<figure><img src="image_28.png" /><figcaption>**Figure 17.** General organization of PCAT transporters associated with T1SS. (A) AaPrtD based on the PDB ID 5L22. Note the highly kinked TM3 and TM6 and the supposed "substrate entry window". Unlike the PCAT transporters discussed previously, the AaPrtD does not present a wide substrate binding cavity and is suggested to transport unfolded cargoes. The ADP moiety (in red spacefill) is found bound to the protomers. Note the lack of CH1 helix at the end of TM2. (B) Homology model of HlyB, highlighting features common to both PCAT1 and PrtD. The CLD-domains are predicted to aid positioning of the cargo into the substrate entry window.</figcaption></figure>

transport cycle at the resetting step from the OF to the IF state. As we will in the later sections, such energy dependency may have parallels with the pump cycle of the RND-transporter-based tripartite assemblies.

**5.6.9. PCATs Associated with T1SS.** The transport mechanisms discussed above, while possibly applicable to bacteriocin transporters in Gram-positive organisms, do not appear to be feasible for the PCAT transporters operating as part of a T1SS in Gram-negative bacteria, as these are often carrying cargoes in excess of 8000 amino-acid long and over 1 MDa in weight,³,¹⁸,⁴⁴⁹ while in S. *enterica*, the giant nonfimbrial adhesin SiiE (595 kDa), exported by the *Pathogenicity Island 4-encoded type I secretion system* (SPI4-T1SS), is the largest protein in the *Salmonella* proteome and consists of 53 repetitive bacterial immunoglobulin domains (Big).⁵²⁶ As known for other T1SS substrates, such as E. *coli* HlyA, Ca²⁺ ions bound by conserved D residues within the Big domains stabilize the protein and facilitate secretion.⁵²⁷,⁵²⁸ Exporting such cargoes would require a departure from the classical "alternate access" mechanism discussed above in favor of some form of continuous threading. Furthermore, several lines of experimental evidence suggest that T1SS substrates must be unfolded in order to be translocation competent,⁵²⁹,⁵³⁰ resulting in the length of an unfolded T1SS substrate that would far exceed the length of the transporter TMD cavity itself. These observations imply that T1SS ABC transporters must operate by a distinct mechanism, where the unfolded polypeptide is likely progressively threaded through the TMD of the transporter over multiple ATP-hydrolysis cycles.³,⁴⁴⁹,⁵¹² These hypotheses were confirmed by the structure of the first ABC transporter associated with T1SS—namely the *AQUIFEX AEOLICUS* PrtD (AaPrtD) coded by the *abcT5* gene (PDB ID: 5L22; UniProtKB: O67184).⁵¹⁵ The PrtD is the ABC transporter within the protease secretion system PrtDEF, best known from the phytopathogen *Dickeya dadantii* (*formerly known as Erwinia chrysanthemi*),⁵³¹ responsible for the secretion of a number of proteases encoded in the same operon—namely, PrtA, PrtB, PrtC, and PrtG.⁵³² Secretion of

the *Dickeya dadantii* protease DdPrtG across the plasma membrane requires a fully assembled PrtDEF complex, and secretion of PrtG *in vivo* requires ATP-hydrolysis by PrtD.⁵¹⁵

The AaPrtD structure was revealed in an occluded, ADP-bound state,⁵¹⁵ and despite lacking C39 or CLD-domains,³ the structure is significant as it provides a blueprint for the wider RTX-associated subfamily of T1SS transporters and it reveals an overall organization, which while similar to the standard PCAT transporters such as the PCAT1 discussed above, has a number of distinct structural features. In contrast to the other PCAT transporter structures available to date, e.g., PCAT1, the hydrophilic TM cavity of which allows at least partial folding of the cargo,²⁷³ the substrate channel of PrtD has been shown to be very narrow and to run throughout the structure for some 40 Å, spanning the entire membrane and ending just shortly before the periplasmic exit. In that it shows analogies to the SecY channel⁵³³ and due to its steric restrictions appears unable to accommodate folded substrate (Figure 17). The structure also suggests a "substrate entry window" just above the NBD domain formed by the TM helices 4 and 6, which is gated by a conserved R307 on the cytoplasmic side,⁵¹⁵ while the periplasmic opening is gated by a hydrophobic seal, similar to the "pore-ring" of the SecY channel.⁵³⁴

AaPrtD also shows a distinct architecture of its TMD, with uniquely kinked TM3 and TM6 helices near the cytosolic solvent-lipid interface. These kinked helices point toward the dimer interface, creating a highly basic concave "bowl" on the PrtD surface formed by TM1, TM3, and TM6. Furthermore, the coupling helix between TM2 and TM3 (CH1), which packs against the NBD of the same subunit, was found to be replaced by an extension of TM2 and an extended loop leading into TM3 presenting a unique architecture among type IV ABC transporters.⁵¹⁵ It is notable that both the "concave bowl" and the aforementioned "substrate entry window" are highly basic, which is speculated to play a role in electrostatic stabilization of the T1SS substrates which tend to be fairly acidic. The absence of the coupling helix leading into TM3 is suggested to reflect substrate entering from the cytosolic
<figure><img src="image_29.png" /><figcaption>**Figure 18.** The function of the tripartite pump MacAB-TolC in toxin secretion and macrolide resistance. MacAB secretes ribosomally synthesized and post-translationally modified peptides (RiPPs), such as enterotoxin STII, that are targeted and exported to the periplasm by the SecY-secretion signal (purple). Following cleavage and disulfide bond formation in the periplasm, the toxin is then secreted out by the MacAB-TolC. Figure modified based on Crow et al.<sup>431</sup></figcaption></figure>

window between TM4 and TM6. Thus, while the *AaPrtD* architecture is similar to the classic 6 + 6 TMD dimer observed in the type IV transporters, the shape and the character of the transporter conduit channel differ markedly from the canonical peptide transporters discussed in the previous sections<sup>515</sup> (*Figure 17*).

The structure of *AaPrtD* also hints at specific adaptations that are relevant to the formation of the tripartite T1SS and shared with other such transporters, including the hemolysin A transporter HlyB and the related *Mannheimia* (formerly *Pasteurella* haemolytica leukotoxin pump-associated ABC transporter LktB*,<sup>535</sup> both of which serve as prototypes pumps for a widely disseminated family of virulence factors, known as the RTX (repeats-in-toxin) cytotoxins. To facilitate the analysis, we have created a hybrid homology model of HlyB based on the *AaPrtD* and PCAT1 structures (*Figure 4* and *Figure 17*).

In comparison with the peptide transporters, the cargo pathway in the T1SS-associated PCATs is strikingly different and much more restrictive, which is achieved by the aforementioned kinks in the TM3 and TM6 leading to constriction of the substrate channel. These kinks are stabilized by conserved sequence motifs, which are shared by the T1SS ABC transporters but not found in peptide PCAT transporters—e.g., the TM3 helix is split into three sections, with the TM3a-TM3b kink being enforced by an “FxT-motif”,<sup>515</sup> which in *AaPrtD* is formed by F128 and T130, while in HlyB the corresponding residues are F264 and T266. A second conserved motif is present on TM6 in the form of a bulky hydrophobic (usually W; W295 in PrtD; W431 in HlyB), followed by a hydrophilic residue (R296 in *AaPrtD*; Q432 in HlyB). These motifs stabilize the kinks via a network of specific inter- and intraprotomer interactions.

