
secretion of so-called repeats-in-toxin (RTX) cargoes,⁴⁴⁹ which
include adhesins,⁴⁹⁷ as well as hemolytic and cytolytic pore-
forming toxins,⁴⁹⁸,⁴⁹⁹ which feature a noncleavable C-terminal
secretion signal.⁵⁰⁰ As their name suggests, the RTX toxins are
defined by the presence of repeated glycine- and aspartate-rich
nonapeptide consensus sequences known as the “GG-repeats”
(GGxGxDxUx; where x is any residue and U is a bulky
hydrophobic residue).⁵⁰¹ Depending on the transported cargo,
the GG-repeat number can vary from less than 10 to over
40.⁴⁴⁹ Concomitant with the secretion of the T1SS substrate
into the extracellular medium, the GG-repeats bind Ca²⁺ ions
with high affinity, triggering cargo-domain folding.⁵⁰⁰,⁵⁰¹

The C39-domain (Interprot ID IPR005074) belongs to the MEROPS⁵⁰² peptidase cysteine-proteinase family CA,⁴⁸³,⁵⁰³ which includes proteins with a papain-like fold. This homology-based prediction was confirmed, when the C32 and H105-residues of the N-terminal C39-domain of CvaB transporter were shown to be critical for the calcium-dependent cysteine protease activity and the secretion of its native cargo colicin V.⁵⁰⁴ The crystal structure of the C39 domain belonging to the bacteriocin-associated ATPase ComA from Streptococcus provided a structural template to the family (PDB ID 3K8U).⁴⁸⁴ The structure revealed a domain consisting of two subdomains with the active site sandwiched between them. The smaller subdomain consists of a bundle of three consecutive N-terminal α-helices, with the catalytic C17 located at the end of the first of them, and is positioned on top of a larger subdomain, that presents a twisted antiparallel β-sheet core (β1-β6) supplemented by two small α-helices (α4 and α5), and is contributing the active site residues H96 and

D112, consistent with a classical catalytic triad for CA cysteine
proteinases.⁴⁸⁴,⁵⁰³ A fourth residue preceding the catalytic
cysteine, in this case Q11, forms a part of an oxyanion hole that
stabilizes the acyl-intermediate formed during catalysis (see
Figure 13).⁴⁸⁴

The recent structure of the protease domain of the PCAT
transporter LahT from a lanthipeptide biosynthetic operon in
the Gram-positive *Lachnospiraceae bacterium* C6A11 in
complex with a leader peptide (PDB ID 6MPZ)⁵⁰⁷ provides
a model for the substrate recognition, where the helical region
of the substrate peptide is oriented in a small groove located
between the C39 domain and the NBD of the transporter,
providing an effective means to lock these domains together. In
addition, the leader peptide may serve as a backstop that
prevents the cleaved cargo from sliding back into the
cytoplasm and thus provides the directionality of transport
upon binding of ATP to the NBDs.

