<figure><img src="image_59.png" /><figcaption>**Figure 45.** Conformational cycling of AcrB/MexB protomer and trimer in dependence of PAP engagement and disengagement. The tree panels present different aspects of the interaction focusing on the PAP-OMF (top) and PAP-RND (middle and bottom). In the *upper panel* the engagement state of the periplasmic adaptor protein conformer 2 (PAP2) is indicated based on the studies by Tsutsumi et al.<sup>306</sup> and Glavier et al.<sup>270</sup> Moreover, as an extension of the previous models,<sup>288,318,335,807</sup> the closed state structure (denoted “C”) found by Glavier et al.<sup>270</sup> is implemented, as well as the structural interpretation by Glavier et al.<sup>270</sup> concerning the engaging role of PAP2 at the AcrB/MexB interprotomer interface. In the *middle panel*, the conformational cycling of a single RND protomer is shown (L-protomer, blue; T, yellow; C, gray; O, red). Whereas all PAPs (PAP1, PAP2) form a stable hexameric arrangement via their β-barrel and lipoyl domains, the membrane proximal domains (MPDs) are anticipated to be in a disengaged or engaged state. The binding of the MPD-domain of the PAP2 is suggested to take place at the interprotomer interface of the RND. The interprotomer crevice is closed between L and L (LL) as well as in LT and OL. Between TT this interface crevice is widened, and in TO, the gap is large. In the *lower panel* the conformational state of the protomer shown in the *upper panel* is given in relation to the states of the other protomers in the trimer (“wheel” representation). The role of the PAP2 MPD engagement is postulated to be supportive for the direction of the cycling (L → T → O → L), preventing the backward sliding of the O-to-T state by acting as a molecular latch. The complete conversion from L to O in the presence of substrate (S) including the protonation of the TMD and the release of the substrate from the O protomer is suggested to be PMF-independent and might explain why the LTO conformations are observed in isolated protein samples upon structural analysis. The return from the O state to the L state, however, is a PMF-dependent step in analogy with the ABC transporter cycle shown in Figure 16. FD, funnel domain; PN1, PN2, PC1, and PC2 are the subdomains of the AcrB/MexB porter domain; TM2 and TM8 are the transducing helices coupling energy transduction from the porter domain to the TMD and vice versa); TMD, transmembrane domain.</figcaption></figure>

“latch” or “ratchet” to prevent backsliding of the conformations during cycling, thus stabilizing the metastable form C/O conformer and allowing the exit of the transported cargo.

We also suggest a two-way communication between the RND transporter and the PAPs. The first stage is an upward signaling from the RND toward the OMF via PAP1, forming the molecular basis for OMF recruitment, which in turn enables the second, downward signaling stage, which commences with the association of the PAP2 with the OMF, resulting in the stabilization of its dilated aperture. This translates into a rearrangement of the α-helical hairpins of the PAP2 sealing the PAP-OMF drug-export conduit and is communicated back to the RND via allosteric transitions within the PAP2 allowing the MP-domain to slot into the T/C interprotomer crevice, that ultimately leads to movement of the “helix-gate” in PN1 (see Figure 43). This latter retrograde communication would correspond to the “activation signal” described as a switch from LTC to LTO in the cryo-EM structure of MexAB OprM.<sup>270</sup>

A summary of the key points of the model is presented in Figure 45, which provides three slices focusing on the PAP-OMF interface (top), the transporter-PAP interface from a side-view (middle), and a top-view rotary diagram (lower panel), taking account of the substrate and proton occupancy.

Under this model, the two PAPs within the RND assembly have clearly delineated and distinct functional roles. PAP1 is primarily associated with the first phase of the efflux cycle and is involved in presentation and feeding of the cargo to the transporter's proximal binding pocket and sensing the successful transition of the drug onto the deep binding pocket, which is associated with the L-to-T transition, while PAP2 is predominantly associated with the second phase, namely detection of the successful docking of the OMF and vetting the release of the drug toward the OMF-funnel by facilitating/stabilizing the T-to-C transition and actively enabling the C-to-O switch, which leads to productive efflux of the drug. The first function requires a close association of the transporter-PAP, as the conformational change associated with the drug-binding and L-to-T switch needs to be communicated “upward” toward
<figure><img src="image_60.png" /><figcaption>**Figure 46.** Proposed model for concerted tripartite assembly along sectional views of the OMF-PAP-RND interaction surface. RND protomers are colored according to their adopted conformation (Loose in blue, Tight in yellow, Closed in gray and Open in red). Six PAPs protomers bind as pairs to the RND trimer. The resting, closed OMF trimer interacts with the PAP hexamer (one pair of PAPs is omitted for clarity). Upon interaction with the OMF periplasmic end, PAP1 destabilizes the lock anchoring the H7–H8 helices to the static H3–H4 helices, resulting in a spontaneous relaxation of the superhelical trajectories of H7–H8 helices as depicted by arrows. Engagement with the OMF is communicated down to the RND via PAP2, allowing the positioning of the MP-domain at the T/C crevice (indicated by the pink arrow), which in turn results in a rearrangement in the underlying PN1 domain leading to the opening the helix gate, denoted by the rotation of the flap symbol, thereby enabling the Closed to Open transition of the protomer.</figcaption></figure>

the OMF, via reorganization of the β-barrel and lipoyl domains, leading to the stitching-up of the helical-hairpin assembly to form a tube and recruit the OMF, which in turn enables the OMF opening and stabilization of the OMF-PAP assembly by PAP2 by completing the interlocking of their respective helical hairpins as suggested by Tsutsumi et al.<sup>306</sup> Indeed, studies of binding of hairpins in isolation to the WT OMF MtrE vs mutationally stabilized open state (MtrE E434 K) indicated approximately 100-fold increase in affinity of the interaction.<sup>719</sup> The significant realignment and tightening of the helical bundle of the PAPs upon OMF binding is associated with a subsequent compression of the lipoyl and β-barrel domain gaskets by ~10 Å completing the formation and the sealing of the tubular conduit.<sup>287,305</sup> The conformational trigger from this compression and realignment is propagated through the PAP2 enabling its MPD to engage with the DN/PN2 of the T-state RND protomer.

Via these protomer-specific interactions, PAP1 and PAP2 provide outward and inward communication from the RND and from the OMF, respectively, utilizing their conformational changes for coupling remote allosteric sites, which are consistent with the models suggested previously.<sup>270,305,306,698,757,758</sup> Intriguingly, we have identified significant differences between the different RND-conformers in the engagement of the DC domain's β-hairpins with the PAP2 (box 6) in the MexAB-trio structures (PBD ID 6TA6),<sup>270</sup> specifically involving the MexB R764 and the main chain of the PAP D278. This interaction may provide an additional level of allosteric communication, as the critically important Y757, which forms part of the MexB drug-exit gate, is located at the base of the same β-hairpin that provides the PAP2-attachment point, and conformational changes as a result of PAP2 binding may be communicated via it to the drug exit gate. Furthermore, the box6-interaction mediated by the PAP2 R277 and the RND transporter E244, which maps to the short helix at the base of the DN-domain hairpin, may convey conformational changes from one RND protomer to the other. While the significance of these interactions needs to be

investigated further, they could help explain the C to O transition trigger and forward communication between the PAP2 and subsequent RND-protomer. It is therefore tempting to suggest that the hexameric PAP β-barrel ring not only provides increased processivity but also enables the one-way PAP2 “latch system”, which in turn gives directionality to the cycle. This system can elegantly explain the lack of cooperativity or directionality observed in individual transporters.<sup>299</sup>

In respect to the PAP-OMF interface our model allows for the first time to tentatively assign a specific role to each PAP conformer within the cycle. As described above in the closed, resting state of the OMF the coiled-coil α-helical hairpins at its tip are stabilized by a network of predominantly ionic interactions, aka “primary gates”, that keep the inner coiled-coil helices (H7/H8) in a tensed supercoiled state relative to the relaxed superhelical trajectories of the outer coiled-coil helices (H3/H4). The disruption of the “primary gates” has been demonstrated to result in a spontaneous relaxation of the super helical trajectories of the “mobile” inner (H7/H8) helices relative to the “static” H3/H4 helices, resulting in dilation of the OMF aperture following an “iris-like” mechanism.<sup>687–689</sup> This interaction is induced by the PAP-OMF interaction and does not require energy input from the transporter.<sup>720</sup> Analysis of the positions of the PAP1 and PAP2 in the zero-degree structures of MexAB-OprM (PDB ID 6IOK,<sup>306</sup> 6TA6)<sup>270</sup> and the corresponding AcrAB-TolC<sup>305</sup> suggests that PAP2 is the main interacting conformer with the OMF-R2 (H7/H8) motif, which is consistent with it being the final recipient (and stabilizer) of its relaxed helical trajectory, the release of which could plausibly be deduced to be triggered by an initial docking of the PAP1. While in the case of MexA and AcrA it is difficult to assign a specific role to each PAP due to their ultimate interchangeability and the quasi-equivalence of the 60° structures, the roles of PAP1 and PAP2 can be established to be distinctly different.

On the example of the TriABC-OpmH system it has been established that PAP1 (TriA) is detrimental to recruitment of
<figure><img src="image_61.png" /><figcaption>**Figure 47.** Qualitative energy diagram of the assembly and cycling of the RND-tripartite assemblies taking into account the role of the PAPs and the newly discovered C-to-O transitions. The symmetrical LLL state is suggested to be a metastable high-energy state of the RND trimer and is prone to spontaneous collapse upon substrate binding. The L to C transitions are suggested to be driven solely by the binding energy between the components and can occur in isolated RND transporters, as witnessed, e.g., by the “mono” structures of MexB.²⁷⁰ The C-to-O transition does not happen spontaneously, suggesting that the C-to-O is an upper energy transition and is offset by the binding of the PAP2 MPD (represented by the lower part of the magenta protomer being colored in orange). PAP2 MPD cycling provides directionality of the cycle and prevents backsliding of the C/O-to-T which in its absence is suggested to be in an unstable equilibrium. The energy from proton release allows for the upper-energy level O-to-L conformational transition, which enables repeat of the cycle.</figcaption></figure>

the OMF and hence likely makes first contact with it, which is supported by mutagenesis,⁸⁰²,⁸⁰³ while the MPD of the PAP2 (TriB) is responsible for the activation of the transporter, which, in light of the latest findings of Glavier et al.,²⁷⁰ is likely mediated via the conformational changes that propagate to the “exit-gate”.

We therefore propose that the PAP1 plays the primary role in sensing the drug-bound state T-state of the transporter and the recruitment of the OMF. Due to the differing helical trajectories of the R1 (H3/H4-associated) and R2 (H7/H8-associated) motifs of the OMF, the primary interaction of PAP1 (based on the TriA data) is suggested to be the R1-motif, although in single-PAP assemblies such as MexAB and AcrAB the interface is quasi-6-fold symmetrized, and 60° rotation of the OMF may possibly produce functional complexes as suggested by the existence of the alternative structures (e.g., PDB ID 6IOL,³⁰⁶ 6TA5²⁷⁰). In the case of a binary AcrAB complex which is transitioning from an LLL state, the drug binding and conversion on L to T would serve as an allosteric signal from PAP1 toward the OMF enabling its attachment.

The initial docking of the PAP1 to the H3/H4 helices of the OMF, which serves as an upward communication signal, triggers at least partial release of the H7/H8-helices, possibly by attacking the interprotomer H8-H4 attachment gate, the full relaxation of which is achieved by breakage of the intraprotomer H7-H4 gate (containing a conserved Y-residue, e.g., Y404 OprM or Y362 TolC) upon subsequent PAP2 docking (see Figure 42D, E and Figure 46). The relaxed

trajectory of H7/H8 is then stabilized by the PAP2 which ends up being attached to the tip of H7-H8 (R2 HTH-motif) with its own RLS-motif, as seen in the recently assigned 0-degree OMF-structures (PDB ID 6I0K,³⁰⁶ 6TA6²⁷⁰) (see Figure 42B and Figure 46). Indeed, the analysis of the tip-to-tip interactions and the possible starting and finishing trajectories of the H7/H8 helices as seen in the 0-degree structures suggests that these are the only arrangements which are compatible with the cross-linking data presented by Ntreh et al.,⁸⁰³ strongly indicating that TriB binds in a compatible fashion to the one presented by PAP2.

## 8.7. RND-PAP Interface and Energetic Considerations

Bridging of the RND transporter and the OMF by the PAP does not appear to require energy as the stable complex assembly can be achieved *in vitro*. Bipartite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to copurify MexB or OprM.⁷⁰⁴ Furthermore, binary interactions between purified proteins probed by surface plasmon resonance (SPR) also suggest a sequential assembly of the RND complex in the absence of energy input,⁶⁹⁷,⁶⁹⁸ with PAP-RND subcomplexes creating a tight formation which appears to be independent of substrate presence. Consistent with this, pump substrate novobiocin and a broad-spectrum pump inhibitor Phe-Arg-β-naphthylamide (PAβN; MC-207,110) were able to bind to the immobilized AcrB but did not affect interactions between the components of the complex.⁶⁹⁸ Crucially, the pump assembly, including the recruitment of the OMF channel, and its opening were found
to be energy independent *in vivo*<sup>719,720</sup> as well. The energy input from cytoplasmic proton release is likely required for resetting the protomer to the initial L-state as evidenced from both *in vivo*<sup>720</sup> and in an isolated controlled system *in vitro*.<sup>705</sup> Based on the above considerations we postulate that the process from the apo-state (L), over drug-bound state (T), to the release of the drug in the O-state is PMF-independent (Figure 47).

Thus, the only energy involved is the binding of the substrate, plus the binding energy of PAP-association, alongside with and the binding of proton(s) to the transporter, with its occurrence in dependence of their concentrations present in the periplasm. On the other hand, the “reset” of the O conformation into the L conformation is suggested to be PMF-dependent. This might explain why the LTO con-formations are observed in isolated protein samples upon structural analysis. The reset of the structure from the O-state to the L-state is suggested to take place upon the substrate release from the drug-binding pocket and the associated release of the proton from D407 and/or D408, which causes a large conformational transition.<sup>352</sup> Upon proton release, the O conformation returns to the L conformation and the TO interface changes into a TL interface which closes the gap between the protomers, and rearrangement of the RND-surface leads to the disengagement of the PAP2 MPD domain.

Our model suggests that the LLL state of the pump is metastable and does not require additional input of energy to undergo L to T transition other than that provided by the substrate binding. Concomitantly, the allosteric transitions associated with the L-to-T transition upon the substrate binding are transmitted to PAP1, which initiates OMF-recruitment (Figure 47).