In addition, HlyB seems to share with the *AaPrtD* a hydrophobic “gasket-ring”, on the periplasmic side (PrtD M43, Y47, I425; PCAT I190, F194, L425/L426; HlyB F174/F175,

M179/L410/I411) that is speculated to prevent dissipation of the proton gradient during transport and the basic-residue gating (PrtD R307/HlyD R443) at the putative substrate entry window. The orientation of the CLD-domain of the HlyB positions its N-terminal tail in direct proximity to the entry of the predicted substrate entry window, which is consistent with the position of the threaded peptide seen in the CtA-bound PCAT1.<sup>505</sup>

In summary, the recent structural snapshots discussed above strongly suggest a common overall cargo-selection and substrate-feeding mechanism between the PCATs associated with peptide transport and those participating in the T1SS. Unlike the peptide-transporting PCATs, the latter do not allow folding of the substrate throughout the single transporter cycle and necessitate multiple processive steps to transport the whole cargo protein, in a mechanism that requires further elucidation.

## 5.7. MacB/FtsX Transporter Family

Initially identified via a bioinformatic search as one of five putative ABC transporter clusters in the *E. coli* genome,<sup>536</sup> the *macAB* genes were originally designated *ybjYZ*. The genes were later renamed to *macAB* (macrolide-specific ABC-type efflux carrier) as the pump was reported to transport several macrolides.<sup>60</sup> However, the relevance of this function *in vivo* remains somewhat questionable, as in *E. coli* it is only pronounced in cells with a deactivated primary AcrAB multidrug efflux pump, and the loss of the orthologous system in *Neisseria gonorrhoeae* did not lead to a pronounced decrease in resistance to azithromycin or erythromycin.<sup>78</sup> In addition, the MacAB requires TolC for its function to form a tripartite system.<sup>60</sup> Related tripartite systems have been identified in a number of Gram-negative organisms, such as *MacBsm* of *Stenotrophomonas maltophilia*<sup>116</sup> and the PseEF pump of *Pseudomonas syringae*.<sup>537</sup>
Strengthening the case for the MacB-family members as bona fide multidrug transporters is the *E. coli* heterooligomeric YbhFSR, which appears to function with the PAP coded by the *ybhF* gene (P0A9U1).<sup>538</sup> This tripartite system can efflux cephalosporins, including the third generation cefoperazone. Recently, Feng et al.<sup>62</sup> characterized the YbhFSR system and found it to export tetracycline, oxytetracycline, chlortetracycline, doxycycline, EtBr, as well as the Hoechst 33342 dye. Furthermore, uniquely for the ABC-family, the findings of Feng et al.<sup>62</sup> suggested that YbhFSR exhibits a dual function both as a MDR efflux pump and as a Na<sup>+</sup> (Li<sup>+</sup>)/H<sup>+</sup> antiporter, although this requires further investigation, especially in light of the rather high homology of the YbhFSR protomers with MATE transporters such as NorM.

**5.7.1. Biological Functions.** In addition to xenobiotics, the MacB-family pumps are associated with the secretion of a wide range of nonantibiotic targets, including the heat-stable enterotoxin II in *E. coli*<sup>154</sup> and the cyclic lipopeptide entolysin in *Pseudomonas entomophila*.<sup>539</sup> The cargoes range in size from ~500 Da for macrolides to ~10 kDa for dispersins,<sup>540</sup> suggesting low selectivity of the MacB pump. Notably, and unlike the typical T1SS which it resembles, the secretion of the heat-stable toxins by MacB-related pumps appears to be Sec-dependent in the first stage and requires a Sec-secretion signal.<sup>154</sup> MacB copurifies with and binds specifically to phosphatidylethanolamine, in an interaction that survives native mass spectrometry in the gas phase,<sup>541</sup> and the related LolCDE system is differentially modulated by different phospholipids<sup>542</sup> indicating possible lipid transport and/or modulation. MacAB appears to be capable of binding to the core lipopolysaccharide, although its exact role is unclear<sup>543</sup> (Figure 18).

In a related system in *Pseudomonas syringae*, PseEF was found to secrete the lipodepsinonapeptides syringomycin (*syrB1*) and syringopeptin (*sypA*),<sup>537</sup> and it was required for the full virulence of the bacterium. The MacAB-TolC pump has also been implicated in the efflux of protoporphyrin IX (PPIX) in *E. coli*, and mutants of macAB or tolC were reported to accumulated PPIX and become sensitive to photo-inactivation.<sup>155</sup> Also, in *Pseudomonas*, an orthologous system known as PvdRT-OpmQ is involved in the secretion of the primary virulence factor pyoverdine, which is a siderophore, acting as both iron-carrier and a virulence-related signaling molecule.<sup>544,545</sup> In enteroaggregative *E. coli* (EAEC), an export system for dispersin, a 10.2 kDa protein which promotes bacterial dispersal across the intestinal mucosa<sup>540</sup> able to convert plasminogen into plasmin,<sup>546</sup> is encoded by a virulence plasmid pAA and has been linked to an MacB-like pump AatPABCD where the AatP is a permease, AatC is an NBD, and AatA is a TolC-like OMF.<sup>547</sup>

In enterotoxigenic *E. coli* (ETEC), the predicted tripartite complex CexPABC, which consists of the MacB homologue CexP (permease)/CexC (NDB), the PAP CexB and the OMF CexA<sup>548</sup> appear to be associated with the export of the coat protein and virulence factor CexE, later found in *Yersinia enterocolitica*, *Providencia alcalifaciens*, and *Citrobacter rodentium*.<sup>549</sup> Intriguingly, despite similarities with the T1SS, this exporter was shown to accept its cargo from the periplasm, where CexE accumulates, and to rely on the general secretory pathway.<sup>548,549</sup> At the time of writing of this review, the roles of the auxiliary transmembrane proteins CexD and AatD are yet to be established. In *Salmonella* Typhimurium, the nonribosomally synthesized siderophore enterobactin is

produced in response to iron starvation and can be processed into salmochelin and several linear derivatives.<sup>550</sup> Trimers of such linearized enterobactin have been shown to protect S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion.<sup>550</sup> Furthermore, in a mouse model of *Salmonella* infection, the deletion of MacB impacted virulence,<sup>101</sup> while MacAB was found to be required for survival of *Salmonella* in cultured macrophages which produce reactive oxygen species (ROS) but was not essential in macrophages which did not generate ROS.<sup>138</sup> Consistent with a stress-response interpretation, expression of *macAB* in *E. coli* and *Salmonella* is inducible and regulated by the two-component system PhoPQ<sup>101,551</sup> which among other things, senses antimicrobial and pH stress, e.g., responding to acidification of the phagosome.