C39-domains have also been revealed in the structures of the
PCAT1 transporter both in the nucleotide free apo-state (PDB
ID 4RY2), in the ATP-analogue occluded state (PDB ID
4S0F),²⁷³ and in complex with the cognate substrate CtA
peptide (PDB ID 6 V9Z),⁵⁰⁵ all showing close correlation with
the one seen in CvaB and confirming the earlier cargo-binding
model derived from the isolated C39-domain⁵⁰⁷ (Figure 14).
As predicted, the peptide signature sequence (Leu(-12)-(X)₃-
Glu(-8)-Leu(-7))⁴⁸⁴ makes a number of specific interactions
based on the conserved residues in the C39-domain which
facilitates its orientation for processing (Figure 14, panel E).
However, intriguingly the peptide-bound structure of PCAT1
presented asymmetric positioning of the cargoes, with one
being fully threaded into the transporter suggesting a
functional asymmetry. The C39-domain appears to orient
<figure><img src="image_26.png" /><figcaption>**Figure 15.** Structure of the ABC transporter nucleotide-binding domains (NBDs). (A) Linear graph from the N- to C-terminus of the typical NBD domain, using HlyB as a template. (B) View of the dimerized NBD based on the crystal structure of the ATP-bound HlyB monomer (PDB ID 1XEF),⁵¹⁶ highlighting the positions of the conserved sequence motifs involved in nucleotide binding. In one subunit, the RecA-like and β-sheet domain is colored pale blue, while the α-helical subdomain, harboring the ABC-motif, is colored yellow. The second NBD protomer is colored wheat. Motifs are presented in colors matching the sequence boxes in panel A. For illustrative purposes, the side chain of the catalytic H662, which is substituted by an alanine in the above structure to stabilize the ATP-bound state, is being modeled in. The side chains of Y477 (loop A; coordinates the adenine ring of ATP); the E631 (the invariant Glu-residue following Walker B, which helps to coordinate the ATP) and H662 (H-loop) are presented as sticks. The Walker B motif usually features four hydrophobic residues that form a β-strand and is terminated by a conserved Asp (in this case D630), which coordinates the catalytic Mg²⁺ cofactor via water-molecule mediation. Two ATP molecules are colored in purple.</figcaption></figure>

the cargo, feeding the C-terminus first into a cleft formed by the TM helices 4 and 6 leading into the large central cavity of the transporter (Figure 14, panel D). Consistent with the biochemical data, the cargo is not seen making specific contacts with the TMD, which may explain the apparent promiscuity of the PCATs.⁵⁰⁵ The energetic implications of the CtA-bound structure are discussed in some detail in the following sections.

A number of Gram-negative PCAT-type transporters seem to possess “C39-like domains” or CLDs, which lack the required proteolytic function,³ especially the ones associated with the export of the so-called “repeats-in-toxin” (RTX) cargos. These CLDs present different noncatalytic amino acids; e.g., in the exotoxin-translocating ATPase PaxB (UniProtKB: Q9RCG7), which is part of the PaxA secreting PaxBD complex, a Y13 is present *in lieu* of a catalytic cysteine. The leukotoxin translocation ATPase LktB from *Mannheimia haemolytica* presents a L10, while an N9-residue is found in place of the cysteine residue expected in the CLD of the hemolysin B (RtxB) transporter from *E. coli* O157:H7 (UniProtKB: Q46717). While this is generally the rule, not all RTX-repeat transporters have disabled C39-domains, e.g., CyaB involved in the transport of calmodulin-sensitive adenylate cyclase-hemolysin (cyclolysin) (UniProtKB: P0DKX5).³³

One of the best-studied examples of a CLD-containing PCATs is HlyB (UniProtKB: P08716), which is part of the T1SS involved in the secretion of α-hemolysin (HlyA).⁵⁰⁸ The N-terminal domain of HlyB shares ~40% homology to C39 peptidases, but critically, this “C39-like domain” or CLD

displays a degenerated proteolytic site, with a catalytically essential cysteine⁵⁰⁴ being replaced by a tyrosine (Y9).⁵⁰⁹ Consistent with this, HlyA does not contain an N-terminal leader peptide or a GG motif and is not cleaved during or after the transport, suggesting an alternative mechanism is in action for its secretion. Compared to the catalytical histidine of ComA-PEP (H96), the equivalent histidine of the HlyB-CLD (H83) is flipped by 180° and the solution NMR structure of the HlyB-CLD (PDB ID 3ZUA)⁵⁰⁶ revealed a π-π stacking between the H83 and W77 residues, which might be a characteristic feature of CLDs, as the tryptophan residue in the equivalent position is conserved across the family. This π-π stacking requirement explains why reintroduction of Cys at position 9 (Y9C) does not restore the proteolytic activity of the CLD. Despite the apparent homology and the similar overall fold between the C39-domain seen in the Streptococcal peptidase ComA,⁴⁸⁴ and the HlyB-CLD, which results in an overall similar fold, the CLD of HlyB represents a particular protein class with a distinct binding site imposing specificity for the RTX-toxin HlyA⁵⁰⁶ with the binding pocket for the latter being located on the opposite side compared to the substrate-binding site of ComA-C39. Notably, all PCAT transporters containing an active C39-peptidase domain appear to transport relatively small substrates with an upper MW limit of ~10 kDa, belonging to the bacteriocin family that possesses an N-terminal, cleavable leader peptide.⁵⁰⁶ In contrast, CLD-containing PCATs are associated with the transport of large cargoes of above 50 kDa, which contain a C-terminal secretion signal and, importantly, belong to the RTX (repeat in toxin)-toxin family. Thus, the existence of an RTX-domain in the
transported substrate can be correlated to the presence of a CLD in the cognate PCAT transporter and *vice versa*.⁵⁰⁶