The binding of the substrate opens the proton access channel in the T-conformer, and the occupation of the proton relay causes the T to C conversion (as demonstrated by a recent MD study).<sup>808</sup> This in turn recruits the PAP2 MP-domain to the interprotomer groove, which by acting as a “door stop” or “latch” stabilizes the C-state, preventing backsliding to the T-conformation, and enables the C-to-O transition. While the occupation of the proton relay facilitates this C-to-O transition *in vivo*, it can be achieved in the absence of PMF in the isolated transporters. Indeed, the energy barrier of the T-to-C transition appears to be relatively small, as additional binding energy provided by the interaction of the MBX3132 inhibitor over native substrate appears to be sufficient to overcome it and stabilize the pump in a quasi-symmetric TTT-state, as seen in the PDB ID 5NGS.<sup>305</sup> The C-to-O step does not occur spontaneously, and in isolation it is likely to represent a higher energy step, which is offset by the binding energy provided from the allosteric association of the MPD of PAP2 with the RND-protomer as suggested by Glavier et al.<sup>809</sup> In agreement with that a peak force of ~8 kcal/(mol Å) was required for the indole to overcome the closed gate barrier,<sup>808</sup> while a smooth energy profile is seen when the gate is open.<sup>810</sup>

In the next step of the cycle the substrate release from C- to the O-state causes the exit gate to collapse, leading to the TM-conformational transition that leads to release of the protons on the cytoplasmic side, which also causes the consumption of the proton gradient. It has to be noted that the MexB-solo incorporated in liposomes<sup>270</sup> in the presence of its substrate Meropenem was not able to achieve any proton transport, which is consistent with the requirement of PAP2 association

for proton release. The collapse of the exit gate, which initiates the O-to-L transition, is also translated to the change of the surface of the PN2 subdomain of the RND-protomer, which lowers the binding affinity of PAP2 to it, leading to its release. Thus, substrate release is sufficient to disengage the molecular “latch” that is the MPD of PAP2, the release of which causes the proton release on the cytoplasmic side, closing the cytoplasmic access (Figure 45, Figure 46, and Figure 47).

The preference of the MP-domain of PAP2 to the substrate-bound T-protomer provides a straightforward control of the pump activity in response to the substrate concentration. However, in the discussed scenario this on-off cycle of PAP2 MPDs is enabled by the β-barrel ring that keeps the tripartite pump assembled through repeated cycles. However, a complete disengagement of the PAPs might be required under certain conditions. It has been previously suggested that a drop in the drug concentrations in the periplasm may drive the adoption of a symmetric LLL-state of the AcrB trimer,<sup>807</sup> which under the current model (Figure 45) could shift the equilibrium toward pump disassembly. While future research will need to provide a definitive answer to the actual mechanism of disassembly, the following findings may provide circumstantial evidence. The PMF is linked to the pH levels in the periplasm,<sup>811</sup> and lower periplasmic pH at ~6.0, which is associated with high PMF, was found to favor pump assembly, while elevated pH at 7.5, corresponding to the decreased PMF, promotes pump disassembly in a PAP-dependent fashion.<sup>698,757,758</sup> Similarly, Ip et al.<sup>812</sup> using site-directed spin-labeling EPR demonstrated that AcrA undergoes not just significant pH-induced conformational transitions but also oligomerization. Thus, the PAPs may also play the role of a PMF-sensor for the assembly.

Such interpretation is consistent with the observation of two-liposome reconstituted MexAB-OprM complexes, where substrate transport was associated with a rapid PMF-consumption<sup>813</sup> followed by a subsequent dissociation of the MexAB and TolC complex.<sup>705</sup> Furthermore, in *E. coli*, the rapid spiking (~1 Hz) of the PMF coincided with the efflux of the potential AcrB substrate tetramethyl rhodamine methyl-ester (TMRM)<sup>814</sup> allowing the suggestion that the transient AcrAB–TolC complex operates in a discontinuous fashion.<sup>807</sup>

In summary, while further functional validation is required to fully confirm the roles of PAP1 and PAP2 at the level of specific OMF-gate opening, this model provides a non-controversial unification of both PAP-OMF and PAP-RND interfaces, allowing us to rationalize the available data, and furthermore provides a testable platform for future studies that we believe will benefit the wider community in the field.

## 8.8. Tripartite Complexes of HME-RND Family

The active role of PAPs in the efflux pump assembly has also been demonstrated in the HME-RND transporters, which are closely homologous to the drug-efflux associated HAE-1 family discussed above and are thus anticipated to form similar tripartite assemblies.

One of two main branches within the HME-family specializes in pumping out divalent ions, e.g. CzcABC, CznABC, and NccCBA involved in Cd<sup>2+</sup>, Zn<sup>2+</sup> and Ni<sup>2+</sup> and Cd<sup>2+</sup>, Co<sup>2+</sup> and Ni<sup>2+</sup> detoxification respectively<sup>45,67,815,816</sup> and with ZneCAB from *Cupriavidus metallidurans* CH34 being a relatively well-studied system.<sup>46,358</sup> In contrast to the HAE-1 RND pumps discussed above, the ZneA transporter does not appear to undergo dramatic LTO conformational transitions,
<figure><img src="image_62.png" /><figcaption>**Figure 48.** Comparison of the binary CuSBA complex and MexAB-trio suggests a possible pathway of conformational reorganization of the PAPs upon engagement with the OMF. (A) Crystal structure of the CuSBA subcomplex as seen in the PDB ID next to the cryo-EM MexAB-OprM structure (PDB ID 6TA6), showing significantly different orientations of the helical hairpins and the difference in packing lipoyl domains of their PAPs. Also, while PAP1 conformer binds at the approximately equivalent interface of the RND surface, the PAP2 conformer, and particularly its MPD domain, appears divergent. (B) Several snapshots from a molecular morphing movie illustrating a possible transition from the OMF-free state to the OMF-engaged state, suggesting possible reorganization of alignment of the helical bundle between the two PAP forms (shown in yellow arrows), as well as the proposed dislocation of the PAP2 MPD within HAE1-systems, which may reflect the engagement of PAP2 in the apo- and OMF-engaged state of the complex.</figcaption></figure>

which can be expected as its ion-cargoes do not require such major reorganizations in order to pass through. There is also a strong indication that unlike other RND transporters discussed here, it may actually be capable of moving the Zn²⁺ from the cytoplasm directly to the extracellular space, in an electrogenic fashion suggesting a proton to Zn²⁺ ratio of greater than 2:1.³⁵⁸ However, little is currently known of the higher order complexes with its participation, although, as mentioned earlier, its cognate PAP ZneB presents an architecture very similar to the PAPs associated with the HAE-1 type RND transporters. ZneB, however, has a well-defined specific metal-binding site, and the crystal structure revealed Zn²⁺-ion coordinated by H220, H284, E328, and a water molecule at the interface between the β-barrel and MP-domain,⁴⁶ yet again highlighting the important substrate-binding role for the PAPs.

The active role of PAPs in the assembly of the tripartite efflux complexes is probably most clearly demonstrated on the example of the Cu⁺/Ag⁺ tripartite transporter CusCBA,³⁵⁹,⁷⁰² where CusA is an RND-pump belonging to the HME-family, CusB is a PAP, and CusC is an OMF.⁸¹⁷ The system is complemented by a periplasmic chaperone, CusF (which is homologous to the C-terminal domain in SilB),⁷⁶⁴ whose role is to transfer the metal ions from the transmembrane ATPase CopA to CusB.⁸¹⁸ This CusB–CusF interaction functions as a switch for the entire Cus-efflux system and facilitates the transfer of copper to the CusA component.³⁶⁰ Binding of

copper is coordinated by a relay of conserved methionine residues, which stretch from the membrane-proximal domain to the helical hairpin of CusB, and importantly, binding to these sites causes CusB to undergo substrate-linked conformational changes.⁷⁶⁸ Electron paramagnetic resonance (EPR) distance measurements revealed that CusB exists as a dimer in solution, which undergoes major structural changes associated with Cu⁺ binding,⁸¹⁹ resulting in a compactification of the apo-structure, to produce a “holo-CusB”. Furthermore, CusB has been demonstrated not just to be the key mental sensing element in the pump but to actively drive pump its assembly in response to metal stress.⁸²⁰ CusB switches the pump on, once excess metal ions are detected, but once the periplasmic ion-concentration goes down, CusB transfers the bound ions back to the periplasmic chaperone CusF, leading to deactivation of the pump and maintaining copper homeostasis.⁸²⁰ Thus, through its interplay CusF, CusB not only plays a central role in substrate binding and pump activation but also functions as a metal sensor controlling the whole system and is directly responsible for complex assembly.

## 8.9. Could the Structure of the Metal Pump CusBA Suggest a Conformation for a Pre-engagement Apo-State for the Transporter–PAP Complex?

Crystal structures of the binary CuSBA complex³⁵⁹ revealed a hexameric assembly of the PAP forming the typical β-barrel and lipoyl domain gaskets on the top of the transporter;
however, CusB forms a clearly delineated trimer of dimers, with each pair being relatively loosely associated with each other. Notably, PAP2 is fully engaged and resolved in the structure, and the electron density is particularly good around the N-terminal segment of PAP2 which appears to be in contact with PAP1 (PDB ID 4DNT,⁶¹⁰ 3NE5³⁵⁹).

The positions of the different PAP protomers, and especially their respective MPDs, are diverging from the one seen in the cryo-EM structures of the HAE-1 assemblies. The orientation of the MPD domain of PAP2 in the CusBA complex is notably different from that in PAP2 seen in HAE-1 assemblies, where it is firmly associated with the interprotomer crevasse and PN2/ DN domains of the RND protomer. The PAP2 features an extended N-terminal tail (in PDB ID 4DNT it is resolved from residue 79 to 96), making extensive contact with the surface of the RND transporter, engaging with the PC1 and PN2 domain of the protomer in an unfolded state. Notably this N-terminal tail of PAP2 also contacts PAP1 directly, making specific contact with the equivalent N-terminal tail of PAP1, which however presents a strikingly different configuration, specifically in the contact range (residues 84–93), where if it forms an α-helix, that contrasts the unfolded region of PAP1.

Importantly, the helical trajectories of the α-hairpins of CusB are pointing away from the center of the pump axis, without participating in helical-bundle tubular formation (PBD ID 3T53).⁶¹⁰ Along with a loose association of the lipoyl domains, such a hairpin orientation of the PAP is inconsistent with sealing of the OMF-channel.

While this may be explained away by functional differences between transporter types, several points suggest that the conformation trapped by the crystal structure of the CusBA complex is one of a pre-engagement LLL, and indeed the complex is trapped in a symmetrized conformation in all the available structures, even those which are supposedly in an extrusion state (e.g., PBD ID 3T53).⁶¹⁰ This symmetrized, pre-engagement state may explain the hairpin domain orientations, as after all, effective sealing of the gasket between the OMF and the short hairpins of the CusB is even more important as it only expels Cu(I) ions, and any leakiness would be nonpermissible.

A speculative morph of the PAP1/PAP2 transitions between the ones observed in the CusBA complex and the MexAB trio complex²⁷⁰ is provided in Figure 48, and the associated video can be found as Supporting Information. The morph highlights the realignment of the hairpin vectors upon engagement of the incoming OMF (shown as yellow arrows), allowing effective sealing of the efflux conduit. Second, the discrepancy of the position of the MPD domains of the respective PAP2 protomers between the HME (left) and HAE1 (right) systems is suggestive of possible structural changes during the peristaltic cycling experienced by the HAE1 transporters, which appears to be lacking in the HME family. It has to be noted that there is no experimental suggestion of unfolding of the hairpin of the HME-PAPs during such a cycle and that a local variation of the height of the periplasmic space could not be excluded.

## 8.10. Additional Level of Regulation of RND Pumps May Be Provided by the Transmembrane Modulators of the AcrZ-Family

A fourth transmembrane component is sometimes present within the RND complexes, e.g., YajC²⁸³ or AcrZ.⁸²¹ These small proteins are entirely α-helical and bind the transporter

within the inner membrane, packing against the TM-helix bundle (Figure 49) in a fashion similar to the γ-subunit of the

<figure><img src="image_63.png" /><figcaption>**Figure 49.** AcrBZ co-structure. The asymmetric AcrB trimer (gray cartoon) with the three protomers in the L (loose), T (tight), and O (open) conformations. The auxiliary transmembrane modulator AcrZ (raspberry cartoon) is located at the surface periphery on each of the AcrB protomers at the transmembrane domain. The X-ray structure display (PDB: 5NC5) was solved in complex with DARPins (not shown) and puromycin (PUY, orange spheres).</figcaption></figure>

SecY-complex.²⁸³,²⁸⁷,³⁰⁵ These proteins appear to be non-essential, playing a modulatory role, possibly changing the efflux profile of the pump in response to environmental stimuli.²⁸³,⁸²¹ Recent cryo-EM structures of the AcrBZ-complex in lipid environments and MD studies indicate that the lipid environment affects the packing of the AcrZ on AcrB, resulting, e.g., in preferential interaction of the cardiolipin with AcrBZ. The removal of AcrZ in combination with cardiolipin deficiency increases the bacterial sensitivity to chloramphenicol,⁸²² suggesting that the combination of AcrZ and the lipid environment works synergistically to allosterically modulate AcrB activity. Furthermore, the interaction has also been seen to differ between the different conformer states of the RND transporter. The role of these small proteins remains unclear, as they are nonessential and are lacking in most studied RND systems, including the MexAB-OprM.