A large group of MacB-like transporters have been described in the multicellular cyanobacteria such as *Anabaena* and *Nostoc* (for recent reviews please consult Shvarev and Maldener<sup>552,553</sup>) that have been found to associate with diverse functions covering heterocyst development and diazotrophic growth (e.g., HgdBCD system, DevBCA system,<sup>554</sup> All0809/8/7); iron metabolism (All2652/1); as well as drug resistance (Alr4280/1/2 and Alr3647/8/9).<sup>553,555</sup> These systems seem to utilize a single OMF HgdD<sup>556</sup> (see section 7 and associated Figure 33A for details). One of the better studied examples includes the DevBCA-HgdD.<sup>557–559</sup> The pump component genes are *devB* (*alr3710*), coding for the PAP protein; *devC* (*alr3711*), coding for TMD; and *devA* (*alr3712*), coding for the NBD, respectively, and they are positioned on a single operon. The *Anabaena* could be considered a multicellular organism, as it forms filamentous nonbranching strings of cells called “trichomes”, which, in the presence of combined nitrogen cells are composed mostly of vegetative cells, but in its absence, the *Anabaena* develops specialized cells, called heterocysts,<sup>560,561</sup> which provide N<sub>2</sub>-fixation capability and transport nitrogen assimilation products to neighboring vegetative cells in exchange for sugars produced by photosynthesis.<sup>562</sup> These heterocysts build an additional envelope around the outer membrane, which is itself composed of two layers: an external heterocyst exopolysaccharide (hep) layer and an underlying heterocyst glycolipid layer (hgl). The hgl layer is made of heterocyst-specific glycolipids (HGLs) and is airtight, restricting oxygen from penetrating into the heterocyst, creating a microoxic environment which is essential for dinitrogen fixation by nitrogenase.<sup>553,559,561</sup> DevBCA forms a heterocyst-specific glycolipid exporter that is essential for the export and development of the hgl layer. To transfer glycolipids through the Gram-negative cell wall, DevBCA has to recruit a TolC-homologue protein called HgdD, that is encoded by the gene *alr2887*.<sup>556,563</sup> The *Anabaena* sp. *hgdD* mutant, like the *devB*, *devC*, and *devA* mutants, does not form the hgl layer and hence cannot grow diazotrophically; that is, they are unable to grow without external sources of fixed nitrogen.<sup>556</sup> The HgdD OMF protein is central to secretion and efflux in *Anabaena*, as it has been shown to pair with at least seven different DevBCA paralogues, with varied functions. While some of these have partially overlapping functions and are involved in heterocyst-specific roles, at least some of these DevBCA paralogues such as the tripartite pumps formed of the gene products of *alr4280/alr4281/alr4282* and *alr3647/alr3648/alr3649* that code for the components of putative MacB-like ABC exporters have been shown to be essential for resistance to a range of different drugs and efflux of ethidium
<figure><img src="image_30.png" /><figcaption>**Figure 19.** Structural organization of MacB. (A) General topology of the single MacB protomer highlighting the N-terminal nucleotide binding domain (NBD; magenta), the connecting helix (CoH), the transmembrane domain (TM domain) with its four helices, as well as the periplasmic core domain (PCD), which is composed of a PD-module and a SABRE subdomain (green). CH1 and CH2, coupling helices 1 and 2, respectively. (B) Two orthogonal orientations of the single protomer of MacB from *A. baumanii* based on PDB ID 5WS4,²⁴ highlighting the principal secondary structure elements. The blue oval delineates the PD. (C) View of the complete MacB-dimer.</figcaption></figure>

bromide, while having no demonstrable role in heterocyst
development.⁵⁶⁴

In addition, the YbbAP is a poorly characterized member of
the MacB-family in *E. coli*,⁴¹⁹ where *ybbA* encodes the
predicted NBD subunit and *ybbP* encodes the predicted
permease subunit of this putative exporter complex. Although

association with heavy metal export has been suggested,⁴¹⁸ this
member of the family remains poorly understood.

**5.7.2. Gram-Positive Homologues of the MacB Transporters.** Within the wider family of MacB-like transporters, there are a number of members from Gram-positive organisms; e.g., in *Bacillus subtilis* the permease YxdM, forming part of the ABC transporter YxdLM, appears to be involved in resistance
to antibacterial cationic peptides.⁵⁶⁵ Also, in *B. subtilis*, YtrF appears to be a permease forming part of the YtrBCDEF complex that plays a role in acetoin utilization.⁵⁶⁶ While very little is known about the structure of these transporters, our bioinformatic analysis suggests that they possess significantly more expansive PCDs than the rest of the MacB-family, possibly presenting a duplicated PD-module/LolC-like extension.

The *yknzWXYZ* operon in *Bacillus subtilis*⁵⁶⁷ has been found to code an unusual four-component transporter system, which is built around the principal YknY (NBD)/YknZ (permease) ABC transporter unit which is homologous to MacB. The system also associates with a cognate PAP (YknX) and a four-TM additional membrane protein (YknW)⁵⁶⁸ to provide protection against sporulation-delaying-protein-induced killing by the 42-residues long disulfide-linked SDP toxin.⁵⁶⁹ The pump appears to protect *B. subtilis* against both endogenous and exogenous SDP, and YknW is critical in the assembly and functionality of the complex, as YknXYZ complex alone does not protect against SDP, while overproduction of YknW alone provides limited protection.

An investigation of the response of *Streptococcus pneumoniae* D39 gene-expression patterns to three antimicrobial peptides—namely nisin, bacitracin, and the human cationic peptide defensin LL-37 identified upregulation of a hitherto uncharacterized operon (*Sp0785*–0787), the deletion of which increased the cell-susceptibility to LL-37 and lincomycin.⁵⁷⁰ Similarly, the PAP, NBD, and translocase coded by the *spr0693-spr0694-spr0695* genes belonging to the same operon in *Streptococcus pneumoniae*⁵⁷⁰,⁵⁷¹ show high similarity to MacAB, with Dpr0693 having 21% identity with the PAP MacA, whereas the Spr0694-0695 complex is 37% sequence identical to MacB and presents a very similar structure.

### 5.7.3. Topology and Structural Organization of MacB Family Members.
The first investigation of the structural organization of the MacB was undertaken by Kobayashi et al.,⁵⁷² who found that the protomers of the MacB/FtsX family are distinct as their NBDs are found at the N-terminus of the transporter, while their TMDs contain four TM helices thus presenting a unique topology. Parallels in the organization of the known tripartite assemblies suggested that MacB-type transporters may organize into higher stoichiometries; however, cross-linking, atomic-force microscopy, and native mass-spectrometry confirmed that the functional unit of the transporter is organized along the universal dimeric pattern characteristic of the wider ABC-superfamily.⁵⁷³

In the monomer of the prototypical family member MacB, the NBD is fused to the N-terminal part of the permease domain of the transporter⁵⁷² and it presents a dimeric architecture; however, a number of other family members have the permease and NBDs coded by separate genes. Examples include the FtsEX complex, which gives the name to the wider MacB/FtsX family. It assembles with a FtsE₂FtsX₂ stoichiometry, with a porter domain formed by the FtsX (UniProtKB P0AC30), while the NBDs are provided by the FtsE (UniProtKB P0A9R7).⁵⁷⁴ It has to be noted that despite the close structural homology, and the apparent ATPase cycles,⁵⁷⁵ this complex does not seem to perform a transporter function but is rather engaged in peptidoglycan remodeling, a role which it exerts by activating the extracellular and periplasmic amidases, as demonstrated by the direct recruitment of the EnvC by the periplasmic domains of FtsX.⁵⁷⁶ Similarly, the connected lipoprotein extrusion complex

LolCDE, while having a heterodimeric permease portion (LolCE), engages a dimeric NDB LoID, to give a general stoichiometry of LolCED₂.⁴³² All of the permease domains of the proteins described above contain the 4 TM-helix architecture of the MacB. However, more exotic architectures have also been described, e.g., the bacitracin-sensing and resistance-associated BceAB-type transporters⁵⁷⁷ that appear to have 10 TM helices arising from a duplication of the 4-TM FtsX fold over TM2–4 and 7–10 and featuring a central helical insertion over TM5–6. These transporters presumably function as monomeric units. Some other members of the family feature this arrangement, e.g. YbbP-YbbA₂ complex;⁴⁴² however, these have been poorly characterized.