Importantly, despite the lack of proteolytic activity, the CLD of HlyB appears necessary for the recruitment and tethering of the unfolded substrate, playing chaperone-like.⁵⁰⁶ The CLD of HlyB was shown to tether its cargo HlyA, preventing its aggregation and/or degradation during secretion, playing the dual role of a cargo recognition as well as a chaperoning unit.⁵⁰⁶ Crucially, combining these data with more recent findings,⁵⁰⁷ suggests that the CLD may also play a role in stabilizing the nucleotide-free state of HlyB by binding the substrate peptide, allowing it to optimally position its cargo for export upon ATP-binding.

In some cases, such as the multifunctional autoprocessing RTX-like (MARTX) (e.g., the *Vibrio cholerae* actin-directed RTX toxin (Q9KS12)⁵¹⁰ and Clostridium sp. glucosylating toxin families the *Vibrio cholerae* actin-directed RTX toxin (Q9KS12)⁵¹⁰), the cargoes themselves display an autoprocessing in cis mediated by an internal C80 peptidase domain, which becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP(6)), present in the target cells.⁵¹¹

Intriguingly, there is also a sizable group of T1SS-associated PCAT-like ABC transporters that lack a CLD or a C39-domain altogether.³ One such example is HasD, which is part of the T1SS of *Serratia marcescens* which secretes the hemophore HasA.¹⁵³,⁵¹² HasA is not cleaved during the transport and contains no RTX domain, and consistent with the observations of Lecher et al.,⁵⁰⁶ HasD does not possess an N-terminal domain. Furthermore, the cellular chaperone SecB is required for the productive HasA secretion by the T1SS by inhibiting its folding and maintaining the unfolded, secretion-competent state of HasA.⁵¹³ Nevertheless, HasA contains primary recognition sites that interact with HasD prior to the secretion, likely within the substrate binding cavity of the transporter, although their exact identity remains obscure.⁵¹² Other notable members include LipB from the *S. marcescens* lipase T1SS (UniProtKB Q54456)⁵¹⁴ and PrtD from the protease secretion system PrtDEF from *Aquifex aeolicus* VF5 (UniProtKB O67184),⁵¹⁵ both of which are discussed below.

**5.6.6. NBDs of PCAT Transporters.** The NBDs of HlyB (PDB ID 1XEF,⁵⁰⁹ PDB ID 2FF7⁵¹⁰), LipB (the ABC transporter involved in the T1SS that secretes the lipase LipA in *S. marcescens*) (PDB ID 5X7K),⁵¹⁴ and PrtD (PDB ID 5L22)⁵¹⁵ have been solved and present a structure that is generally identical to that of the canonical type IV ABC transporters. ATP-binding promotes dimerization of the HlyB-NBD.⁵¹⁷ Indeed, the HlyB NBDs has been solved in an ATP-bound state (PDB ID 1XEF),⁵⁰⁹ presenting a dimer in which two intact ATP molecules are bound at the interface of the Walker A motif and the C-loop provided by the opposite protomers (Figure 15). Biochemical data suggests a mechanism of substrate-assisted catalysis, rather than that general base catalysis might operate in the HlyB-type ABC-ATPases. Two highly conserved residues, namely E631 and H662, form a catalytic dyad, with H662 playing the role of a “linchpin”, that coordinates a complex network of intermolecular interactions, and were found to be required for interprotomer communication, that covers ATP, water molecules, as well as Mg²⁺ ions and amino-acids both in cis and trans orientation (Figure 15).⁵⁰⁹