## 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

Understanding of the molecular basis of pump function and especially the finer modulation and regulatory aspects of the RND tripartite systems requires the use of membrane mimics that enable their characterization in physiologically relevant
environments. Methodologies for the structural study of
detergent-free membrane protein stabilization involve scaffold-
ing of membrane fragments around the proteins of interest
using engineered lipid-avid peptides (nanodiscs), fatty amines,
or styrene-grafted amphipathic polymers (amphipols or
SMALPs, respectively).⁸²³⁻⁸²⁵ All these chemical tools allow
for a more physiological environment around the protein.
Lipids have an effect on the activity of membrane proteins
because of the so-called “bulk” effect that reflects the physical
properties of the membrane: bilayer thickness, physical state,
curvature, and surface tension can significantly influence the
behavior of membrane proteins. For instance, as membrane
protein secondary transporters exploit the electrochemical
membrane potential to energize the transport of their
substrates, the composition of the membrane is obviously
crucial. In addition, there are also increasing clues for specific
phospholipid-protein interactions where bound lipids act as
chemical partners with a prominent role of the lipid
headgroup. Overall, the effect of the membrane composition
on the function of bacterial efflux pumps has never been
systematically explored, but emerging evidence that it plays a
significant role is described below. A major drawback is the use
of detergents, and it is questionable whether the structures
determined in such an environment are representative of what
is found in vivo. It has been suggested that in some cases, the
use of detergent can lead to artifactual structures and to their
misinterpretation.⁸²⁶ Youzhong Guo realized that the structure
of D407A AcrB obtained in detergent³²⁶ was significantly
different when obtained by cryo-EM after reconstitution into
polymer-wrapped lipid particles.³³⁰ Compared to the wild-type
protein, the AcrB D407A mutant in detergent shows a
significant shrinkage of the central cavity that was interpreted
as a mechanistic transport feature.³²⁶ The narrowing of the
central cavity turned out to be impossible in the presence of
lipids because of steric constraints. Following similar lines, it
was shown that the affinity of AcrB for rhodamine 6G (R6G),
determined by fluorescence polarization, is 100× higher in
SMALPs⁸²⁵ than in detergent.⁸²⁷ As further evidence for the
importance of lipids in maintaining a functional assembly, Qiu
reconstituted the AcrB transporter also in SMALPs and cryo-
EM experiments are conducted on these AcrB nanoparticles
surrounded by lipids from the in vivo *E. coli* membrane. The
resulting structures (R = 3.2 Å) show a trimer of the AcrB
transporter with additional densities in the transmembrane
part of the protein. A lipid patch within the central cavity of the
AcrB trimer and isolated lipids around the transmembrane
domains that may belong to the lipid belt were described.³³⁰
This is the first time that such a patch structure is described. It
is proposed that this lipid patch is directly involved in the
transport mechanism of AcrB by allowing the harmonization of
the trans-conformational LTO changes occurring within the
three protomers of AcrB. The membrane patch consists of 24
lipids modeled as phosphatidylethanolamine (PE),³³⁰ the
major lipid in the *E. coli* membrane.⁸²⁸ Molecular dynamics
has been performed on a membrane representative of the one
mentioned above with AcrB inserted in this membrane. At the
beginning of the simulation, the transporter only interacts with
PE molecules; the CL and PG molecules are far away from the
protein. During the simulation, the latter 2 lipids come closer
to the protein suggesting a role of PG and CL in the
stabilization of the AcrB trimer.⁸²² Thus, the membrane patch
would consist of a more complex mixture than just PEs, with
the addition of the lipids PG and CL. The role of PE was also

revealed thanks to X-ray structures of the AcrB complex in
complex with fusidic acid. Both fusidic acid and DDM were
identified at a TM1/TM2 groove of AcrB that was postulated
to be an access path for carboxylated substrates. The possible
presence of physiological phospholipids at this site was
confirmed by performing ensemble docking calculations
using structures of AcrB as well as molecular dynamic
simulations of the protein embedded in a model POPE
bilayer.³³⁴

Mass spectrometry has developed significantly over the past
decade and allows the identification of specific interactions
between a membrane protein and a phospholipid which are
now maintained in the use of this method.⁸²⁹,⁸³⁰ This
technique could be envisaged to study AcrB in SMALPs and
its lipid composition in a very precise way. The latest advances
even make it possible to study the vesicles of the inner and
outer membranes of *E. coli* and determine their protein
components and associated lipids.⁸³¹ More generally it is
believed that it is important to consider the endogenous
membranous environment as a pool of potential regulators,
local effectors at the membrane, or allosteric ligands. Of
foremost importance, AcrZ is a transmembrane protein that
was shown to potentiate the activity of AcrAB-TolC against
some antibiotics, but the molecular explanation for this effect is
lacking.

In vitro proteoliposome assays have been shown to be
excellent and tunable tools for the characterization of
transporters at the molecular level. They offer the possibility
of studying purified proteins isolated from the complexity of *in
vivo* systems, without pleiotropic and off-target effects, which
complicate measurements and interpretations from experi-
ments performed in whole cells. In addition, they allow for a
compartmentalization of the protein and hence make it
possible to tackle the difficult challenge of determining their
catalytic parameters. The path toward the successful
reconstitution of a tripartite pump is interspersed with many
pitfalls, and several laboratories have contributed to reach a
sensible framework to reach this goal. The milestones, major
results along this path, and possible improvements are
reviewed in refs 832 and 833. For a long time, protocols
were based on the reconstitution of the RND pump in the
presence or absence of its periplasmic adaptor. As major inputs
in the understanding of efflux pumps, reconstitution assays
demonstrated that the PAP is mandatory for active trans-
port³¹¹,⁷⁰⁶ probably because its presence is required to make
the substrate/proton antiport perfectly coupled.⁷⁰⁷ In vitro
studies also allowed a better understanding of the path
experienced by the substrate through the transporter³¹¹,³⁵⁴ or
the possible cross reactivity between unrelated pumps.⁷⁹⁴
Ultimately, such methodologies should eventually allow
measurement of orders of magnitude of substrate affinities
and rates of transport. In their system, Helen Zgurskaya and
Hiroshi Nikaido relied on the use of fluorescent phospholipids
which are trapped by protein-free acceptor vesicles upon
transport. When known substrates of AcrB were added to the
test buffer, the efflux of fluorescent phospholipid was inhibited.
Measurements of competition of the binding at the equilibrium
of the fluorescent lipid by various potential substrates
(antibiotics, bile salts, etc.) resulted in IC₅₀ values in the
order of ten micromolar.⁷⁰⁶ Reconstitution of another RND,
CzcA from the efflux pump CzcB(2)A, responsible for heavy
metal resistance in *Ralstonia spp.*,⁸¹⁵ yielded for the first time a
rate constant of 385 s⁻¹ and a sigmoidal kinetics with a Hill
<figure><img src="image_64.png" /><figcaption>**Figure 50.** MacAB-association highlighting the differential binding of PAP protomers based on PDB ID 5NIK.<sup>272</sup> Different PAP protomers can be named PAP1-PAP3 based on their interaction with the MacB transporter in an analogy to the RND transporter complexes. PAP1 can be seen as an intraprotomer relative to the transporter, while PAP2 interacts exclusively with the SABRE subdomain of the same transporter protomer. PAP3 appears to occupy an interprotomer position extending over the cleft between the neighboring transporter protomers. (A) Top view looking down at the interprotomer interface of MacB. (B) Side view showing the position of the β-barrel gasket of the PAP hexamer. (C) Different PAP conformers make inequivalent contacts with the crown of the transporter, and PAP3 in particular, which is interprotomer-bound, is loosely associated with the complex.</figcaption></figure>

coefficient of 2. However, caution should be taken regarding the physiological relevance of these data because the reported Michaelis–Menten constant (of 6.6 mM) seems to be abnormally high. The overall order of magnitude for the velocity of transport is a matter of heated debate: Aires and Nikaido concluded that ~0.3 protons are consumed per trimer of AcrD and per second,<sup>311</sup> and by contrast, in another study, EtBr transport by MexB gave rise to an impressive turnover rate of 500 s<sup>-1</sup> based on a number of pumps previously estimated by immunoblotting methods. In 2015, it was possible for the first time to measure *in vitro* the transport by the MexAB-OprM efflux pump of *P. aeruginosa* entirely reconstituted in proteoliposomes.<sup>813</sup> In this setup, a functional reconstitution of the MexAB–OprM system made it possible to trace the respective transport of a fluorescent substrate and protons via a pH-sensitive fluorescent reporter. This system promises insight into the largely unanswered question of H<sup>+</sup>/drug stoichiometry, substrate specificity, and measurement of the catalytic constants of the various pumps.

## 9. TRIPARTITE ASSEMBLY OF MACAB-TOLC

The lack of clear substrate membrane access channels, the very narrow TM-helical bundle, and the reported involvement in the transport of Sec-dependent peptide cargoes<sup>154,548,550</sup> strongly imply that MacB operates differently from the canonical type IV ABC transporters and floppases. The latter operate on a two-step alternate access model, providing transmembrane shuttling either from the inner-leaflet of the cytoplasmic membrane or directly from the cytoplasm,<sup>390,465,524</sup> while the cargo of the MacB/FtsX transporters is acquired from the periplasm only. In that respect there is a functional overlap of the family with that of the tripartite RND transporters, which are suggested to work as a universal

"second stage" supplementing the single-component transporters that remove the xenobiotics from the cytoplasm.<sup>834</sup>

A reconstruction of the complete *E. coli* MacAB-TolC pump produced from docking of MacB into a hybrid cryo-EM map with varying local resolution (from 3 to 8 Å), derived from the analysis of the fusion-stabilized *EcMacB-MacA* MPD-domains and of a higher resolution disulfide-stabilized MacA-TolC subcomplex, has been reported by Fitzpatrick et al.<sup>272</sup> The assembled structure revealed MacB:MacA:TolC protomers in a 2:6:3 stoichiometry consistent with the earlier biochemical and biophysical essays.<sup>573</sup> The complex presents dimensions comparable to the ones observed in the RND-based assemblies measuring some 320 Å along its long axis (membrane to membrane) to span the whole periplasm.<sup>272</sup>

The PAPs associating with the MacB-FtsX like transporters, such as MacA, similar to the PAPs of RND systems, have a strong propensity to form hexamers in isolation,<sup>759</sup> that are stable under native-MS conditions<sup>573</sup> and can be readily crystallized.<sup>571,737,746</sup> Functional studies have shown that MacA has a role in regulating the function of MacB, increasing its affinity for both erythromycin and ATP.<sup>573</sup> While the MacA–MacB complex is formed with a nanomolar affinity even in the absence of ATP, the affinity of interaction further increases in its presence,<sup>835</sup> with the PAP acting as a switch for allowing ATP-hydrolysis by MacB.<sup>836</sup>

Studies by Tikhonova et al.<sup>743</sup> demonstrated the functional dependency of MacB on MacA, showing that after reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the
<figure><img src="image_65.png" /><figcaption>**Figure 51.** Comparison of the assembly of MacAB with the MlaEF-complex reveals a common organization. Both transporter complexes are stabilized by a hexameric β-barrel gasket (colored green and red), formed of their respective periplasmic partner proteins, which, although structurally nonhomologous, provide an identical solution to binding to a dimeric transpoter interface. Left, a homology model of the MacAB subcomplex based on the PDB ID 5NIK, taking into account the transmembrane portions of the MacA which were unresolved in the cryo-EM structure, indicates that these are of sufficient length to reach the connecting helix (CoH) of MacB and could provide additional allosteric coupling across the membrane, as established for the MlaEF-D complex (on the right, experimental structure (PDB 6XBD)).</figcaption></figure>

presence of macrolides suggesting that MacA forms a
functional subunit with the MacB transporter.⁷⁴³

Notably, the lipoyl domains of the PAPs in these assemblies form tight hexameric gaskets, similar to the ones formed by the PAPs in isolation⁷³⁷ with an extended flexible loop donated from each protomer creating a “gating ring”,²⁷²,⁵⁷¹ which is suggested to prevent backflow, favoring unidirectional substrate efflux in an outward direction (Figure 35C). In the hexameric structure of *E. coli* MacA, the lipoyl domain loops (G56-L72; T199-I214) protrude into the cavity delineated by the assembled hexamer, providing a constriction of the lumen, which is narrowest at residue N209, which in the stand-alone structure (PDB ID 3FPP)⁷³⁷ is effectively sealed (Figure 35C in section 7.3).

Similarly, in the PAP Spr0693 (PDB ID 5XU0), which pairs with the Gram-positive MacB-homologue Spr0694-0695, the hexameric α-helical barrel has an inner diameter of ~31 Å. A gating ring is formed by the flexible loops from the lipoyl domains (N211–V224), with six G220 residues at the innermost site restricting the internal diameter to around ~7 Å.⁵⁷¹

The β-barrel and MP-domains of MacA mediate the interaction with the periplasmic domain of MacB, in agreement with the reported functional data⁶⁰,⁵⁷³,⁸³⁶ and the observation that the MP domain is required for MacA and MacB to associate in solution (with nanomolar dissociation constant).⁸³⁶

In the reconstructed tripartite assembly,²⁷² three MacA MPDs are found to bind to a single MacB protomer, with one MPD contacting the PD-sub domain and two contacting the SABRE subdomain. Located above the PCD-crown of the transporter, the β-barrel domains of MacA form what we refer to as a head-to-tail hexameric “gasket”-ring, which docks to the periplasmic domain of MacB (Figure 50 and Figure 40—ATPase rings). Stabilization of this β-barrel gasket-ring is mostly due to the interprotomer interactions of three residues (which in the *EcMacA* correspond to T293, Y275, and E231), that are strictly conserved across the MacA-family indicating a common mechanism.⁷³⁷

The β-barrel domains of the three MacA protomers make nonequivalent contacts with the PCDs of the transporter, which could, in an analogy to the PAP1 and PAP2 discussed in the context of RND transporters, be named PAP1–PAP3, respectively (Figure 50). Of these, PAP1, similar to the PAP1 in the RND assembly, makes intraprotomer interactions only relative to the transporter (e.g., using the chain identifiers from the PDB ID 5NIK).²⁷² PAP1 could be assigned to chain D, and both its MP-domain and β-barrel domain interact with the PD-base of the PCD domain of the MacB (chain J), while PAP2 (chain F) interacts exclusively with the SABRE domain of the same transporter protomer (chain J), and the last, PAP3 (chain G), appears to occupy an interprotomer position extending over the cleft between the neighboring transporter protomers. However, in contrast to the tight association of the RND–PAP2 with the interprotomer space in the RND
transporters, the “inter-protomer” PAP3 in MacB appears to
make very few direct contacts with the underlying MacB using
its β-barrel domain, and its association is mainly maintained via
lateral contacts with the other PAP mediated via the β-barrel
domain loops. Similarly, while the PAP3 MPD does contact
the SABRE subdomain of the PCD; this contact, at least in the
conformation observed in the cryo-EM structure, is restricted
to a single loop. Such limited contact of this PAP3-protomer
with the transporter makes this site particularly susceptible to
allosteric modulation and may provide a natural switch for
assembly/disassembly of the complex.²⁷² Furthermore, the
asymmetric interactions of the MPDs as well as the β-barrel
domains of MacA with the underlying transporter result in
distortion of the hexameric β-barrel ring relative to the shape
formed by the MacA MPDs in isolation, allowing it to
accommodate the 2-fold symmetry of the MacB PCDs.²⁷²

In an organization which mirrors that observed in PAPs
participating in the RND transporter assemblies, the lipoyl
domains of MacA form an additional hexameric ring that stacks
above the β-barrel gasket but does not contact MacB. The self-
association of the “lipoyl-gasket” clearly plays a role in
stabilization of the whole PAP hexamer, as can be seen in
the example of DevB–DevAC interaction.²⁶⁶ From the lipoyl
domain-gasket up, the hairpin arrangement of the PAPs from
the MacA-family and that of the AcrA-family follow similar
trajectories and present a unified interface to the OMF, which
is further supported by the aforementioned chimeric-protein
studies, swapping the hairpins between MexA and MacA (PDB
ID 4DK1).⁷⁵¹ Consistent with this the MacA-family also shares
the conserved RLS-motif at the tip of their α-hairpin.