The first glimpse of the 3D structural organization came from the periplasmic domain of the *Aggregatibacter* (previously *Actinobacillus actinomycetemcomitans* (PDB ID 3FTJ)),⁵⁷⁸ which revealed a structural organization distinct from the then available ABC transporter structures. In the past decade, the structural and conformational space for type VII transporters has expanded dramatically to include the complete MacB from several different species of Gram-negative bacteria,²⁴,²⁷²,⁴³¹ as well as the Gram-positive homologues *Spr0694*–0695 from *Streptococcus pneumoniae* (PDB ID 5XU1)⁵⁷¹ and *YknWXYZ* from *Bacillus amyloliquefaciens* (PDB ID 5F9Q).⁵⁷⁹ In addition, the related heterodimeric LolCDE lipoprotein sorting system (LolC, PDB ID 5NAA, 6F49;⁴³² LolE, PDB ID 5UDF (Abendroth et al., to be published)) and the FtsX family members (PFAM family FtsX (PDB ID 4N8N, 6HEE, 6HFX)⁴³³,⁴³⁴ have also been determined. Importantly, the complete assembled structure of the tripartite MacAB-TolC from *E. coli* has also become available (PDB ID 5NIL, 5NIK),²⁷² allowing a direct comparison with the RND-based assemblies.

### 5.7.4. General Topology of MacB/FtsX Family and TM Domains.
The recent determination of a number of MacB/FtsX family transporter structures²⁴,²⁷²,⁴³¹,⁵⁷¹ has revealed a number of unique features within the TM region of these transporters, and these have been the subject of several excellent reviews (see Greene et al.⁴⁴² and Murakami et al.⁵⁸⁰).

First and foremost, the TM-region is composed of only 4 TM helices, with a dimer forming a 4 + 4 helical bundle, which is a stark contrast from the typical 6 + 6 architecture seen in the prototypical ABC family members, such as the type IV family (e.g., Sav1866 and MsbA).⁴²⁸,⁵⁸¹ Second, TM helices 1 and 2 are longer than the TM3 and TM4 and protrude above the membrane plane into the periplasm, where they are extended into a “stalk-like” structure crowned with a sizable globular periplasmic domain (Figure 19), known as the periplasmic core domain” or PCD,⁵⁷⁸ formed from the large periplasmic loop which is spliced into the periplasmic extensions of the stalk-helices TM1 and TM2, and in this respect resemble somewhat the RND transporters.⁵⁷²

The TM3 and TM4 are notably shorter than the stalk-helices and pack outside of the helical bundle formed by them. They do not protrude above the membrane plane and are linked by a short periplasmic loop, which is sometimes referred to as a “shoulder”.⁴³¹ The cytoplasmic, N-terminal part of TM1 is linked to the NBDs by an amphipathic helix, that runs parallel to the membrane surface and a “skirting loop” (Figure 19). The former helix is equivalent to the “elbow helix” seen in the type IV transporters but is commonly referred to as a connecting helix (CoH)²⁴ due to its linkage with similar helices in type V ABC transporters, e.g. ABCG5/ABCG8⁴²⁸,⁵⁸²
Finally, the NBD domains are located much closer to the membrane than in the typical type IV ABC transporters, which allows close association of the connecting helices with them, providing additional allosteric control. Each MacB TMD provides two coupling helices (CH1 and CH2) for TMD-NBD interdomain communication. The major coupling helix (CH1) is located on the cytoplasmic loop connecting TM2 and TM3 and provides a means of communicating conformational changes from the NBDs to the TMDs, while a second, “minor coupling helix” (CH2) is located after the TM4 at the extreme C-terminus. Deletion of the CH2 in A. baumannii MacB had a notable impact on the macrolide resistance²⁴ but only a minor effect on the pump activity in the *EcMacB*.⁴³¹

In a further departure from the architecture of type IV transporters where CH1 and CH2 from one-half-transporter contact the NBDs of the opposing protomer, the coupling helices (CH1 and CH2) are not shared between the protomers; i.e., the coupling helices of MacB/FtsX only engage the NBD of their cognate protomers²⁴,⁴³¹ (see Figure 12 in the PCAT section and Figure 19).

**5.7.5. Periplasmic Core Domains (PCDs).** As mentioned above, the bulk of the periplasmic portion of the type VII ABC transporters is contributed by a globular “periplasmic core domain” or PCD.⁵⁷⁸ It forms a conserved module, which is presented across the MacB and also FtsX-type transporters⁵⁷⁴,⁵⁷⁵ allowing to unite them into a separate PFAM group (PF02687) and giving the name to the whole family, so it can often be referred to as MacB/FtsX. The PCD domain could be split into an “upper” and “lower” subdomain,⁵⁷⁸ which have also been named periplasmic subdomain C (PSC), and periplasmic subdomain N (PSN), respectively²⁷² (Figure 20), with the lower portion presenting a mixed α-β sandwich of a general (β-α-β-β-α-β) configuration, sharing high structural homology to the porter (PD) domains of the RND transporters,⁵⁷⁸ while the “upper subdomain”, which has become known as the “SABRE” (an acronym for *small, alpha/beta rich, extracytoplasmic*), is unique to the wider FtsX family and can vary significantly in structure. In some of the recent literature, the PD and SABRE subdomains have also been referred to as periplasmic subdomain N (PSN) and periplasmic subdomain C (PSC), respectively,²⁷² but to avoid confusion, we will stick to the PD and SABRE nomenclature (Figure 20).

Due to its variability, the SABRE subdomain can be used as a differentiator within the wider MacB/FtsX family, and based on this it is possible to subdivide the group into several cohorts. It is notable that in the prototypical members of the MacB family, including the *Aggregatibacter actinomycetemcomitans* AaMacB,⁴³¹,⁵⁷⁸ *Acinetobacter baumannii* AbMacB,²⁴ *E. coli* EcMacB,²⁷² and the Gram-positive transporters belonging to the family, such as the *Streptococcal* permease Spr0695 (PDB ID 5XU1),⁵⁷¹ the SABRE domain presents a continuous entity, which is spliced into the middle of the “lower subdomain” (PD), splitting it evenly into two β-α-β motifs. Within this “canonical” MacB group, the SABRE forms a pseudocontinuous extension of the PD β-sheet, presenting a semirolled β-barrel.

A number of MacB-related proteins share the extended architecture above, with their PD modules superposing with low RMSD,⁴³¹ and they differ predominantly in the organization of their SABRE subdomains which display considerable diversity; however, as they do not form tripartite

<figure><img src="image_31.png" /><figcaption>**Figure 20.** Comparison of the porter domain of RND transporters and PCD of MacB. (A) The lower part of the periplasmic domain (PCD) of MacB shares structural homology with the porter domains (PD) of the RND transporters, as can be seen on the example of the AaMacB (PDB ID 3FTJ)⁵⁷⁸ (right) and the PC1 PD module of AcrB (PDB ID 2DHH)²⁸⁸ (left). (B) The topology diagram of the domain indicates that the SABRE subdomain is spliced within the core PD module between the β2 and β3 strands.</figcaption></figure>

complexes, we will not discuss them at length, highlighting some key members and respective PCD-domain peculiarities.