A couple of follow-up studies⁵¹⁶,⁵¹⁸ revealed the kinetics of ATP-driven NBD association alongside the structures of the

ADP-bound form of HlyB NBD, in wild-type (PDB ID 2FF7), as well as in mutant forms of the transporter (PDB ID 2FFA; 2FFB) allowing us to complete the functional cycle of the transporter. Cavity analysis of dimeric ATP-bound NBDs indicates that inorganic phosphate should not be able to diffuse freely out of the binding site, and its exit requires an additional conformational change in the domain.⁵¹⁶ Intriguingly, these structures also revealed a structural asymmetry within the ATP-bound NBD dimer in the form of one open and one closed phosphate exit tunnel. This tunnel gate is controlled by a salt bridge between D551 and a R611-residue, the side chain of which flips open to allow diffusion of the inorganic phosphate. Analysis of the structures, and the loss of cooperativity upon mutation of these conserved residues, strongly suggests a sequential ATP-hydrolysis is taking place;⁵¹⁶ however, whether this sequential release of ADP and Pi may be connected to the processivity of the transporter remains unclear. ATP-hydrolysis (~-30 to -35 kJ/mol) and NBD dimerization (-33.4 kJ/mol) are comparable from an energetic point of view, and thus either step could represent the potential “power stroke” during the reaction cycle of the ABC transporters. In addition, both ATP binding and ADP-Pi dissociation could contribute to the power stroke.⁵¹⁶,⁵¹⁹ In the case of HlyB, the cycle has been suggested to take two distinct steps, with the *mechanical energy* being derived from the rigid-body motion of the α-helical subdomain and the rearrangement of the N-terminus of the Walker A motif associated with its binding of ATP and associated NBD-dimerization. This energy is transmitted to the TMDs likely used as a power-stroke. The second stage is suggested to employ the *chemical energy* of sequential ATP-hydrolysis, which is used for the recycling of the dimer by enabling the ADP dissociation. Analysis of the ADP-bound state of the HlyB-NBD revealed a distortion of helix-6, and it has been suggested that the tilting of this helix is used as an enthalpic energy storage device, with the elastic energy being possibly used for programmed release of ADP from dissociated NBD protomers and additional transmission to the TMDs to complete substrate translocation.

The archaeal ABC transporter MJ0796 from *Methanocaldococcus jannaschii*⁵²⁰ has been linked to the LoID from the LoLCDE system and is the closest NBD homologue to the *E. coli* LoID, with ~50% identity and 70% similarity between the two. Due to the close sequence similarity, its NBD may be a representative of MacB-family transporters, and intriguingly the dissociation of the NBDs upon ATP-hydrolysis seems to differ from that observed in the HlyB-family. While in the HlyB no significant increase in negative charges accompanying ATP-hydrolysis was detected within the ATP-binding site of HlyB;⁵¹⁶ the dissociation of the NBDs in the LoID/MacB family appears to be driven primarily by the active electrostatic repulsion.⁵²⁰ This discrepancy may reflect the different mode of substrate engagement between the two respective transporter families, with LoID/MacB operating a discontinuous, single-step extraction of the substrate, while the HlyB is required to progressively thread its substrate over multiple cycles.