Thus, the arrangement of PAPs around the MacB-dimer
differs substantially from the organization of the RND
transporter complexes, showing a much-lighter engagement
with the transporter, and the PAP–MacB interactions are
restricted to within the same transporter protomer, with any
interprotomer contacts being mediated by PAP–PAP inter-
actions instead. This loose association, along with an exposed
proposed substrate location site and the apparent lack of
enclosure provided by the two stalk helices, poses some
questions regarding sealing of the assembled complex and
cross-membrane communication with the NBDs, which are
suggested to provide remote allostery to the complex.

An unexpected analogy to the PAP-MacB communication
may be found in the function of the maintenance of the
phospholipid asymmetry (Mla) MlaFEDB complex, the
structure of which has recently become available (PDB ID
7CGN; 7CH0; 7CGE; 6XBD).437–439 In the sections above
we mentioned the close topological connection of the MacB
(and other type VII ABC transporters) with the new MlaE-
family, which is suggestive of common operational mecha-
nisms. It is thus even more notable that the same stoichiometry
of 2:6 as in the MacB–Mac complex is also observed in the
complex between the transporter MlaEF and the periplasmic
protein MlaD, which belongs to the mammalian cell-entry
(MCE) family, which while phylogenetically not connected to
the PAPs,837 forms analogous hexameric assemblies with
participation of their respective β-barrel domains (Figure 51).
Similar to MacA–MacA interaction, three MlaD protomers
associate with a single MlaE protomer.

Within this complex, a striking arrangement of TM-helices
emanating from MlaD can be seen in the MlaED complex,
where it is tempting to suggest the N-terminal helices of MlaD

are facilitating the substrate transfer and activity of the
transporter.

The TM-helices of different MlaD protomers make distinct
contacts with the TMD and connecting helix (CoH) of the
transporter MlaE (also referred to as elbow or interfacial helix-
1 (IF1),⁴³⁹ with the TMs of D1- and D2-protomers of MlaD
forming close hydrophobic contacts with the elbow CoH of
MlaE, while D3-protomer packs closely against the TM1 and
TM3 of MlaE. Mutagenesis of the respective contact residues
in MlaE rendered the bacteria sensitive to the treatment of
SDS/EDTA,⁴³⁷ and correspondingly, mutations in the MlaD-
TM domain also rendered the transporter nonfunctional.⁴³⁹
Furthermore, truncations of the coupling helix (IF1) of MlaE
also abolish transporter function, highlighting the importance
of this structural element. The existence of the CoH is a
notable feature of MacB, and similar to MlaE, it only has a
single coupling helix, with its NBDs being connected to only
one porter domain subunit, which suggests a similar
mechanism of action. Intriguingly, in the absence of nucleotide
MlaE adopts an outward facing conformation.

While the TM-domains of MacA are not resolved in the
cryo-EM structure of the complex,²⁷² modeling the full-length
MacA (see Figure 51) suggests that, similar to the MlaEF–
MlaD interaction, these TM-helices could reach and plausibly
contact the CoH and/or intramembrane helices of MacB, thus
providing direct coupling of the conformational transitions in
the periplasm to the transporter and its NBDs. Consistent with
such a model and interpretation, while MacB is able to interact
with a MacA, which lacks the TM-helix,⁵⁷³ this TM-helix is
required for activation of the ATPase activity in vitro, and its
deletion obliterates the MacAB-mediated macrolide resistance
in vivo.⁷⁴³ This also holds true for the MacAB homologues
from the Gram-positive organisms, as the ATPase activity of
the Streptococcal Spr0694–0695 MacB-like transporter was
activated by full-length, but not by the N-terminally truncated
version, of the cognate PAP Spr0693 when reconstituted in
proteoliposome,⁵⁷¹ suggesting that the intramembrane com-
munication between the PAP and the transporter is essential
for the general function of the MacB-like assemblies and not
restricted to tripartite complexes with their participation.

Furthermore, evidence from the related DevBAC system in
Anabaena shows that N-terminal cytoplasmic domains in the
PAPs of MacB-group play an important role in activation of
the ATPase activity.²⁰⁶ There, a variant of the PAP DevB,
lacking 22 N-terminal residues completely abolished the
recognition of the glycolipid substrate by the DevAC ATPase
despite not having an impaired binding to the transporter.
Similarly, the DevB homologue, All0809, is not able to
promote a substrate-dependent reaction of the ATPase activity
of DevAC, although it binds to DevAC,⁵⁵⁵ suggesting a further
layer of the underappreciated PAP role in the process may be
present. Importantly, besides the wild-type reference DevB,
none of the PAP variants harboring hairpin deletions tested
(with small exception) could mediate the substrate-dependent
ATPase activity,²⁰⁶ suggesting that formation of the hexameric
PAP-assembly is critical for the activation of the pump, which
is in agreement with an earlier-reported PAP hairpin-targeting
mutation causing similar defects.⁵⁵⁹

The above observations suggest that full understanding of
the functional cycle of the MacB-based pumps could not be
achieved by looking at the transporter in isolation, as similar to
the MlaE₂F₂D₆ which presents a complex 10 + 6 TM-assembly,
the MacB₂–MacA₆ complex should be interpreted as an
<figure><img src="image_66.png" /><figcaption>**Figure 52.** Modified molecular bellows mechanism for the MacAB-TolC pump. MacA (green-cyan) and MacB (blue) undergo allosteric conformational transitions (symbolized by orange arrows) upon binding of substrate and ATP. Similar to type IV ABC transporters, substrate binding is suggested to bring a conformational transition that enables the nucleotide binding domains (NBDs) to engage with ATP. Substrate is expelled through TolC (orange) upon ATP binding. ATP-hydrolysis (symbolized by the small orange lightning bolts) resets the pump, in order for another cycle of efflux to proceed. Interactions within the membrane between the TM-domains of the MacA and MacB are suggested to provide an additional level of transmembrane communication. See text for further details.</figcaption></figure>

integrated 8 + 6 TM-system, with the MacA-protomers forming an active part in the presentation of the cargo as well as communication of allosteric changes across the membrane while also providing an additional level of cooperativity.

While such communication would be analogous to the
vertical communication from the PAPs to the TMD of the
RND transporters discussed in the preceding section, its trans-
membrane nature involving the MacB-CoH, and PAP-TM-
helices is suggested to enable the periplasm-to-inner-
membrane signaling leading to the activation of the NBD-
ATPase activity on the opposite side of the membrane. This is
in stark contrast to the RND transporter assemblies, where
major allosteric communications, as well as energy coupling
access (in the form of opening of the proton-accessible channel
upon substrate-driven L-to-T transition) are restricted to the
periplasmic side of the membrane. This is providing a further
suggestion that the unique organization of the MacB
transporters and their piston-like TM-assembly suggest that
their ATPase cycle may differ significantly from that of the
classical “clam”-like type IV ABC transporters and the classical
alternating access model.

## 9.1. Functional Cycle of the Tripartite MacB-Pumps

The significant conformational changes of the PD/SABRE
domains observed in the different apo- and nucleotide-bound
structures, as well as the close proximity of the NBDs in some
of the nucleotide occupied structures,²⁴,²⁷²,⁴³¹ hint at a
possible efflux model, which has been dubbed the “molecular
bellows model” in analogy to the blowing of the compressed
air in the macroscopic analogue. These parallels are enhanced
by the existence of the one-way-valve operation of the gating
ring of MacA,²⁷² with asymmetric energy need for the drug
passage in an outward and inward direction on the example of
erythromycin MD. Such an arrangement is supposed to
prevent backflow from the OMF and suggests that the gating
ring is always engaged. Under the emerging model discussed

here, the engagement of PAPs with the OMF could be
communicated downward to the lipoyl ring, which experiences
a twisting motion settling down, and this causes the gating ring
to dilate, allowing simultaneous cargo export. While the
“bellows model” agrees that the stitching of the “stalk helices”
(leading to the formation of the TM1-TM2 bundle) and the
associated compression of the supposed substrate binding site
is associated with the efflux of the cargo, the exact nature of the
power-stroke has been less clear.

Taking into account the ABC transporter cycles discussed in
section 5.6.7, we believe that a very modest modification of the
initially proposed “bellows” model can unify the operation of
the MacB and type IV ABC systems. In parallel to the
differential binding of PAP1 and PAP2 to the different RND-
conformers, the differential affinity of MacA to the substrate-
occupied and empty states of the MacB PCDs could be a
driving force for tripartite complex assembly. Such a model
may also explain how separation of the two PCD domains of
the transporter upon the ATP-hydrolysis may, due to the loose
binding of the PAP3, result in a dissociation of the tripartite
assembly. Given the existence of the stable crystal and cryo-EM
complexes of MacA-TolC, it is tempting to suggest that in
contrast to RND complexes, where the PAP-RND forms a
more-stable complex, in the MacB-based pumps the PAP-
OMF subcomplex may be more stable. In such case a “top-
down” assembly of the PAP-OMF onto the MacB may be
possible, with this being contingent with the MacB being
already substrate-occupied.

Under our modified bellows model, illustrated in Figure 52,
the substrate binding is the primary driving force of tripartite
assembly, and similar to the situation discussed in the RND-
PAP association, the MPDs of the MacA-type PAPs are
suggested to have a strong preference to the substrate-bound
conformer of the PCDs, with such binding likely being
associated with the recruitment of the OMF.
Indeed, based on the structural homologies discussed above (see the PD-domain connection section), it is plausible that the PCDs may undergo significant conformational changes upon substrate binding, e.g., similar to the PD-transitions seen in the Super F to I -form of the SecDF transporter.³⁶⁶ This initial substrate-binding is suggested to bring the PCDs closer together, creating a conformation analogous to the substrate-bound occluded conformation seen in the type IV transporters, e.g., during the TmrA cycle.⁵²⁴ This allosteric transition is communicated via the stalk helices downward across the membrane, enabling the binding of ATP by the NBDs.

In turn, the dimerization and allosteric changes in the NBDs, caused by ATP-binding, result in further tightening of the transmembrane helical bundle, which is communicated back to the PCDs and likely provides the power stroke that leads to the ejection of the substrate into the PAP-OMF cavity above the crown of the transporter, by stabilizing the outer-facing conformation of the PCDs in agreement with the previously proposed models.⁴³¹ Under this scenario, the substrate engagement is the main driver of PCD rearrangement, leading to the initial stitching of the TM1 and TM2, facilitating the ATP-engagement by the NBDs, with the power-stroke being associated with the ATP-binding, rather than ATP-hydrolysis.

The departure of the substrate causes the closure of the outer-facing conformation and produces a stable, ATP-bound, occluded conformation of the transporter, that requires ATP-hydrolysis to drive the NBDs apart. We further suggest that the rearrangement of the PCDs and the TMD of the transporter is sensed by the MPDs and TM-domains of the PAPs, providing additional allosteric signaling. Our modeling suggests that upon ATP-binding by the NBDs, the rearrangement of the transporter TMD allows the MacA-TMs to pack better against the helical core of the TMD, which is likely associated with their ability to convey an ATPase-stimulatory signal (possibly via their interaction with the connecting helix (CoH)), which would explain the functional data.⁵⁷³,⁷⁴,⁸³⁵

Under such a scenario, there could be a possibility of a single ATP-hydrolysis independent cycle of the transporter. Indeed, exactly such an event has been reported in the closely related LolCDE system, the ABC-components of which are virtually identical to MacB.⁸³⁸ Uniquely, LolCDE can be purified in a liganded form, which is an intermediate of the lipoprotein release reaction, and it has been demonstrated that the lipoprotein transfer from liganded LolCDE to the periplasmic chaperone LoLA in a detergent solution does not require ATP-hydrolysis and a single transfer event can be stimulated by vanadate. The close homology of the two systems suggests that a similar operation may be in place with MacB-based transporters which will support the ATP-binding power-stroke hypothesis.

However, an alternative mode of operation could not be fully ruled out, as recent analysis of MacAB-TolC incorporated into liposomes containing quantum-dots suggested close synchronization of the substrate efflux with ATP-hydrolysis. Furthermore, using the same system did not detect any transfer in the vanadate-treated sample.⁸³⁹ While a power-stroke associated with ATP-hydrolysis may be an explanation for such behavior, a closer analysis of the MacAB-assembly provides one intriguing alternative. As mentioned previously, the crown formed of the β-barrel gasket and the lipoyl domain gasket is much-more loosely associated with the transporter than in the case of RND-based assemblies, and it presents a domelike structure, which along with the gating-loops of the

lipoyl domain (Figure 50 and Figure 52) creates a “waiting room” type of cavity, where the substrate would plausibly be transferred after the PCD-contraction. The ATP-binding causing the “occluded” substrate-free state of the PCD is suggested to cause contraction of the cavity, squeezing the substrate through the “one-way valve” formed by the gating loops of the lipoyl domain,²⁷² and may serve as the trigger initiating the NBD-activation and resetting of the complex. However, taking into account the additional allosteric communication between the PAP TM-domains and the NBDs, their engagement with the ATP-bound NBDs may cause additional twist in the lipoyl domain, which may facilitate the passage of the cargo through the gate. Upon ATP-hydrolysis, the gating loops equilibrate back into the closed state, preventing substrate back-sliding.

Thus, to answer all these questions conclusively, a bona fide substrate-bound structure of MacB in complex with the PAP is required, along with functional analysis, and given the rapid progress achieved in the past few years, we are hopeful that this riddle will be resolved soon.

## 10. TRIPARTITE ASSEMBLY OF THE MFS-BASED SYSTEMS

Tripartite assemblies based on the MFS transporters remain the least-well studied and correspondingly least-well understood, with even complex stoichiometry being a matter of debate. Similar to other PAPs, *E. coli* EmrA in isolation was found to form dimers, and as the dimerization was more pronounced in the full-length, membrane bound protein, it was suggested to take place via a leucine zipper domain located within the N-terminal part of the protein, covering part of the TM-helix domain.⁷⁶⁰ Removal of the N-terminal portion of the protein impacted dimerization even when a major part of TM-helix and membrane anchoring was preserved, suggesting the cytoplasmic N-terminal tail is essential. Later surface plasmon resonance (SPR) measurements⁶⁹⁷ have suggested a trimer formation in agreement with the sequential trimerization (ST)-model, with participation of preassembled dimers with a $K_d$ of 1.87 µM. The EmrA trimers were also found to be the prevalent cross-linked species.