The second group of proteins that give the joint-name to the MacB/FtsX family, the aforementioned FtsEX complex,⁵⁷⁴ is central for peptidoglycan remodeling, a role which it exerts by activating the extracellular and periplasmic amidases, via direct recruitment of the EnvC by the PCD of FtsX.⁵⁷⁶ FtsEX shares similar TM-architecture to MacB, featuring 4 TM-helices and a large periplasmic loop, and it appears to function as a dimeric assembly of (FtsE)₂(FtsX)₂ stoichiometry.

In *Streptococcus pneumoniae*, FtsEX interacts with modular peptidoglycan hydrolase PcsB (PDB ID 4CGK),⁵⁸³ and the activation of the ATPase activity by FtsE triggers trans-membrane activation of PcsB.⁵⁸⁴ PcsB is a dimer in which the V-shaped coiled-coil (CC) domain of each protomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. It has been suggested that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, and FtsX PCD (referred to as extracellular loop 1 or ECL1) has been shown to interact directly with the coiled coils of the PscB.⁴³⁴ The PCD of FtsX reveals an upper extended β-hairpin and a lower α-helical lobe, with each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB.

Within the classical FtsX family transporters the mycobacterial homologues (e.g., PDB ID 4N8N)⁴³³ could be considered a basal group, and there the SABRE domain is presented by a simple helical extension, while the FtsX from *Streptococcus pneumoniae* (PDB ID 6HFX; 6HEE; 6HE6)⁴³⁴ presents an
<figure><img src="image_32.png" /><figcaption>**Figure 21.** Diversity of PCD domains in the FtsX/MacB family and porter domain connections. (A) The basic PD-module of the PCD is conserved across the MacB/FtsX family; however, there are multiple variations on the theme. The architecture of the module is expandable, but intriguingly, it features conserved regions of insertion, which are shared not only between Gram-positive and Gram-negative bacteria but also with eukaryotic organisms. The PDB codes for presented structures are as follows: Mycobacterial FtsX (4N8N); SpFtsX (6HE6); Spr0695 (5XU1); LolC (5NNA); NPC1 (5U74); DISP (6XE6). (B) PD domains present in different proteins outside of the MacB-family (see text for more details).</figcaption></figure>

additional *β*-hairpin insertion between *β*1 and *α*1 (see Figure 21 for a comparison of SABRE domains). Intriguingly, exactly the same expansion of the basic PD-module can be found in the C-terminal domain (CTD), the PD1-domains of the eukaryotic RND-related cholesterol transporter NPC1 (e.g., PDB ID 5U74,⁵⁸⁵,⁵⁸⁶ 6W5R,⁵⁸⁷ and 6UOX⁵⁸⁸), as well as the second extracellular domain (ECD-II) of the Hedgehog-signaling associated Patched1 (PDB ID 6OEU)⁵⁸⁹ and the recently resolved Dispatched (DISP1) (PDB ID 6XE6)⁵⁹⁰ showing the topological versatility of the basic PD-fold.

The recent structure of the EnvC bound to the periplasmic domain of FtsX (PDB ID 6TPI)⁵⁹¹ reveals an EnvC-FtsX complex with 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled-coil domain of EnvC, showing that the FtsX-PD modules could be used not just to provide lipid- and PG-binding but can mediate protein-protein interactions.

In Gram-negative bacteria the inner-membrane-associated LolCDE ABC transporter, the LolA chaperone, and the LolB outer-membrane receptor form an essential system for membrane extraction and transportation of the newly produced lipoproteins from the outer leaflet of the plasma membrane to the inner leaflet of the outer membrane.⁵⁹²

The permease unit of the transporter is formed by the heterodimer of LolC and LolE, both of which have MacB/FtsX-like structure, and in *E. coli* the LolC-LolE heterodimer cooperates with the NBDs of the ATPase LoID, which provides energy for the lipoprotein extraction. A very similar system is present in *A. baumannii*, where a homodimeric LoIF binds to a dimer of LoID.⁵⁹³ The structures of the PCD of LolC have been revealed, both in isolation (PDB ID 5NAA⁴³¹ and 6F49⁴³²) and bound to the periplasmic chaperone LoLA (PDB ID 6F3Z)⁴³² which reveals how a solvent-exposed *β*-hairpin loop (termed the “Hook”) and trio of surface residues

(the “Pad”) of LolC are essential for recruiting LoA from the periplasm and priming it to receive lipoproteins (PDB ID 6F49).⁴³²

The PCD of LolC presents a departure from the prototypical MacB PCD fold, and its SABRE domain has a distinctive *β*-hairpin structure formed by residues P167–P179 (full-length LolC numbering), which has been named the “hook”, which is not observed in the MacB, and which, along with a trio of charged residues from the external side of the SABRE *β*-barrel, named the “Pad”, participates in the LoLA recruitment⁴³² (see Figure 21 above). A very similar arrangement is seen in the structure of the related *A. baumannii* LoIF (PDB ID 5UDF) generated from a structural genomics effort

*Bacillus subtilis* and *B. amyloliquefaciens* produce sporulation-delaying protein (SDP), which is a peptide toxin capable of killing cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. SDP producing cells protect themselves against the endogenous SDP via the action of the four-component transporter YknWXYZ which acts as a major SDP efflux pump.⁵⁶⁸ Within this assembly, the YknYZ forms a noncanonical ABC transporter, where YknZ is the permease, while YknY is the NDB. Heterodimeric assembly of YknY₂Z₂ forms a transporter remarkably similar to MacB,⁵⁷⁹ while the PAP YknX assembles into a hexameric pattern very similar to MacA.⁵⁹⁴ Despite the sequence identity of around 20%, the extracellular domain (ED) of the *Ba*YknZ reveals a structure which is virtually identical to that of the PCD of the *Aa*MacB with DaliLite server bringing a Z-score of 20.1 and structural superposition returning an RMSD of just 1.6 Å over the C-*α* backbone of the corresponding domain.⁵⁹⁴

Similarly, the structure of the related Gram-positive pump Spr0694–0695, where Spr0695 is the permease and Spr0694
<figure><img src="image_33.png" /><figcaption>**Figure 22.** Topological connections of the basal RND PD-module and the docking/funnel subdomain in RND transporters. (A) The upper panel presents the topological diagrams, demonstrating the DN domain could be derived from the basal PD-module by flipping out the β2/β3 hairpin which forms part of the central β-sheet. This disrupted central sheet is complemented by the corresponding β2/β3 hairpin of the neighboring subunit by β-augmentation. Note that the antiparallel character of the central sheet changes as a result. PD1 AcrB denotes the organization seen in PN1/PC1 subdomains. (B) The crystal structures of the corresponding modules on the example of the AcrB PC1 subdomain (PDB ID 2GIF) and the DN1 subdomain of CusA (PDB ID 2DHH).</figcaption></figure>

provides the ATPase function, is surprisingly similar to the Gram-negative equivalent (PDB ID 5XU1)⁵⁷¹ and presents PCDs which are virtually identical to those of MacB, suggesting communality of operation, despite the lack of OMF engagement in the respective complex. One small exception is the presence of a well-defined helical loop inserted in the SABRE domain (residues 202–216), which seems to form specific contacts with the TM helices of the transporter and could provide additional conformational coupling or, as suggested by the authors, plays the role of a gating loop.⁵⁷¹