**5.6.7. Structure of AMS/PCAT Family Transporters.** Structurally, the most studied member of the PCAT-family is the PCAT1 from the thermophilic Gram-positive bacterium *Clostridium thermocellum* (aka. *Hungateiclostridium thermocellum*) (UniProtKB - A3DCU1), which is responsible for the secretion of the small cargo protein CtA. CtA is encoded in the same operon as the main transporter and represents a 90-
<figure><img src="image_27.png" /><figcaption>**Figure 16.** Modified alternating-access mechanism of PCAT1. CtA is recruited and cleaved in the *inward-facing* (IF) conformation. In the model by Kieuvongngam et al.,⁵⁰⁵ the substrate specificity of the transporter is conferred primarily, if not exclusively, by the interaction of the C39-domains with the leader peptide, and the transporter TMD essentially does not otherwise interact with the cargo. In a modification of the cycle proposed by Rachman and Mchaourab,⁵²² the cargo is at the center of the interaction, as cargo interactions have been observed in transporter variants lacking C39-domains. In both cases, the interaction of the cargo proteins with the TMD leads to subsequent cleavage of the leader peptide, and a transient *inward closed conformation* (IC), similar to that observed in the McjD,⁴⁵⁹ is created, which enables the ATP binding. ATP-binding drives conformational changes in the TMD leading to occlusion of the cargo protein binding chamber, known as the *outward occluded state* (not shown). ATP binding stabilizes the *outward-facing* (OF) conformation in which the PEP domains are disengaged. After cargo release, TMDs isomerize to form an *occluded state* (OCC) in the absence of cargo. The energy of ATP-hydrolysis resets PCAT1 back to the inward-facing conformation, allowing PEP to dock into the TMD–NBD interface. Padlock indicates the closure of the outer “gate” of the transporter.</figcaption></figure>

residue polypeptide, containing an N-terminal signal sequence (leader peptide), which is cleaved by the C39 domain of the PCAT1, to produce a mature, transport-competent cargo of 66 residues²⁷³,⁵⁰⁵ (Figure 14).

Each PCAT1 protomer consists of an N-terminal C39-domain, a transmembrane domain (TMD) containing 6 TM helices, and a C-terminal NBD, containing the conserved signature sequence-motifs for ATP-binding and hydrolysis.²⁷³ The ATPase activity of PCAT1 was found to be low and rather insensitive to the presence of substrate, while its peptidase activity was found to be negatively impacted by the addition of ATP (ATPγS). Furthermore, at the same enzyme/substrate ratio, the isolated C39-domain appeared to cleave the substrate 80% more slowly than the nucleotide-free full-length PCAT1, while the addition of ATP inhibited the proteolytic activity of the full-length PCAT1 by 90%, to a level comparable with the isolated C39-domain.²⁷³

The structure of the PCAT1 shows that the two TMDs from the functional dimer form a large α-helical barrel, with a rhomboidal cross-section and an area of approximately 440 Å² traversing nearly the entire lipid bilayer. Its size is large enough to allow for a small protein cargo such as CtA to be accommodated even in a folded state. Consistent with that, the inside of the translocation pathway is lined with charged residues creating a *hydrophilic* environment for the cargo protein.²⁷³ The two C39 cysteine-protease domains are positioned at the opposite ends of the helical-core at the entryway of the TM-barrel and are loosely associated with TM3, TM4, and TM6 and make weak contacts with the NBDs (Figure 12B, D and Figure 14).

The apo-structure of PCAT1, obtained in the absence of ATP, reveals an inward facing (IF) conformation, with its two NBDs in a loosely packed dimer primed for ATP binding, while the cargo-binding cavity is open to the cytosol and accessible to the cargo at the exact site where the C39-peptidase domain is docked.²⁷³ The structure of the catalytically inactive E648Q PCAT mutant bound to ATPγS, reported by the same group, shows an occluded TMD cavity and

presents a supposed catalytic intermediate highlighting the conformational changes associated with ATP-binding. It reveals rearrangements of the TMDs that involve the movement of TM3 and TM6 toward the center of the α-barrel, resulting in disruption of the binding interfaces of the C39-domains and their displacement and disengagement from the translocation pathway.²⁷³ Importantly, the PCAT1 has been shown to be proteolytically active in the absence of ATP, suggesting that in the apo-conformation, while the C-terminal region of the substrate is threaded into the translocation pathway, the N-terminal leader peptide may remain associated with the C39-peptidase domain. Such orientation of the substrate is speculated to be optimal for proteolytic cleavage to free the cargo from the leader peptide. Once cleavage has taken place, ATP binding would induce closure of the cytoplasmic opening and dissociation of the peptidase domains. Consistent with such an interpretation, the lower protease activity in the ATP-bound form, in which the C39-domains are disengaged from the translocation pathway, could prevent cleavage of the substrate when it is not in position for translocation.²⁷³