Although an experimental structure of EmrB is not available at present, reasonably reliable topological estimates could be obtained from the analysis of the structures of the related MFS pumps such as YajR,³⁸² EmrD (PDB ID 2GFP),³⁸⁰ and PepTSo (PDB ID 4UVM).²⁷¹ The C-terminal part of EmrB likely presents a helical arrangement equivalent of the TM14 of the PepTSo,²⁷¹ and earlier studies suggested that this TM region of the MFS transporter may be involved in a leucin-zipper like interaction with the TM-domain of the PAP EmrA, forming a TM-helical bundle.⁷⁶⁰ A similar suggestion was extended to the N-terminal portion of the transporter, covering a major part of TM1, with the implication of a 2:1 PAP to transporter subunit interaction,⁷⁶⁰ and although current structural data on the organization of TM-helices in the known MFS transporters does not lend itself to a straightforward model of such an assembly, as it requires another PAP-binding site per MFS-protomer, such overall arrangement is appealing as (assuming a dimeric transporter MFS) it gives rise to six PAPs and a 34 TM-helical model (six from PAPs and 28 from the transporter-dimer), which is roughly equivalent to the 36 TM-section observed in the RND-assemblies. Despite their low resolution, the latest EM data on the complete reconstituted EmrAB-TolC⁹ is
<figure><img src="image_67.png" /><figcaption>**Figure 53.** Principal modes of T1SS depending on the cargo. (A) Typical RTX-toxin secretion system based on the HlyABD-TolC. The complex is assembled upon substrate binding and remains stable through a number of iterative ABC-cycles by the HlyB transporter. There is no periplasmic intermediate of transport and no processing of the cargo (HlyA), with the CLD-domains of HlyB (in yellow) playing a cargo-recruitment and chaperoning role. (B) An example of the transport by the bacterial transglutaminase-like cysteine proteinase (BTLCP)-associated transporter family on the example of the LapBCDEG system. Notably, the complex disassembles upon association of the cargo (LapA) with the OMF, where it may be anchored for extended periods by its N-terminal “plug” domain, creating a periplasmic intermediate. While the CLD of the LapB does not proteolytically process the cargo, this is achieved by a periplasmic protease LapG, which is controlled by a cyclic di-GMP receptor LapD in response to environmental stimuli providing control of the cell adhesion. (C) The microcin-based secretion system is exemplified by the CvaABC. The PCAT transporters associated with this type of secretion have catalytically active C39 domains. (D) The HasDEF system of *Serratia marcescens* involved in the secretion of the hemophore HasA presents a departure from the common pattern presented in panels A–C, where the cargoes are fed C-terminus first and instead HasA is threaded N-terminus first. Furthermore, the system lacks both C39 and CLD domains and relies on the chaperoning function of the SecB. Additional details are provided in the main text. Figure modified based on Smith et al.⁶ and Masi and Wandersman.⁵¹²</figcaption></figure>

compatible with such an arrangement, and earlier EM data based on isolated EmrAB has suggested that it forms a “dimer of dimers” in a back-to-back formation;⁸⁴⁰ however, at present a model with a monomeric transporter with six PAPs giving a 20 TM-helix assembly could not be excluded entirely.⁷³⁸ A possible hint for such an assembly, where one EmrA is being permanently associated with the EmrB and an additional pair is being recruited, is suggested from the SPR studies which resulted in the ST-model of trimerization mentioned above.⁶⁹⁷

There is limited data available on the potential substrate role in the assembly; however, PAPs from MFS-associated systems are capable of binding drug specifically, providing substrate differentiation as demonstrated for EmrA and HmrA (46.2% identity), and binding to native pump substrates, e.g., CCCP, DNP, and nalidixic acid, provokes conformational changes within the EmrA, which however does not respond to erythromycin, which causes a similar effect in the erythromycin efflux-competent HmrA.⁷⁶⁰ Cargo-specific effects have also been demonstrated for the VceA, a PAP component in the equivalent MFS-based assembly VceABC in Vibrio.¹²² This binding occurs in the periplasmic domain and appears to be dependent on the presence of the TM-domains, as no binding was detected to the soluble, TM-truncated versions of the EeEmrA and AeEmrA tested,⁷³⁸ providing strong indication that functional TM-domains are needed for such a function. Thus, the existence of extended TM-domains in this class of

PAP and their implied engagement in transporter association provide a plausible model for allosteric control and activation.

While future work is needed to elucidate the exact nature of the PAP-MFS interaction, it is clear that the mode of operation of these assemblies is markedly different from the RND- and MacB-based systems, due to the lack of extended periplasmic domains. Consistent with this, the PAPs engaged with the MFS transporters also lack MDP domains, and their TM regions are essential for dimerization and engagement with the transporter.⁷⁶⁰ The rigidity of the extended α-helical hairpin domains also likely provides less allosteric control of the cycle, with the stability of these assemblies being higher than the corresponding RND systems,⁶⁹⁷ and they might be engaged in a constitutive low-level efflux, rather than being engaged temporarily for high-intensity stress-related responses, which is also consistent with their much lower copy numbers in the cell.⁶⁹⁶

## 11. ASSEMBLY OF TYPE 1 SECRETION SYSTEMS

The cargoes for the type 1 secretion systems (T1SSs) are secreted in a Sec-independent fashion⁶,⁸⁴¹ and correspondingly lack the N-terminal signal-sequence typical of periplasmic bound cargoes of the Sec-system.⁸⁴² There are two broad ranges of cargoes secreted by the T1SSs.⁸⁴³,⁸⁴⁴ The first includes relatively small molecules with a molecular weight in the sub-10 kDa range that are secreted in a single step, such as bacteriocins (in the context of Gram-negative bacteria,
microcins), e.g., colicin V (aka microcin V; CvaC).⁸⁴⁵
Microcin secretion is facilitated by an N-terminal signal, which is recognized by the C39-domain of the cognate PCAT-type ATPase forming the core of the transporter complex and is then proteolytically processed concomitantly with the secretion of the cargo. This has been discussed in the preceding sections on the example of PCAT1,⁵⁰⁵ so to avoid repetition we will not discuss this group further in great detail. A schematic of their transport is presented in Figure 53, panel C.

The second diverse group of cargoes are generally larger
proteins, some of which are among the largest coded within
the bacterial proteome and include a number of toxins,
adhesins, lipases, and proteases.⁴⁴⁹,⁸⁴⁶,⁸⁴⁷ Rather uniquely, the
targeting signal for the cargo secretion in this second group is
located within the 50–60 residues of the C-terminal.⁸⁴⁸–⁸⁵⁰ As
the nature of this signal is quite diverse and reveals no clear
sequence consensus, it has been suggested that secondary
structure is at least partially involved.⁸⁵¹ It appears to present a
set of two short α-helical fragments bridged by a linker peptide,
with a downstream low-complexity region.⁸⁵²,⁸⁵³ The secretion
of some of the cargoes within this group relies solely on this C-
terminal signal, e.g., the well-studied heme acquisition protein
HasA.⁵²⁹,⁸⁵⁴ A major group of the cargoes secreted by the
T1SS present additional Ca²⁺ binding motifs known as RTX-
motifs or GG-repeats—GGXGXDXUX (where X is any amino
acid and U is a hydrophobic amino acid)—precede the C-
terminal secretion signal in some of the passenger proteins,
including HlyA and LipA.⁴⁴⁹,⁴⁹⁷ These play an important role
in folding of the cargo, and X-ray structures reveal that they
form a β-roll motif (e.g., PDB ID 1KAP⁵⁰⁰,⁵⁰¹). Within the
RTX-group, there is significant diversity of cargoes, which can
be ranked in the order of organizational complexity. The
hemolysin/cytolysin family of pore-forming toxins includes
hemolysin A (HlyA), in which the region N-terminal of the
glycine-repeats code just one functional domain—the pore
forming toxin itself²¹⁴ and the CyaA from *Bordetella
pertussis*,⁸⁵⁵ which apart from the toxin domain, possesses an
adenylate cyclase module which affects the cAMP levels upon
entering the target host cell. More complex architectures are
present in the so-called MARTX (multifunctional autoprocess-
ing repeats in toxins), which contain protease domains and
target eukaryotic cells, where they undergo autoprocessing
upon binding to inositol hexakisphosphate (InsP6).⁸⁴⁷,⁸⁵⁵,⁸⁵⁶

Another large group is the large repetitive RTX adhesins (e.g., FrhA of Vibrio cholerae, RtxA of Legionella pneumophila, and SiiE of Salmonella enterica), which function in biofilm development and cellular adherence and attain very large sizes, sometimes in excess of 1.5 MDa, e.g., in the case of the *MpIBP* of Marinomonas primoryensis.⁴⁴⁹,⁸⁵⁶,⁸⁵⁷ These are critical molecules for biofilm formation, and they feature a number of repetitive domains, in addition to the RTX repeats; for example, the *MpIBP* possesses some 120 bacterial immunoglobulin-like domains (B1g) and a unique ice-binding domain, which is associated with the lifestyle of this bacterium.⁸⁵⁷

The majority of the T1SS appear to translocate their cargoes
from the cytoplasm to the extracellular space in a single
translocation event,⁸⁵⁸ without disassembly of the complex or
any periplasmic intermediates. While the details of the
secretion are still not fully confirmed and vary between
different assemblies, in a first approximation the process
features the following key elements (Figure 53, panel A). The
typical RTX cargo (e.g., α-hemolysin) is fully synthesized prior

to secretion and is threaded in an unfolded state, C-terminus
first. The C-terminal part of the protein carrying the RTX
motifs emerges on the cell surface and binds to Ca²⁺ ions to
facilitate the folding of the jellyroll RTX domains, which
contributes to the secretion process.²¹⁴

As a first step, the RTX-cargo is recruited by the ATPase, and the secretion motif is recognized by the catalytically inactive C39-like (CLD) domain of the PCAT transporter,⁵⁰⁶ which feeds the C-terminus of the cargo into the substrate-binding cavity of the transporter, keeping it unfolded. This association leads to the assembly of the complete translocase and causes a conformational switch in the transporter, allowing for the outer-facing conformation to be attained, which brings the NDBs together, allowing them to bind the ATP²⁷¹ (Figure 53, panel A). ATP-hydrolysis in turn resets the system back for another cycle, continuously threading the cargo, and these cycles continue until the N-terminal end is reached. Following the release of the cargo outside of the cell, the complex disassembles. This is in contrast to the secretion of the microcins (Figure 53, panel C), where the PCAT has an active C39 domain and the secretion signal is N-terminally located on the cargo, which also gets proteolytically processed. Importantly, and in common with the RTX-toxins depicted in panel A, the transfer of the cargo takes a single step.

Recently, a group of RTX-containing proteins related to the
RTX-adhesion proteins has been identified and separated into
its own class, named, due to linkage to the bacterial
transglutaminase-like cysteine proteinase (BTLCP),⁸⁵⁹
BTLCP-adhesins.⁶,⁸⁶⁰ The prototype of this BTLCP-adhesin
group, LapA of *Pseudomonas fluorescens* Pf0-1, also possesses a
number of B1g motifs but also a novel, specialized N-terminal
"retention module" to anchor itself at the cell surface as a
secretion-intermediate threaded through a dedicated OMF
LapE (Figure 53, panel B). In addition, they often feature von
Willebrand factor A domains (vWFA), which are peptide-
binding folds, also found in *Pseudomonas putida* LapA and
LapF.⁸⁶⁰,⁸⁶¹ The first stage of the transport involves the
association of the cargo with the CLDs of the LapB ABC
transporter, thus resembling the HlyBCD system. However, a
startling discovery has been made, showing that these adhesins
break the dogma of "one-stop-transport" which has been
associated with T1SS, and instead their translocons are able to
dissociate before the adhesin has left the OMF barrel, releasing
the PAP transporter pair⁸⁶⁰ (Figure 53, panel B).

Indeed, the very nature of the “retention module” or “plug
domain” is to embed the adhesin within the OMF-channel,
where it may stay permanently, unless processed by a
periplasmic protease, LapG. The stalled LapA functions as a
biofilm-promoting factor, which is dependent on the cyclic-di-
GMP levels, as the LapG protease is sequestered in the
presence of cyclic-di-GMP by a membrane-anchored cyclic-di-
GMP receptor LapD but can be released once the levels of
linear di-GMP (pGpG) increase⁸⁶² (Figure 53, panel B). Thus,
while the actual transporters associated with the T1SS of
BTLCP-linked adhesins do not possess active proteolytic sites,
their cargoes are proteolytically processed in response to
environmental stimuli.

Intriguingly, all the PCAT transporters associated with three
groups of T1SS presented so far have N-terminal domains,
which are implicated in substrate binding. However, while the
CvaB transporter has a C39-domain with an active catalytic
site, both the transporters associated with the transport for
pore-forming cytolysins (HlyB-type PCATs) and LapB-type
transporters associated with the secretion of the BTLCP-linked
adhesins have catalytically inactive CLDs. These however differ
dramatically in their consensus sequences, suggesting a
different mode of operation and cargo specificity.⁶

A surprisingly similar arrangement was also discovered in the
secretion of the giant adhesin MpIBP,⁸⁵⁷ where two dedicated
modules were found to anchor the adhesin to the inside of the
OMF-channel, as a stable periplasmic intermediate, suggesting
that this is a more widespread mechanism than previously
thought.