From the data available on the PCD domains, it is clear that they are predominantly engaged in peptide binding and protein–protein interactions, and furthermore, they display major conformational changes between different structures. It is a bit surprising that the PCD domains do not present high dimerization propensity on their own.⁵⁷⁸ However, in the available apo-EcMacB and the nucleotide bound structures, as well as the PCDs of the ADP-bound structures of AbMacB,²⁴ they appear to undergo significant conformational rearrangements, which have been suggested to arise from the NBD-liked allostery.⁴⁴²

**5.7.6. Versatility of PD Fold outside of the MacB/FtsX Family. Modularity of Assembly, Phylogenetic Connections, and Topological Linkage between Different Transporter Groups.** The porter domains are the central building blocks of the periplasmic part of RND transporters. Numbering these for an isolated PD domain from the N- to

the C-terminus, by the respective secondary structure elements, provides a β₁-α₁-β₂-β₃-α₁-β₄ configuration, which we refer to as the “standard PD” domain, which is observed in the AcrB PC1 domain²⁷⁹ (Figure 22).

The standard PD domain of RND transporters is a version of the ferredoxin-like fold,⁵⁹⁵ which is composed of 2 α-helices and 4 β-strands, in an α-β sandwich configuration, with the α-helices being spliced between the β1/β2 and β3/β4, respectively, and the 4 β-strands forming an antiparallel β-sheet. The fold has been initially described from the activation domains of pro-carboxypeptidases (e.g., PDB ID 1NSA⁵⁹⁶ and 1AYE⁵⁹⁷) and a number of RNA-binding proteins, including small nuclear ribonuclear proteins, e.g. U1A small spliceosomal protein (PDB ID 1URN)⁵⁹⁸ and ribosomal protein S6 (PDB ID 1RIS),⁵⁹⁹,⁶⁰⁰ with the latter showing a β-roll transition of the central sheet.

The pore domains of prototypical HAE-1 type transporter such as AcrB,²⁷⁹ as well as the HME group transporters, such as CusA,³⁵⁴ contain four variants of the PD fold, namely the PN1, PN2 and PC1, PC2 domains (Figure 5 and Figure 6), with PN1 and PC1 being continuous folds, while PN2 and PC2 harbor insertions between their respective β2 and β3 strands, comprising the funnel (FN) domains.²⁷⁹ Furthermore, while PN2 and PC2 present a 4 stranded β-sheet, the PN1 and PC1 show an extended 5-stranded sheet provided by β-augmentation in trans from the Cβ13 and N/β13 strands, respectively²⁷⁹ (Figure 5, Figure 6, and Figure 22).
<figure><img src="image_34.png" /><figcaption>**Figure 23.** Different families of RND transporters and related proteins. The representatives of the SecDF family, as well as the prokaryotic MmpL and Hopanoid transporter groups are shown, as well as the known structures of eukaryotic RND transporters. The PDB ID codes are given in parentheses. Due to the poor resolution and disorder, the missing loops of the DISP1 have been modeled for completeness. See text for more details.</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>**Figure 24.** Structural transitions within the PD-domains of SecDF are linked to the functional cycle of the transporter. The PD modules in the periplasmic domains of SecDF (P4 and P1-base) undergo a conformational transition from β-barrel to β-sheet between the Super F form (as seen in the PDB ID YHF) and the F-form (PDB ID 3AQP). Modified based on Furukawa et al.⁶⁶ These are linked to the substrate engagement by the P1-head domain of the transporter, and similar transitions may occur within other PD-containing transporters, e.g., the PD-domains of the MacB/FtsX family.</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>**Figure 25.** PD domains provide a flexible modular architecture which is shared across a number of different transporters. (A) Topological diagram showing the decoration of PD domains in different transporter classes. Where the transporters are not homooligomers with a single PD module per domain, the PD1 and PD2 domains of respective subunits are presented. The insertion points are limited and conserved across the known PD-domains, with notable splicing events taking place between β2 and β3 of the central β-sheet. Minor β-hairpin decorations are seen in the FtsX family and eukaryotic RND transporters. In addition, the SecDF family displays a unique insertion between α2 and β4. (B) Gallery of the PD-domains of the notable members of the transporter families discussed above. The domain abbreviation and PDB ID code of the respective structures are provided in parentheses. Abbreviations: MLD, middle luminal domain; ECD1/2, extracellular domain 1/2; CTD, C-terminal luminal domain or Cys-rich domain.</figcaption></figure>

Our analysis of the fold of the globular subdomains that form the funnel or docking domain of the RND transporters reveals that they could be derived from the “standard” PD domain topology by an additional β-hairpin insertion following α2-helix and a flip of the β-hairpin formed by the β2 and β3 strands of the central β-sheet, leading to its disruption. The central β-sheet is then supplemented during oligomerization, by the equivalent β2-β3 hairpin from the neighboring protomer, providing interconnectivity between the subunits of the functional trimer. As a result of this “hairpin swap”, the central continuous β-sheet of the “standard PD domain” is replaced, e.g., in the DN1 domain of AcrB, by a

pseudocontinuous β-sheet, where, due to the outward flip of the β2-β3 hairpin, the antiparallel interface between the cognate β1- and β3-strands is substituted by a parallel β1- and β3'-strand arrangement (Figure 22).

A number of RND and related transporters present variations on such an architecture including the HAE-2 family, with representatives such as MmpL3 (PDB ID 6AJF);²⁷⁷ HAE-3 HpnN (PDB ID 5KHN);²⁷⁸ and the more distantly related SecDF translocases (e.g., PDB ID 3AQP³⁶⁴ and PDB ID 5YHF³⁶⁶), as well as representatives from eukaryotic groups, such as the human cholesterol transport associated Niemann–Pick disease type C protein 1 (NPC1) (PDB ID: 5JNX⁶⁰¹ and
6W5R$^{587}$), mouse cholesterol translocator Patched (PDB ID 6MG8),⁶⁰² and the human Patched 1 (PDB ID 6OEU$^{589}$ and 6DMB$^{603}$) (Figure 23).