### 5.6.8. Models of the Mechanism of Cargo Transport of the PCAT Type ATPases.
The cryo-EM structure of the PCAT1–CtA complex, solved to 3.4 Å resolution, reveals two copies of CtAs to be bound to the transporter C39-domains via their N-terminal leader peptides, but only one of these presents density extending into the TMD cavity of the transporter and hence is positioned for productive cleavage and translocation⁵⁰⁵ (Figure 14A, B, D). This structure has provided a possible mechanistic model of the complex assembly and cargo processing, which is consistent with the general “alternating access” allosteric model of ABC transporter action,³⁹⁰ exemplified by canonical type IV ABC transporters such as P-glycoprotein (ABCB1),⁴⁶⁵ TM287/288,⁴⁷⁶ and MsbA.⁴⁶⁴

In the model proposed by Kieuvongngam et al.,⁵⁰⁵ the substrate cargo-protein is recognized by the C39-domain of the transporter via specific interaction with its leader sequence with the transporter being present in an inward facing conformation. This allows the cargo-portion of the CtA to
get recruited into the transporter cavity, following which the cleavage of the signal occurs, possibly concomitantly with the binding of the ATP, which causes the outward-facing conformation of the transporter to be stabilized, allowing the now cleaved cargo to leave the cavity. Following cargo release, the TMDs isomerize to create an “occluded form” of the cavity, which is sealed from both sides. Subsequent ATP-hydrolysis resets the transporter conformation to inward-facing (IF), releasing the prebound signal peptides and allowing the C39-domain to redock at the TMD–NBD interface where it can recruit another cargo.

Importantly, this newly proposed model⁵⁰⁵ implies that the substrate specificity of the transporter is conferred primarily, if not exclusively, by the interaction of the C39-domains with the leader peptide, and the transporter TMD essentially does not otherwise interact with the cargo. In that, the model of Kieuvongngam et al.⁵⁰⁵ differs from the ones described for other classical type IV ABC exporters, such as P-glycoprotein (Pgp/ABCB1), which show that the TMDs of the transporter clearly interact with their cargoes, e.g. as evidenced by the substate-bound structure of Pgp.⁵²¹ It has to be noted that the model proposed by Kieuvongngam et al.⁵⁰⁵ has also been contradicted by the recent findings of Rahman and McHaourab,⁵²² who used spectroscopic detection of the fluorescent (bimane) and spin-labeled protein substrate of the PCAT1 as it passes through the transporter. These results suggested that the cargo protein binds specifically to the core of the transporter in the inward-facing (IF) conformation and is modulated by ATP binding. This resulted in a different, “cargo-centric” model of transport by PCAT1,⁵²² in which the recognition and binding of the leader peptide of the cargo-protein by the C39-domain enables interactions of the transported fragment of the cargo protein with the TMD of the transporter, which is subsequent to cleavage of the leader peptide. Importantly, binding of the cargo to the TMD was reportedly detected in transporter variants lacking C39 proteinase domains, suggesting that while the C39-domain facilitates cargo recognition and orientation the interaction of the cargo with the TMD is the main contributor to affinity. This alternative model of transport suggests that there is an ATP-dependent equilibrium binding of the cargo to the TMB domain, which is speculated to transition to an outward-facing (OF) conformation of the TMB leading to a release of the cargo.

We have attempted to summarize the above data and the proposed models into a noncontradictory general model for PCAT action below, which unites both viewpoints and also takes into account the possible role of the extracellular gate closure below (Figure 16).