This is achieved, as in contrast to the RTX modules, which
require a Ca²⁺ concentration of above 3 mM (typically
associated with the extracellular medium) to fold, the N-
terminal “retention modules” of LapA, and the corresponding
N-terminal “plug” domains, and β-barrel spanning domains
seen in the MpIBP are structured in the absence of Ca²⁺ and
capable of folding during the secretion process, allowing them
to engage and anchor themselves with the OMF partner
proteins.⁶,⁴⁹⁷

While markedly divergent, the different T1SSs discussed
above present a uniting feature, which is that all of their
cargoes are transporter C-terminal first. The implication of this
characteristic is that the cargo polypeptide needs to be
synthesized in its entirety prior to secretion and may be
expected to become at least partially folded. Unless active steps
are taken to the contrary by chaperoning systems, and as
discussed in the earlier section on the PCAT transporters, this
is provided by both the transporter cavities themselves and
their associated CLD-domains and in some cases auxiliary
chaperones.²¹⁴

In addition to the cargo interaction with the PCAT
transporter itself, which is at the heart of the T1SS and was
covered in the preceding sections, the second major-driving
force behind the assembly of a functional T1SS is the PAPs.
Their role appears to be rather multifaceted and involves direct
binding of the cargo, stimulation of the assembly process, and
recruitment of the OMF, as well as possibly participation in the
folding of cargoes. Distinct regions of the PAPs seem to play a
different role in the process. Many of the studies into the role
of the PAPs in T1SSs derive from the hemolysin A (HlyA)
secretion system in E. coli, which involves the PCAT-type
ATPase HlyB and its cognate PAP HlyD and the OMF
TolC.⁶¹

As mentioned in the previous sections, the transport of HlyA
does not involve proteolytic processing,⁸⁴⁹ and correspond-
ingly, the C39-domain of the HlyB is catalytically inactive and
is referred to as a CLD.⁵⁰⁶ The regions covering the N-
terminal section of the α-helical hairpin domain (residues 127
to 170) and the C-terminal 33 residues of HlyD, respectively,
were shown to be required for *in vivo* secretion.⁸⁶³ HlyD was
also shown to interact directly with the toxin substrate by
direct cross-linking, and in addition, masking its C-terminal
fragment by in-frame fusion to a c-Myc epitope blocked cargo
translocation,⁷²¹,⁸⁶³ suggesting an active role of the PAP in
substrate recognition and its presentation to the transporter.

Furthermore, the N-terminal 59 residues of HlyD form a
small cytoplasmic domain,⁷³¹,⁸⁶⁴ truncation of which abolished
the secretion of HlyA.⁸⁶⁵ A more detailed analysis of the
region, including a dissection of the residues (1–25) forming a
predicted amphiphilic helix and a downstream charged region
(residues 26–38), demonstrated that these regions are
necessary for direct interaction of the PAP with its cargo
(HlyA) and subsequent recruitment of the OMF into the

functional translocator but not required for either the
oligomerization of HlyD or the formation of the PAP/ABC
transporter complex.⁸⁶⁶ This data also suggests that HlyA
binding to the cytosolic domain of HlyD promotes an outward
trans-membrane conformational change, propagated to the
periplasmic domain of HlyD, leading to the recruitment of the
OMF.

The periplasmic portion of the PAP has also been implicated
in cargo-related function, and analysis of random point
mutations in PAPs, in particular HlyD⁷⁵⁵ and related PAP
CvaA, involved in colicin V secretion⁷⁵⁴ demonstrated that
they result in misfolding of the substrates, even when their
secretion rate was not affected. These mutations map to the
lipoyl domain and the α-helical hairpin of HlyD, and their
impact of cargo-folding suggests that the α-barrel of the
hexamerised PAP may provide a chaperoning function.⁷⁵⁵
Similarly, TolC barrel mutations have been reported to result
in incorrect folding of substrate HlyA affecting its hemolytic
activity,⁸⁶⁷ which could be restored by refolding *in vitro*.
Furthermore, by analyzing a variety of passenger proteins
spliced in front of the HlyA C-terminal signal sequence and
introducing folding mutations into the cargo, Bakkes et al.⁵³⁰
were able to demonstrate that the efficiency of secretion of
HlyA by the *E. coli* HlyBD-TolC is dictated by the folding rate
of the substrate.

Importantly, the formation of the inner-membrane HlyBD
translocase complex is not just initiated by the substrate but is
also ATP-independent, as demonstrated by the use of a HlyB
mutant competent in ATP-binding but not in ATP-
hydrolysis.⁷²¹ This mutant was capable of ATPase-PAP
assembly, substrate recruitment, and bridging of the OMF;
however, HlyA stalled in the channel. A similar result was
obtained when the PAP HlyD C-terminus was masked. This
leads to the conclusion that HlyA export likely occurs via the
assembly of a contiguous double-membrane channel which is
formed without the requirement for ATP-hydrolysis by the
PCAT transporter, by substrate-induced, transient, and
reversible bridging of the HlyBD-translocase to the OMF.⁷²¹

In the aforementioned protease secretion system PrtDEF
(see PCAT ABC transporter section 5.6) from Dickeya
dadantii (formerly Erwinia chrysanthemi),⁵³¹ responsible for
the secretion of a number of proteases, including PrtA, PrtB,
PrtC, and PrtG,⁵³² the ABC transporter PrtD displays a basal
ATPase activity that is specifically inhibited (half-inhibition at
0.1 µM) upon encountering a cognate C-terminal secretion
signal of its substrates (PrtB and PrtG metalloproteases) but
not by the C-terminal sequences from nonrelated cargoes.⁸⁶⁸ It
is thought that this inhibition may take the form of a
stericprevention of NBD closure in a manner similar to the
inhibition of eukaryotic TAP1/2 by the viral peptide ICP47,⁸⁶⁹
which forms a long-helical hairpin plugging the translocation
pathway of TAP from the cytoplasmic side stalling the
system.⁵¹⁵ This inhibited conformation of PrtD binds the
PAP PrtE, with the complex of PrtD and PrtE recruiting the
OMF PrtF.⁴⁹² Thus, this system presents a clear ordered
sequence of assembly, where the substrate first recognizes the
PCAT-ABC transporter, which then binds the PAP assembly,
which in turn recruits the OMF component.⁴⁹² Notably, while
substrate binding was shown to be required for assembly of the
three components, there was no evidence for ATP being a
driver of the assembly.

A related T1SS in *Serratia marcescens*, formed around the
PCAT type transporter HasD, and the PAP HasE are involved
in the secretion of the heme acquisition protein HasA<sup>153,870</sup>
(Figure 53D). In *E. coli*, the reconstituted HasA secretion
system was dependent on the presence of an OMF channel,
which could be either TolC<sup>871</sup> or the recombinant PrtF, the
OMF from the *Dickeya dadantii* T1SS PrtDEF. The analysis of
the secretion of the *Serratia marcescens* HasA protein by the
HasD indicated a strong coupling between synthesis and
secretion in the type 1 secretion pathway<sup>529</sup> due to slow
threading of the substrate, suggesting active maintenance of the
cargo folding is involved, and indeed folded HasA has been
found to inhibit its own secretion. Primary interactions
between HasD and newly synthesized HasA have been
suggested to be driven by linear sites which are sequentially
exposed on the unfolded cargoes.<sup>512</sup> ATP-binding promotes
dimerization of the NBDs, facilitating and stabilizing the
outward facing transporter conformation, and the substrate is
then fed into the recruited OMF N-terminus first, while
presumably the C-terminus stays associated with the trans-
porter until the threading is complete. These data support a
model of type 1 secretion involving a multistep interaction
between the substrate and the PCAT transporter that stabilizes
the assembled secretion system until the C-terminus of the
cargo is presented, and such interaction is suggested to
underwrite the multistep processivity required for translocation
of large, unfolded cargoes. Crucially, the C-terminal signal of
the cargo hemophore HasA was shown to trigger ATP-
hydrolysis, driven by the PCAT ABC transporter HasD,
leading to disassembly of the complex and separation of OMF
(HasF) to its pre-engagement state.<sup>512</sup> In agreement with this,
it was reported that the C-terminal peptide of HasA may also
possess an intermolecular activity that is capable of triggering
complex dissociation *in vivo* when it is provided as a distinct
peptide.<sup>872</sup> This appears to be a departure from the general
model of type 1 secretion discussed above (Figure 53A–C),
where the C-terminal part is driving the secretion with a
contribution from folding of the RTX-motifs. Furthermore,
HasA is not proteolytically processed and its cognate ATPase
HasB does not possess a CLD-domain. While at 19 kDa HasA
presents a relatively small cargo, it still is necessary for it to be
transported in an unfolded state, which is facilitated by SecB
on the cytoplasmic side.<sup>873</sup> Thus, HasA secretion appears to
present a distinct mode of T1SS as presented in Figure 53D.

Despite their considerable variety, an emerging common
pattern across all the T1SS assemblies discussed above is that
the ATP-hydrolysis does not appear to be required for the
formation an ABC transporter-PAP-OMF complex, which
instead is substrate and PAP-driven,⁵¹²,⁷²¹,⁸⁶⁶ while the C-
terminal signal of HasA triggers HasD-driven ATP-hydrolysis,
leading to disassembly of the T1SS complex.⁵¹² Such energy
depending also provides further parallels between the
assembly and disassembly of the PCAT-based T1SS tripartite
complexes and the tripartite complexes arranged around RND
transporters discussed above,⁷⁰⁵,⁷²⁰,⁸¹³ with the interprotomer
association being driven by protein-protein interaction and
substrate-binding/cargo folding without the requirement of
energy input, while the energy-dependent step is likely
required for resetting and disassembly of the complex. As
discussed in the previous section, similar energy-dependency
for complex dissociation has also been described for a MacB-
related LolCDE complex, where membrane-extracted lip-
oproteins are passed over to the periplasmic chaperone
LoLA,⁸³⁸,⁸⁷⁴ in a process that requires ATP-binding and
hydrolysis but not the conformational change associated with

the release of the ADP and Pi (as it could be trapped by
vanadate), suggesting that a full cycle of ATP-hydrolysis is
similarly required for complex disassembly.

Despite the recent advances in the understanding of the
functional organization of the T1SS, the structural data on
these systems lags compared to other tripartite assemblies.
However, the recent availability of all principal components of
the T1SS machinery allow for some tentative reconstructions
of the complete assembly.

Previous attempts stopped short of modeling the complete
assembly, and to gain some insights into its possible
organization, here we have extended the modeling of the
HlyAB-TolC system to include both the full HlyD transporter
(based on the homology with PrtD and PCAT1 struc-
tures)⁵⁰⁵,⁵¹⁵ and the β-barrel and the transmembrane domains
of HlyD, which have not been resolved in the crystal
structure<sup>739</sup> (Figure 54). The overall assembly is compatible

<figure><img src="image_68.png" /><figcaption>**Figure 54.** Qualitative model of the complete tripartite T1SS assembly based on the HlyBC-TolC. The assembly is notable for the extended tubular architecture, which presents a unique break in the supercoiling pattern, associated with the architecture of the T1SS PAPs. The hexameric β-barrel ring of HlyD binds closely to the membrane due to absence of the MP-domains, and the PAP-associated transmembrane helices could provide interactions within the membrane. The approximate membrane boundaries are shown with the blue rectangle. See the text for more details.</figcaption></figure>

with the available functional data⁷⁵²,⁷⁵⁵ and suggests a unique
helical trajectory of the HlyD, which mimics the helical paths
of the TolC, and arrangement that is facilitated by the helical
breaks in the hairpin domain of HlyD. The modeling of the
extended TM-helices of HlyD suggests, similar to the MacAB-
model presented earlier, that they may be able to participate in
direct intermembrane interactions with the TMD of the HlyB,
possibly providing additional allosteric control and modulation
of the ATPase activity which could explain the previously
accumulated functional data. While the near future will no
doubt provide experimental proof of such models, they provide
the best current approximation of the function of the T1SS.

## 12. TARGETING TRIPARTITE ASSEMBLIES—PUMP INHIBITORS AND DISRUPTORS

The growing threat of antibiotic resistance has significantly
reduced the efficacy of existing antibiotics. Therefore, there is
an urgent need to develop new therapeutic approaches to treat
infections caused by MDR bacteria.⁸⁷⁵ The manifold roles of
tripartite efflux systems in Gram-negative bacteria make them
attractive drug targets; therefore, efflux inhibition is seen as a
prospective strategy to overcome efflux-mediated resistance.
Compounds that inhibit the function of efflux pumps directly
or indirectly are known as efflux inhibitors (EIs). Although the
term efflux pump inhibitor is also used in the literature, EIs is
deemed more accurate since not all compounds interact
directly with efflux systems to inhibit efflux. The primary
rationale for using EIs is to restore the susceptibility of
multidrug resistant (MDR) bacteria to antibiotics by
preventing their extrusion, thereby increasing intracellular
concentrations of antibiotics.⁸⁷⁶ EIs could also be used to
revive the effectiveness of older antibiotics that have fallen out
of use due to their loss of efficacy against MDR bacteria.
Furthermore, due to the reported roles of tripartite efflux
systems in biofilm formation and virulence, EIs could also be
employed as antibiofilm and antivirulence agents.¹⁸⁵,⁸⁷⁷ For a
compound to be classified as an EI, it must fulfill two
important criteria. First, an EI should not exhibit antibacterial
activity, but when used in tandem with an antibiotic, it should
potentiate the activity of that particular antibiotic. Second, an
EI should not possess activity against a strain that lacks the
efflux pump that it is supposed to inhibit. Lastly, an EI should
not be a substrate of efflux pumps.⁸⁷⁸ Importantly, EIs should
be able to pass across the outer membrane into the periplasm
to exert their activity. The chemical diversity of identifed EIs
has been recently reviewed in detail elsewhere.⁸⁷⁹,⁸⁸⁰ Studies
have found that EIs can exhibit different mechanisms of action
(Figure 55), and here we review the literature on this basis.

### 12.1. Targeting the Inner Membrane Transporter

The inner membrane component of tripartite systems plays an
important role in energy transduction, substrate recognition,
and uptake. Therefore, the majority of EIs described in the
literature are posited to target the inner membrane transporter,
such as AcrB. One of the first EIs to be characterized was
phenylalanine-arginine β-naphthylamide (PAβN, 1; Figure
56), a synthetic peptidomimetic inhibitor of the RND family
tripartite efflux systems MexAB-OprM, MexCD-OprJ, and
MexEF-OprN of P. aeruginosa.⁸⁸¹ Since then, PAβN has been
shown to be a broad-spectrum inhibitor of several different
tripartite RND efflux systems in Gram-negative bacteria,
including AcrAB-TolC from S. Typhimurium, and CmeABC
from C. jejuni and C. coli.⁵⁰,⁸⁸² PAβN has been suggested to act
as a competitive inhibitor of RND efflux systems by binding to
the distal binding pocket, specifically the hydrophobic trap, to
prevent the binding of substrates.³⁴³ Surface plasmon
resonance (SPR) studies have shown that PAβN binds to
both AcrA and AcrB with a micromolar affinity.⁸⁸³ Lamers et
al.⁸⁸⁴ also reported that PAβN can permeabilize the outer
membrane of Gram-negative bacteria, indicating an additional
mechanism of action for PAβN. A recent study reported that

<figure><img src="image_69.png" /></figure>

**Figure 55.** Schematic diagram of the different mechanisms of action of efflux inhibitors. Inhibition of gene expression prevents transcription of efflux genes, thereby reducing efflux pump expression. Compounds that inhibit the functional assembly work by interfering with the interaction surfaces between the components of the tripartite efflux systems. Energy decoupling agents facilitate flow of protons, thereby dissipating the electrochemical gradient, so that it cannot be exploited as an energy source by efflux pumps. Inhibitors of substrate efflux can work as a competitive inhibitor by actively competing with the binding site to prevent substrate binding or by blocking conformational change to prohibit enzymatic catalysis. Inhibitors of periplasmic adaptor proteins (PAP) are thought to work by preventing binding to the inner membrane transporter and by preventing self-association of the PAP protomers. Outer membrane factor (OMF) inhibitors block the periplasmic entrance site to prevent translocation of substrates across the outer membrane. PAP, periplasmic adaptor protein; OMF, outer membrane factor.