### 5.7.6.1. MmpLs.
In *Mycobacterium tuberculosis* the 14 members of the MmpL (mycobacterial membrane protein large) transporter family form a group of proton-driven carriers, that translocate complex (glyco)lipids and side-rophores across the cell envelope.⁶⁰⁴,⁶⁰⁵ Unlike the RND transporters of the HAE-1 and HME families, these transporters operate as dimers, although some earlier data on the orthologous Corynebacterial CmpLs,⁶⁰⁶ namely CmpL1, has suggested a possibility of a trimeric assembly.⁶⁰⁵ As these transporters are subject to a detailed review in the current issue of *Chemical Reviews*,⁶⁰⁷ we will give only a cursory note about them here. Phylogenetic analysis has suggested that the group shares high homology to the classic HAE/HME RND family and similarly presents an internal gene duplication with resulting 12 TM-helices,⁶⁰⁸ with extracellular porter-domains D1 and D2 homologous to the PD-domains being spliced between the TM1/TM2 and TM7/TM8, respectively⁶⁰⁹ (see Figures 23 and 24 and Figure 25A). Two broad clusters of MmpLs can be derived based on the topology of their PD domains, a more basal group, called cluster II, including MmpL 3, 11, and 13, which possess two standard PD domains (with a β₁-α₁-β₂-β₃-α₁-β₄ configuration), “cluster I” which includes the rest of the family that have a standard N-terminal PD domain, and an expanded C-terminal D2 domain featuring an insertion between the β₂-β₃ (β₁-α₁-β₂-X-β₃-α₁-β₄),⁶⁰⁹ thus presenting a PD-architecture equivalent to that observed in the MacB/FtsX family discussed above. Consistent with the prediction, the structure of the isolated D2 domain from the MmpL11 (PDB ID 4YOL)⁶⁰⁹ showed high homology to the PD domains of classical RND transporters. Cross-linking experiments suggest that the orientation of the PD domains in MmpL 11⁶⁰⁹ is similar to the PC1/PN2 domain orientation on the AcrB, e.g. (PDB ID 2GIF)³¹⁸ or CusA (PDB ID: 4DNT).⁶¹⁰ However, later structures of the MmpL3 from *M. smegmatis* (PDB IDs 6AJF-6AJJ,²⁷⁷ 6OR2⁶¹¹) display a distinct domelike arrangement. In addition, the PD2 domain of MmpL3 (PDB ID 6OR2) does appear to have a small insert, after β2 (see Figure 25B). At present there are no experimental structures of PD domains belonging to cluster I MmpLs, although our bioinformatic analysis suggests their insertions are not related to the ones observed in the funnel domains of RND transporters and may be closer related to the α-helical domains observed in the HAE-3.

### 5.7.6.2. Hopanoid Transporters.
The hopanoid transporters, which are representatives of the HAE-3 group and are responsible for the transport of the eponymous pentacyclic triterpenoids with a sterol-like function in the bacterial membrane,²⁷⁸ also exhibit high similarity to the RND transporters, consistent with that present for PD-like domains. The crystal structure of the HpnN (PDB ID 5KHN)²⁷⁸ (see Figure 23) revealed a dimeric assembly of the transporter, which presented four PD domains, with PD1 and PD2 being equivalent to the classic PD domains observed in HAE-1 and HME RND transporters, but in addition, they present helical inserts forming additional PD3/PD4 domains, with no relation to any hitherto known domains. The splicing of the PD3/PD4 into the PD1 and PD2, respectively, produces the same topology (β₁-α₁-β₂-X-β₃-α₁-β₄) as discussed above for

MmpL and the MacB/FtsX proteins suggesting a general pattern for expanding the architecture (see Figure 25, panel B).

#### 5.7.6.3. SecDF.
Working in conjunction with the major secretory complex SecYEG/SecA⁶¹² that is driven by SecA ATP-hydrolysis, the proton motive force powered SecDF complex facilitates the threading of the unfolded protein cargoes and plays a chaperone-like function.³⁶⁵,⁶¹³,⁶¹⁴ In *Thermus thermophilus* SecDF, the SecD and SecF are fused into a single polypeptide chain, and its structure (PDB ID 3AQP)³⁶⁴ revealed that similar to the group I MmpLs discussed above, SecDF presents a pseudosymmetrical, 12-helix transmembrane domain belonging to the RND super-family and two homologous major periplasmic domains, P1 and P4 (Figure 24 and Figure 25). While the P4 domain shows a classic β₁-α₁-β₂-β₃-α₁-β₄ organization, similar to the PD-domains in HAE-1 PD and MmpL3, the P1 domain presents a β₁-α₁-β₂-X-β₃-α₁-β₄ organization, where “X” is a large insert forming the so-called “head” subdomain, while the core PD β-strands form the cores of both the P1 and P4 form a pseudosymmetrical 8-stranded antiparallel β-sheet that covers the TM region of SecDF and is planar in the most structures, including P1-membrane facing “F” forms and P1-outward facing “I”-forms;³⁶⁴,⁶¹⁵ however, unexpectedly, the PD-domains of the transporter were also revealed in a β-barrel form (known as Super F-form) (PDB ID 5YHF).³⁶⁶ The β-barrelled super F-form is speculated to engage with the emergent preprotein coming out of the SecYEG on the periplasmic side of the membrane, which is bound to the P1-head. The conformational transition from super F-to-F and planification of the β-sheet is speculated to be associated with substrate dragging, and consistent with such interpretation, fixing the β-sheet by cross-linking between the interface of the P1-base and P4 abolished the translocation activity of SecDF, suggesting that β-sheet rearrangement occurs during protein translocation.⁶¹⁵ Further transition of the F to I-form is associated with the formation of a proton-accessible tunnel in the TM-part of the protein (as seen in PDB ID 5XAN) and subsequent release of the cargo, leaving the P1-head outward facing in the cargo-disengaged “I without tunnel form”³⁶⁶ (Figure 24).

In a possible analogy to the efflux RND transporter cycle, the substrate binding causes conformational transitions, that allow the access and binding of the protons to the TM-domains, with actual release of the protons on the cytoplasmic side being associated with the cargo release and thus, recycling of the complex. Despite the similarities, the proton relay network in SecDF does not appear to function the same way as in canonical RND transporters such as AcrB. Notably, while there is still a tight interplay between the residues from TM4 (esAspIV; “es”—standing for “essential”) and TM10 (Tyr), unlike the AcrB, the proton-relay is supported by critical residues located on TM11 (esArgXI).³⁶⁶

#### 5.7.6.4. PD-Domains within the Eukaryotic RND Transporters.
Members of the eukaryotic sterol transporter family include the Niemann–Pick type C protein 1 (NPC1) and the Niemann–Pick type C protein 1 like 1 (NPC1L1). The first is a lysosomal membrane protein essential for cholesterol homeostasis. Defects of NPC1 are associated with hereditary Niemann–Pick disease leading to a fatal accumulation of low-density lipoprotein (LDL)-derived cholesterol.⁶¹⁶ Active as a monomer with 13 TM helices and an extended N-terminal domain, NPC1 presumably acts as a receptor/transporter for the cholesterol-loaded NPC2 soluble carrier via the N-terminal
<figure><img src="image_37.png" /><figcaption>**Figure 26.** (A) Schematic representation of the TM helix packing at the dimer interface between different classes of ABC transporters shows close connections of the MacB architecture to the type VI and type VIII (MlaEF) transporters. Topologies are derived from the structures of MacB (PDB ID 5GKO),²⁴ MlaE (PDB ID 6XBD),⁴³⁹ LptB2FGC complex (PDB ID 6S8N),⁶³⁵ Wzm-WznT (PDB ID 6M96),⁴²⁹ and ABCG2 (PDB ID 5NJG).⁶³² Top-down view from the periplasm. Note: due to the intertwisting of the helices in the type IV transporters, the diagram depends on the level at which the slice is cut through the TM; the provided view is representative to the middle of TM segment, based on the ABCB1 structure (PDB ID 6QEX).⁵²¹ (B) Topological connections within the ABC transporter families. The ABC transporter families share a common core architecture, which can be derived from the four TM helices seen in the type VII (MacB/FtsX) family. The other main families of exporters are presented, which could be seen as derived from MacB by addition of C-terminal helical fragments. Homologous helices are colored consistently from the N-terminal to the C-terminal. The intracellular coupling helices (CH1 and CH2) are presented in green and red. CoH = connecting helix. Substrate binding domains are shown as gray boxes (PCD, periplasmic core domain; PH, pore helix; PGH, periplasmic gate helix; ECD1,2, extracellular domains 1, 2; β-JR, β jelly roll domains). Adopted with modifications based on Schaeffer et al.⁶³⁴</figcaption></figure>