The actual role and timing of the ATP-hydrolysis within the functional cycle of the ABC transporters remains an open question, with significant spontaneous conformational transitions from IF-to-OF being observed via an occluded intermediate, e.g., in unbiased molecular dynamics simulations.⁴⁷⁶ Recent data derived from the heterodimeric ABC exporter TM287/288 from the thermophilic bacterium *Thermotoga maritima* suggests that ATP binding alone is sufficient for the IF-OF conversion to occur, which is presumably associated with the active transport of one substrate molecule.⁴⁷⁶ Directionality of transport is suggested to be achieved by an affinity switch of the substrate binding site, which unavoidably undergoes a conformational transition due to rearrangements of TM-helices during the switch from

an IF to an OF conformation. Using a synthetic single domain antibody as conformational probe and a combination of spin-label, it was shown that the ATP-occlusion firmly traps the transporter in the OF state, preventing transporter cycling.⁴⁷⁶ They further demonstrate that efficient extracellular gate closure, which is based on interactions of charged residues from TM1s of the opposing protomers, is required to dissociate the NBD dimer after ATP-hydrolysis to reset the transporter back to its inward-facing state. In that, the ATP-hydrolysis appears to be strictly required to initiate dissociation of the closed NBD dimer, and once the ATPs are hydrolyzed the mechanical force of this firmly sealed extracellular gate (termed closed D-lock) is required to dissociate the NBDs.⁴⁷⁶

In the extreme thermophile Gram-negative bacterium *Thermus themophilus*, only a single T1SS has been identified, which is based around the heterodimeric *Thermus thermophilus* multidrug resistance proteins A and B (TmrAB), in which the TmrB component has a degenerate NBD-binding site.⁴⁷⁸ E523 from the NBD of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding residue in TmrB (D500) was found to play only a regulatory role.⁴⁷⁸ Swap of active residues between the NBDs did not result in rescue of the function, suggesting a built-in asymmetry of the complex. The prevalence of heterodimeric ABC half-transporters, especially among eukaryotic organisms, suggests an underlying functional principle of NBD asymmetry. TmrAB not only shares structural homology with the antigen translocation complex TAP but is also able to complement antigen processing in human TAP-deficient cells.⁵²³ Recent cryo-EM structures of the TmrAB in different states and conformations,⁴⁷⁹ which could be attributed to two inward-facing (IF), four outward-facing (OF), and two asymmetric posthydrolysis states, have provided significant insight into the general conformational cycling of the ABC transporters. Using single-turnover assays, Stefan et al.⁵²⁴ have been able to further analyze the cycle of the TmrAB reconstituted in lipid nanodiscs at single-liposome resolution. Strikingly, the analysis of the data suggests that a single conformational switch by ATP-binding drives unidirectional substrate translocation. Under this new model, the ATP-binding initiates the IF-to-OF transition and is stoichiometrically coupled to substrate translocation in a single step. Along with this “power stroke” caused by occlusion of two ATP molecules, the substrate-binding site is being remodeled, priming substrate release on the extracellular site. Following this “power stroke”, ATP-hydrolysis and subsequent phosphate release launch the conformational transition of OF-to-IF and hence the return to the resting state, which facilitates nucleotide exchange and thus in turn primes a new round of substrate binding and translocation.⁵²⁴ This model contrasts the previously proposed steady-state models, as the utilization of the single-turnover assays reveals a power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines. Furthermore, the recent structure of the lipid-linked oligosaccharide (LLO)-flippase PglK essential for asparagine-linked protein glycosylation in *Campylobacter jejuni*, in the outward facing conformation, strongly suggests that the release of LLO to the outside occurs before ATP-hydrolysis and is followed by the closing of the periplasmic cavity of PglK.⁵²⁵

While further details are needed to fully populate the conformational cycles of the ABC transporters, these results strongly suggest that ATP-hydrolysis is required to drive the