PAβN exerts its EI activity in AcrB by restraining drug-binding
pocket dynamics rather than acting as a competitive inhibitor.
Computational and experimental data suggested that cipro-
floxacin and PAβN bind to AcrB simultaneously at different
subsites within the distal binding pocket and that PAβN
inhibits efflux by perturbing the substrate translocation
pathway.⁸⁸⁵ PAβN was tested *in vivo* to be cytotoxic.⁸⁸⁶
However, it is still one of the most commonly used inhibitors
in assays to restore the activity of various antibiotics. One of
the other early broad-spectrum EIs to be identified was the
synthetic compound 1-(1-naphthylmethyl)-piperazine (NMP,
2; Figure 55), which was shown to potentiate the activity of
several antibiotics, including chloramphenicol, levofloxacin,
and linezolid, in *E. coli* strains that were overexpressing *acrAB*
or *acrEF*.⁸⁸⁷ In addition, NMP has been shown to potentiate
the activity of antibiotics in several other Gram-negative
bacteria, such as *A. baumannii*, *K. pneumoniae*, *C. freundii*, and
*K. aerogenes*.⁸⁸⁸⁻⁸⁹⁰ SPR studies have shown that NMP binds
to AcrB with a higher affinity than PAβN, even though they act
as EIs at the same concentration,⁸⁹¹ which could be explained
by the additional membrane-permeabilizing effects of PAβN.
To date, the exact mechanism of action of NMP has not been
elucidated. However, molecular dynamics simulations suggest
that NMP binds to the outer face of the distal binding pocket
to straddle the G-loop of AcrB, thereby reducing its flexibility
and impairing the proper binding of substrates.³⁴³,³⁴⁹ Further
development of this compound was discontinued due to its
low potency and structural similarity with serotonin
<figure><img src="image_70.png" /><figcaption>**Figure 56.** Structural formulas of representative efflux inhibitors that have been discussed in this review.</figcaption></figure>

agonists.⁸⁹² Another strategy to inhibit inner membrane transporters is by depleting the energy source required for efflux. Carbonyl cyanide *m*-chlorophenylhydrazone (CCCP, 3; Figure 56) is a protonophore that dissipates the proton motive force, thus inhibiting all secondary active transporters. Due to its toxicity, CCCP has no therapeutic potential; however, it is a useful experimental tool as it is widely used in efflux assays.⁸⁹³

Certain clinically approved drugs have also been reported to display EI activity. The psychotropic drug chlorpromazine (4; Figure 56) has been reported to inhibit efflux in Gram-negative bacteria.⁸⁹⁴ In S. Typhimurium and several other serovars, chlorpromazine was shown to significantly potentiate the activities of various antibiotics. In the same study, chlorpro-

mazine was reported to repress *acrB* expression, suggesting that it may inhibit efflux by reducing efflux gene expression.⁸⁹⁵ Chlorpromazine can also affect cellular morphology and replication, membrane permeability, and energy generation, suggesting additional mechanisms of action.⁸⁹⁴ A recent study has shed some light into the mechanism of how chlorpromazine acts as an EI. Grimsey et al.⁸⁹⁶ reported chlorpromazine and amitriptyline to be substrates and inhibitors of the AcrB pump. Exposure to subinhibitory concentrations of chlorpromazine in *E. coli* and S. Typhimurium was found to select for *ramR*- and *marR* mutants, which encode for the regulators of the AcrAB-TolC system. Additionally, it was reported that when S. Typhimurium cells with a nonfunctional AcrB pump
(D408A mutation) were exposed to subinhibitory chlorpromazine and amitriptyline concentrations, the D408A *acrB* mutants were found to revert to a wild-type *acrB* allele, suggesting that both compounds act as substrates of AcrB. Lastly, molecular docking and dynamics simulations and free energy calculations were shown to demonstrate that both chlorpromazine and amitriptyline bind to the hydrophobic trap within the distal binding pocket of AcrB.

Through high-throughput drug screening and rational drug design, new and more potent EIs have been identified. A pyridopyrimidine (D13-9001, 5; Figure 56) derivative acts as an inhibitor against the MexAB-OprM pump in *P. aeruginosa*³⁴⁷ and AcrAB-TolC of *E. coli* and was co-crystallized with AcrB (*E. coli*) and MexB (*P. aeruginosa*).²⁹¹ This compound was shown to bind to the deep binding pocket area, which was designated the hydrophobic trap due to the enrichment of phenylalanine residues in this region. Furthermore, isothermal titration calorimetry was used to show that D13-9001 binds to AcrB and MexB with binding affinities of 1.15 µM and 3.5 µM, respectively.²⁹¹ MBX2319 (6; Figure 56) is another pyranopyridine inhibitor of the AcrAB-TolC system which was identified through high-throughput screening of 183,400 compounds. It was shown to potentiate the activity of multiple antibiotics against *E. coli*.³⁴⁸ MBX2319 binds tightly to AcrB;³⁴⁹ however, it does not display any detectable binding affinity to AcrA as verified by SPR.⁸⁸³ Later, molecular docking studies led to the development of more potent derivatives of MBX2319, such as MBX3135 (7; Figure 56). Pyranopyridine derivatives (MBX2319, MBX2931, MBX-3132, and MBX-3135) were found to be highly potent in the inhibition of efflux pumps in *E. coli*, *E. aerogenes*, *K. pneumoniae*, and *S. enterica*.³⁴⁸,³⁴⁹,⁸⁹⁷

Crystal structures of the MBX inhibitors (MBX2319, MBX2931, MBX-3132, and MBX-3135) were obtained with the soluble periplasmic domain of AcrB (AcrBper) and were found to bind in the proximity of the same region in the hydrophobic trap.³⁴¹ The inhibitory effect was suggested to be due to the competitive and overlapping binding of the inhibitors to the deep binding pocket of the T protomer, and due to the tight interaction, it possibly halts the conformational LTO cycling. Interestingly, single-particle cryo-EM structures revealed an LTO trimeric setup in the presence of substrate (puromycin), whereas in the presence of MBX-3135, mostly particles in the TTT conformation were found.²⁸⁷,³⁰⁵ A recent study reported the identification of 43 novel EIs of Gram-negative bacteria from a high-throughput drug screening of the Prestwick drug library and the proprietary compound library from Roche. It was found that the majority of identified EIs had a specific EI-antibiotic combination and/or species-specific synergy with antibiotics. Of the EIs identified, auranofin (8, Figure 56), an antirheumatic agent, increased ethidium bromide accumulation and potentiated antibiotic activity in checkerboard assays.⁸⁹⁸ While most drug screening efforts have focused on looking at inhibitors of RND inner membrane transporters, a recent study identified an inhibitor of the ABC type efflux pump MacAB in S. Typhimurium. Compound 9 (Figure 56) was identified from screening a drug library for compounds that inhibited the growth of a macAB-overexpressing strain in the presence of erythromycin. It was reported to increase the susceptibility of this strain to several different macrolides, although its mechanism of action remains to be explored.⁸⁹⁹

The EIs discussed so far, and indeed the majority of the EIs designed and tested so far, target the transporter component of the system, notably the RND transporters, within which the majority of the substrate specificity is thought to reside. However, there is growing evidence for the involvement of PAP and OMF partners in contributing to the selectivity, making them possible targets for competitive inhibition. A recent study by Marshall and Bavro⁷¹⁸ found several antibiotic-specific phenotypes associated with novel point mutations in TolC from *E. coli*. For example, Q129L and Q352E mutations in TolC were shown to increase susceptibility to only tetracycline and acriflavine, respectively. Another recent study found that the MexXY RND pump of *P. aeruginosa* exports anionic β-lactams, such as carbenicillin and sulbenicillin, when it forms a tripartite complex with OprA but not with OprM. Interestingly, both MexXY-OprA and MexXY-OprM tripartite complexes can extrude aminoglycosides, whereas OprA confers an expanded substrate profile.⁶⁶⁵ This suggests a new role for OMF components of tripartite efflux systems in the vetting of substrates. In other studies, the PAPs ZneB and CusB of the tripartite ZneCAB and CusABC RND efflux systems, respectively, have been reported to contribute to the metal ion specificity of the two systems.⁴⁶,³⁶⁰ Thus, these components may also present themselves as possible chemotherapeutic targets.

## 12.2. Targeting the Periplasm Adaptor Protein

Although the bulk of EIs described in the literature are reported to target inner membrane transporters, the PAP component of tripartite systems has also been shown to be a viable target for efflux inhibition. Abdali et al.⁸⁸³ carried out the parallel experimental and structure-based screening of two drug libraries to identify inhibitors of AcrA, the PAP component of AcrAB-TolC. Several EIs were identified and shown to bind to AcrA, change its structure in vivo, and potentiate the activities of several antibiotics in *E. coli* and other Gram-negative bacteria. Additionally, the identified EIs, including NSC 60339 (10; Figure 56), were found to require AcrA binding to inhibit efflux activity. Further optimization efforts of compound 9 lead to more potent derivatives, such as SLUPP-225 (11; Figure 56), which was shown to penetrate the outer membrane of *E. coli* cells and exhibit more potent efflux inhibition activity.⁹⁰⁰ Later, Darzynkiewicz et al.⁹⁰¹ investigated the potential binding sites of compound 9 and found that it likely binds to a site situated between the lipoyl and β-barrel domains of AcrA. The β-barrel domain of AcrA has been previously shown to be involved in RND binding, and the lipoyl domain is involved in self-association; therefore, AcrA inhibitors may function to prevent the functional assembly of AcrAB-TolC.⁹⁰² In a recent study, Green et al.⁹⁰³ searched the ZINC15 database for compounds with physicochemical properties matching those of existing EIs and Gram-negative antibiotics. Subsequently, compounds with matching criteria were computationally docked onto AcrA of *E. coli* at the previously reported binding site of compound 9, as well as several other sites that when disrupted have been reported to disrupt efflux. The top 34 hits from all docking poses were then analyzed using a combination of *in vitro* binding assays to measure binding affinity to AcrA and AcrB and *in vivo* antibiotic potentiation assays. They identified six compounds (12a–f, Figure 56) with a shared scaffold that bound to AcrA within the millimolar range as verified by SPR. Given that the compounds had weak binding affinities, it is
possible that they bind to AcrA weakly at multiple sites. Promisingly, the six compounds also potentiated the activities of erythromycin and novobiocin in hyperporinated *E. coli* cells, as well as in wild-type *A. baumannii* and *K. pneumoniae*. However, PAPs are promiscuous, which must be taken into account when targeting PAPs to inhibit efflux. McNeil et al.⁹⁰² demonstrated that in *S. enterica* inactivation of AcrA results in AcrE compensating for its loss, indicating interchangeability between the two PAPs. Therefore, a successful PAP inhibitor would need to target both AcrA and AcrE in *S. enterica* to inhibit efflux.

## 12.3. Targeting the Outer Membrane Factor

The OMF component of tripartite efflux systems plays an important role in expelling substrates across the outer membrane to the external environment. As a paradigm OMF, TolC is rather promiscuous and functions with several different tripartite efflux systems, including T1SS, RND, ABC, and MFS efflux systems. Despite being an attractive drug target, TolC inhibition is relatively under-researched. Higgins et al.⁹⁰³ reported the crystal structure of TolC in complex with hexaamminecobalt (HC), a trivalent cation, and showed that it blocked the periplasmic entrance of TolC by forming salt bridges with D374 of each TolC monomer. Later, Gilardi et al.⁹⁰⁴ investigated the biophysical interaction of HC with TolC and found that it binds to TolC with a high affinity and fast association and dissociation rates. Furthermore, HC was found to not exhibit any intrinsic antimicrobial activity nor potentiate antibiotic activity in *E. coli*. Thus, further research is required to identify more potent TolC inhibitors capable of inhibiting efflux and potentiating antibiotic activity. However, TolC inhibition may not be a good target for efflux inhibition in practice. Krishnamoorthy et al.⁶⁵³ found that the activity and assembly of the AcrAB-TolC complex in *E. coli* can tolerate substantial fluctuations in the amount of TolC available. Furthermore, they reported that only a small fraction of intracellular TolC is necessary for the efflux requirements of *E. coli* cells, indicating that significant depletion of TolC is well tolerated in bacterial cells.

## 12.4. Other Strategies to Inhibit Efflux

Although chemical inhibition of efflux is predominantly reported in the literature, there have been recent developments in inhibiting efflux through different strategies. One example is using antisense oligomers to reduce or inhibit efflux pump protein translation in a sequence specific manner. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are an example of synthetic nucleotide oligomers that inhibit antisense mRNA translation. Ayhan et al.⁹⁰⁵ developed and identified an *acrA-PPMO* capable of increasing the efficacy of several antibiotics, such as cefotaxime, chloramphenicol, clindamycin, and fusidic acid, by up to 40-fold and preventing AcrA protein translation in a dose-dependent manner in *E. coli*. Additionally, the *acrA-PPMO* was shown to be effective in *K. pneumoniae* and *S. enterica* by significantly increasing the activity of piperacillin-tazobactam. More recently, an optimized *acrA-PPMO* was shown to potentiate the activity of azithromycin and levofloxacin in several clinical Enterobacteriaceae strains. Crucially, the *acrA-PPMO* was demonstrated to increase the efficacy of azithromycin *in vivo* in a *K. pneumoniae* septicemia murine model. Inhibition of MexAB-OprM, the homologous RND system in *P. aeruginosa*, was also reported to enhance the efficacy of several antibiotics. PPMOs targeting *mexA* and

*mexB* were found to enhance the activities of azithromycin, cefotaxime, and piperacillin-tazobactam by at least 4-fold in the majority of tested clinical *P. aeruginosa* isolates.⁹⁰⁶ In a different study, antisense oligonucleotides were developed to inhibit the translation of the *cmeA* gene, which encodes for the periplasmic adaptor protein CmeA of CmeABC in *C. jejuni*. The *cmeA* antisense oligonucleotide was reported to sensitize *C. jejuni* cells to ciprofloxacin and erythromycin at very low concentrations.⁹⁰⁷

Another strategy for inhibiting efflux is through CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRIPSR-associated protein)-mediated genome editing. Owing to the versatility of CRISPR-Cas, it has also been demonstrated to sensitize bacteria to antibiotics by targeting antibiotic resistance genes, such as *β*-lactamases.⁹⁰⁸ A recent study demonstrated that CRIPSR-Cas can also be used to delete efflux genes. As a proof of principle, Xu et al.⁹⁰⁹ used native CRISPR-Cas in *P. aeruginosa* to delete the *mexB* efflux gene, thereby sensitizing cells to several antipseudomonal antibiotics. Although in its infancy, the CRIPSR-Cas system has the potential to specifically target the sequence of bacterial efflux pump genes, thereby avoiding the problem of toxicity associated with off-target effects of classical EIs. Peptide-based approaches have also been explored as a means to inhibit tripartite efflux systems. By exploiting the transmembrane AcrB TM1–TM8 protein–protein interface, Jesin et al.⁹¹⁰ designed synthetic peptides to mimic TM1 or TM8 of AcrB to interfere with AcrB trimerization. Using Nile Red efflux assays and antibiotic susceptibility testing, TM1 and TM8 peptides were shown to significantly reduce efflux activity and potentiate the activity of antimicrobials. Targeting protein–protein interactions between components of tripartite efflux systems to prevent complete assembly could be another strategy to inhibit efflux.

## 13. CONCLUSION

The Gram-negative double membrane is a formidable barrier across which many molecules must be transported. As shown above, bacteria have addressed this challenge by evolving a diverse array of tripartite efflux pumps and type 1 secretion systems, many of which play important roles in the export of xenobiotics and toxic compounds, including antibiotics and a number of other cargoes, including very large virulence factors and adhesion molecules.

We have shown that despite the apparent diversity in structure, function, and role between the complexes there are commonalities between seemingly disparate systems. In particular, our comparative analysis has highlighted the roles of the periplasmic adaptor proteins, indicating that far from being passive connectors they in fact are active architects of the assembly and couple allosteric transitions between the inner membrane transporters and outer membrane factors.

In the case of the tripartite systems which utilize proton motive force, the association of the PAPs with the respective RND- and MFS-based transporters allows these assemblies to combine two different electrochemical gradients across two separate semipermeable membranes in a unique way. This is in stark contrast to classical transporters that perform transport of substrate and counter-transport of ions in opposite directions by coupling their respective electrochemical gradients created across a single membrane.

Unifying the wealth of functional data with the recent advances in the structural biology of tripartite assemblies
allows us to propose an integrated model of their assembly and
functional cycling. The importance of many of these tripartite
systems in resistance to antibiotics and also in virulence makes
them attractive targets for the development of inhibitors. While
much progress has been made in this field, we believe that a
better understanding of the structure–function relationship of
individual efflux systems alongside an integrated view across
families of these systems will accelerate progress toward finding
clinically useful inhibitors.

14. REMAINING CHALLENGES AND FUTURE DIRECTIONS

Despite recent progress, there are still some persistent
questions that remain unanswered within the field. As our
understanding of the basic structure of the isolated
components of tripartite pumps has grown, the research
focus direction has been shifting toward the complete
assemblies, including specifically the control of their assembly
and disassembly, which could be targeted for therapeutic
purposes.

One clear tendency is moving away from *in vitro* studies toward understanding the more dynamic aspects of their function and regulation, especially in the context of the cell. This includes on one hand the structural aspect, e.g., where the potential of future use of cryo-electron tomography has been recently demonstrated and the resolution of the reported *in situ* protein assemblies is rapidly increasing. On the other hand, it also applies to the functional characterization of these assemblies, where significant headway has been made by creation of functional reconstitution assays in native membrane environments. In combination with the rapidly developing field of transportomics, these developments promise to accelerate the characterization of substrate specificity, as well as the screening of potential efflux inhibitors (EIs) targeting these assemblies.

These technologies also promise to address the remaining
questions in the energetics of the pump assembly and cycling;
as e.g. the H⁺/drug stoichiometries of RND assemblies remain
to be established and are difficult to address *in vivo*, they thus
would benefit from the controlled environments and sensitivity
afforded by such studies. Similarly, the exact nature of the
power-stroke and efficiency of the cycle for ABC transporters,
which remain a much-discussed subject, could hopefully be
addressed by a combination of structural studies of trapped
intermediates and functional analysis.

Modularity of the assemblies, in the form of pumps
presenting e.g. heterooligomeric IMPs or PAP components,
could potentially provide a way to impose regulation on the
efflux systems. This is most likely kinetic regulation. While
cooperative kinetics have been shown for AcrAB-TolC, its
implication for the cell physiology is obscure. One possible
explanation for that could be the high energy consumption of a
permanently active pump, resulting in wasteful drainage of the
proton gradient. In the case of lower concentrations of the
drug, a more moderate pace would save energy, whereas at
high drug concentrations the pump is fully activated. However,
clear experimental proof of the above model is still awaiting.

While the increased understanding of interprotomer
interactions within the RND complexes has allowed the
development of the integrated model above, the exact roles of
PAP1 and PAP2 protomers remain to be fully validated
experimentally. The existence of systems such as TriABC,
which utilize separate PAPs, also poses the question of their

biological function and the benefits that these provide for the
bacterium. While our current models of the tripartite complex
suggests that the transporters and OMFs do not interact
directly and that such interaction is solely moderated by the
PAPs, it is difficult to reconcile these models with the existence
of PAPs such as BesA which lack their hairpin altogether,
suggesting alternative modes of interaction and assembly are
possible, if even in just a subset of tripartite assemblies.

Also, while understandably the RND transporters have
attracted the lion's share of research interest over the past
decade, the understanding of the mechanisms of the other
tripartite assemblies is still lagging behind, and notably the
questions of the transmembrane allostery in the T1SSs and
MacB-based systems needs to be elucidated. As shown above,
full understanding of the transport cycles of these assemblies
could be achieved by studying the transporters in isolation due
to the close involvement of the PAP in their function, which
has been largely ignored to date.

Given the broad substrate specificity of the multidrug efflux
pumps, it is unclear why they are presented in so many
versions in the bacterial genomes. The overlapping specificities
and joint action of the pumps, especially in the context of
multiple antibiotic stress, need to be clarified. The under-
standing the synergistic effects of the activity of the members
of the different (super)families is also a rapidly developing field
and will likely benefit from the wider deployment of the
technologies listed above.

Antimicrobial resistance is a current global health
er emergency, and tripartite efflux pumps are an important
mediator of this resistance. Efflux pumps underpin many
mechanisms of resistance; for example, they not only directly
lower the intracellular concentration of antibiotic drugs but
also reduce the frequency at which mutations conferring
resistance via other mechanisms are selected. One significant
future challenge will be in developing new drugs that are not
subject to resistance via efflux. There has been significant
progress in understanding which drugs can be transported by
respective efflux systems and the specific residues involved in
their transport. In the future, a complete understanding of
substrate export will be needed to enable development of drugs
that cannot be transported by major efflux pumps and for
which selection of simple mutations does not permit
development of resistance.

The concept of inhibitors to potentiate the use of existing
and novel antibiotics holds a lot of promise, and there is also
evidence they may be useful as antivirulence or antibiofilm
compounds. However, no EIs have yet successfully reached the
clinic largely due to issues with either potency or toxicity.
Development of effective and safe EIs is an enormous future
challenge, but better understanding of the structure, assembly,
and mechanism of these systems will aid rational drug design
approaches and rationalize the mechanism of compounds
detected through high-throughput screening approaches. As
much of the current effort has been targeting transporters, the
promise of inhibitors targeting either OMFs or PAPs or
interfering with assembly of the functional pump has yet to be
fulfilled.

Efflux pumps and secretion systems are increasingly
important for biotechnological and industrial applications.
Efflux pumps are critical for increased export of synthesized
products to aid isolation and in other cases for providing
resistance to toxic products which can then increase yield. In
contrast to the medically related future challenges, in this
sphere, work to optimize transport of specific products is needed to improve the efficiency of industrial and biotechnological applications, and this will rely on a future better understanding of substrate export. Better understanding of the molecular determinants of the interprotomer recognition and the promiscuity of the assembly may allow the creation of “designer pumps” with a priori desired specificities.

## ASSOCIATED CONTENT

### Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrev.1c00055.

Molecular morphing movie illustrating a possible transition from the OMF-free state to the OMF-engaged state (zip)

## AUTHOR INFORMATION

### Corresponding Authors

Jessica M. A. Blair – Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom; ● orcid.org/0000-0001-6904-4253; Email: j.m.a.blair@bham.ac.uk

Vassiliy N. Bavro – School of Life Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom; ● orcid.org/0000-0003-2330-8924; Email: v.bavro@essex.ac.uk

### Authors

Ilyas Alav – Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

Jessica Kobylka – Institute of Biochemistry, Biocenter, Goethe Universität Frankfurt, D-60438 Frankfurt, Germany

Miriam S. Kuth – Institute of Biochemistry, Biocenter, Goethe Universität Frankfurt, D-60438 Frankfurt, Germany

Klaas M. Pos – Institute of Biochemistry, Biocenter, Goethe Universität Frankfurt, D-60438 Frankfurt, Germany

Martin Picard – Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, CNRS UMR 7099, Université de Paris, 75005 Paris, France; Fondation Edmond de Rothschild pour le développement de la recherche Scientifique, Institut de Biologie Physico-Chimique, 75005 Paris, France; ● orcid.org/0000-0002-6518-8900

Complete contact information is available at: https://pubs.acs.org/10.1021/acs.chemrev.1c00055

### Author Contributions

J.K. and M.S.K. contributed equally

### Notes

The authors declare no competing financial interest.

### Biographies

Ilyas Alav is a Ph.D. student in Microbiology and Infection at the University of Birmingham. He received his B.Sc. (Hons) in Biochemistry from the University of Nottingham in 2016. He then went on to obtain his M.Sc. in Pharmacology from King's College London in 2017. He joined the group of Jessica Blair for his Ph.D. thesis in 2018. His current research focuses on targeting efflux pumps to combat antibiotic resistance in *Salmonella* Typhimurium, with a particular interest in periplasmic adaptor proteins. His Ph.D. position

is funded by the Midlands Integrative Biosciences Training Partnership.

Jessica Kobylka graduated with a diploma in Chemistry from Johannes Gutenberg University in Mainz in 2017. She joined the Pos lab in 2016 for her diploma thesis and started with her Ph.D. thesis in 2018. Her current research interest is the functional analysis of RND transporters from Gram-negative bacteria.

Miriam S. Kuth received her B.Sc. in Biochemistry in 2016 and her M.Sc. in Chemistry in 2018 from the Goethe-University Frankfurt/ Main. She joined the Pos lab in 2019 for her Ph.D. thesis in Biochemistry. Her current research focuses on functional and structural analysis of RND transporters. Her position is funded by the Friedrich Naumann Foundation for Freedom (https://fnst.org/).

Klaas M. Pos is Professor of Membrane Transport Machineries at the Institute of Biochemistry, Goethe University Frankfurt. His research program includes the structural and functional elucidation of Gram-negative efflux pumps. These pumps are involved in many essential processes of the Gram-negative cell, including the efflux of antibiotics. The Pos lab has keen interest into the molecular determinants of drug transport and the energy-coupling, as well as inhibitor binding to the RND pumps.

Martin Picard is a CNRS Research Director in the Laboratory of Physico-Chemical Biology of Membrane Proteins at IBPC, Université de Paris. He tackles the problem of membrane transport as an interdisciplinary approach by combining structural information and biophysical and biochemical techniques. His group has developed innovative *in vitro* tools to monitor the assembly and function of multipartite bacterial transporters in proteoliposomes.

Jessica Blair is a BBSRC David Phillips Fellow and Lecturer in Antimicrobial Resistance at the University of Birmingham, UK. Jess got her undergraduate degree in Biological Sciences from the University of Oxford in 2006 and a Ph.D. in Microbiology from the University of Birmingham in 2010. She runs a research group in the Institute of Microbiology and Infection investigating the molecular mechanisms of antibiotic resistance with a focus on efflux pumps.

Vassiliy N. Bavro is a Reader (Associate Professor) of membrane protein science at the University of Essex, Colchester, UK. He received his Ph.D. with Winfried Weissenhorn at European Molecular Biology Laboratory (EMBL), Grenoble, France, in 2003 and was a postdoctoral and Marie Curie Fellow with Ben Luisi at Dept. of Biochemistry, University of Cambridge, UK (2003–2007), studying the mechanisms of the TolC-family. Following that he was a senior postdoctoral fellow with Stephen J. Tucker at the Dept. of Physics, University of Oxford, UK (2007–2012), working on ion-channels. He started his own group as a Birmingham Fellow at the University of Birmingham, UK in 2012 focusing on the structural biology of tripartite efflux systems, with particular interest in the contribution of PAP proteins to the assembly, and his group continued in this direction after moving to the School of Life Sciences at the University of Essex in 2016.

## ACKNOWLEDGMENTS

We thank Dr. Martyn Symmons for discussion, advice, and help with topology diagrams throughout and William Batista Dos Santos for input on the OMF section. The research in the Bavro lab was partially supported by funding from BBSRC (BB/N002776/1). Work in the Pos lab is supported by the German Research Foundation (DFG-SFB 807, Transport and Communication across Biological Membranes and DFG-FOR2251, Adaptation and persistence of the emerging
pathogen *Acinetobacter baumannii*). M. S. Kuth is a recipient of a fellowship from the Friedrich-Naumann-Stiftung für die Freiheit. Work in the Picard lab is supported by the 'Initiative d'Excellence' program from the French State (Grant 'DYNAMO', ANR-11-LABEX-0011-01) and by ANR (ANR-19-CE44-0015-01). Work in the Blair lab is supported by a BBSRC David Phillips Fellowship to J.M.A.B. (BB/M02623X/1). I.A. was funded by the MIBTP2 BBSRC BB/M01116X/1 at the University of Birmingham. Molecular graphics in Figures 1, 4, 7, and 9 were created with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. Figures 2, 3, 11, 16, 18, 33, 53, and 55 were created with BioRender.com. All other molecular graphics were produced with the help of Open-Source Pymol, The PyMOL Molecular Graphics System, Version 2.4 Schrödinger, LLC.

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