soluble loop.²⁷⁴,⁵⁸⁶ It is also the target of the Ebola virus acting as an intracellular receptor. Structures for the full-length NPC1 and in complex with the cleaved glycoprotein of the Ebola virus have been published (PBD ID: 5JNX,⁶⁰¹ 6W5R⁵⁸⁷). NPC1L1 is an intestinal membrane protein involved in the absorption of cholesterol and a target of the therapeutic compound ezetimibe, for which recently cryo-EM structures have been solved.⁶¹⁷ Patched (PTCH1) and Dispatched (DISP) belong to the eukaryotic dispatched RND transporter family. Both are involved in the hedgehog signaling pathway, during embryogenesis and tissue homeostasis, where Dispatched exports the N-terminal portion of sonic hedgehog, with C-terminally linked cholesterol,²⁷⁴,⁶¹⁸,⁶¹⁹ and PTCH1 acts as a receptor of the lipidated sonic hedgehog protein, binding of which releases the downstream suppression of Smoothened (SMO). Cryo-EM structures of monomeric human PTCH1 resemble the architecture of NPC1 (PDB ID 6OEU, 6OEV;⁵⁸⁹ 6DMB⁶⁰³), presenting an RMSD of ~2.5 Å, and recently the structure of the human Dispatched-1 (DISP1) homologue has been obtained (PDB ID 6XE6.⁵⁹⁰ These eukaryotic homologues possess two separate PD domains, which are decorated in a similar fashion to the ones discussed above for the Cluster I MmpLs and Hopanoid transporters. Notably, the two PD-

like domains of NPC1, named the middle luminal domain (MLD) and C-terminal luminal domain or Cys-rich domain (CTD), present large insertions between the $β_2-β_3$ (PDB ID 5U74),⁵⁸⁶ an organization which is also shared by the Patched structure.⁵⁸⁹,⁶⁰³ In addition, the NPC1 MLD-domain (PD1) contains a β-hairpin insertion between $β_1$ and $α_1$, which mimics the ones observed in the FtsX members such as discussed previously (Figure 25 and Figure 21). A very similar insertion is seen in the extracellular domain 2 (ECD2/PD2) of the DISP1 (PDB ID 6XE6)⁵⁹⁰ but not in the PTCH1.

Taken together, the analysis of the PD domains of the RND-like transporters presented, as well as the FtsX/MacB family, displays a clear pattern of usage and modification of the PD modules, with two “hotspots” of insertion being utilized—the first one is located at the tip of the $β_2-β_3$ hairpin of the central PD-$β$ sheet and allows for splicing of large subdomains, e.g., the SABRE domain in the case of the FtsX/MacB family, or the large helical subdomain inserts in the case of PD3/PD4 domains of the hopanoid transporters and the eukaryotic RND-like transporters (NPC1/DISP1/PTCH1). The second insertion point accommodates smaller splice-ins in the form of an additional β-hairpin insertion, as seen in the *S. pneumoniae* FtsX (PDB ID 6HE6); the ECD2 of DISP (PDB ID 6XE6),
and the MLD domain of NPC1. Finally, the SecDF presents a unique insertion in its first PD-domain, with the P1-head domain being spliced after α2 (see Figure 25).

Notably the architecture seen in the PD domains is also present in a number of additional periplasmic proteins which lack transporter function, e.g., the two-component signaling modulator family of MzrA proteins (PDB ID 4PWU),⁶²⁰ as well as more distantly related proteins including the archaean Tk-subtilisin chaperone pro-peptides (PDB ID 3A3O) and,⁶²¹ similarly, chaperoning activation domains of carboxypeptidases (PDB ID 1PBA),⁶²² as well as acylphosphatases (e.g., mtACP PDB ID 1APS)⁶²³ and the aforementioned small nuclear ribonuclear proteins such as U1-SNRP A (PDB ID 3G8T,⁶²⁴ 3UCU⁶²⁵) (see Figure 21B).

### 5.7.7. Relations of the Membrane Porter Domains of MacB/FtsX to Other ABC Transporter Families.
At first sight, there appears to be limited similarity between the porter domains of MacB/FtsX containing 4-TMs and the more typical ABC transporters, which contain 6-TMs as standard. However, closer inspection of the MacB-TM regions utilizing DALI pairwise fold-comparison software²⁴ and comparison against different members of the type IV family, such as ABCB1 (PDB ID 4M1M,⁶²⁶ 3G60⁶²⁷), heterodimeric TmrAB (PDB ID 5MKK), and the ADP-bound form of the lipid-linked oligosaccharide flippase PgIK (PDB ID: 5NBD),⁶²⁸ reveal unexpectedly high similarity of the MacB/FtsX family to this family, with the ABCB1 returning a Z-score of 8.7 (PDB ID 3G60), based on the superposition of the first three N-terminal helices (TM 1–3) of the type IV transporters showing high structural similarity to the MacB TM helices 1–3. Despite this similarity, it has to be noted that the dimer interface of MacB is not matched with the dimer interface presented by the structures of type IV ABC transporters, e.g. in the MacB/FtsX TM helices 1–3 do not form part of the substrate binding cavity and are hence more likely to play a role in energy transduction, rather than substrate interactions, while in type IV helices 1–3 face away from the central cavity toward the lipid bilayer²⁴ (Figure 26, panel A, showing the packing in ABCB1 6QEX⁶²¹).

An expanded topological analysis of the type VII ABC-family topological relationships, which in addition to the type IV transporters discussed above also included type I importers (e.g., MetNI(Q) (PDB ID 3DHW),⁶²⁹ YjgP/YjgQ (LptBFG; type VI family)),⁶³⁰,⁶³¹ as well as the members of type V exporters, such as the ABCG⁵⁸²,⁶³² and ABCA⁶³³ involved in eukaryotic lipid export, revealed unexpected similarities, suggesting that they might be evolutionarily related.⁶³⁴ Based on these similarities, an alternative classification of ABC transporter topologies has been proposed, with the core of these transporters' structures consisting of the four-TM core segments (1–4), as observed in the MacB/FtsX family (e.g., PDB ID 5WS4), and it has been proposed that MacB/FtsX (Group VII) may be a basal group of transporters from which the other topologies covering groups V, VI, and even IV are derived. Uniquely, the MacB/FtsX family additionally possesses a second coupling helix (CH2) at the C-terminal end of the fourth core TM segment (Figure 26, panel B; TM-segment is colored red), which is not present in the TMDs of LptBFG (YjgP/YjgQ; type VI) or in group V (ABCG and ABCA) transporters.⁶³⁴

Intriguingly, the complete topology of type IV floppase/ exporters (formerly type I exporters) could also be derived when using MacB/FtsX as a basis, as TM segments 1, 2, 3, and

6 can be structurally aligned to the TMs 1–4 of MacB. Insertion of TM helices 4–5 carrying a canonical coupling helix between TM3 and 4 of MacB/FtsX produces a topology equivalent to the type IV floppase/exporters. Consistent with this, DALI superposition of MacB (PDB ID 5WS4) with type IV heterodimeric exporter TmrAB (PDB ID 5MKK),⁵²³ which is a homologue of the antigen translocation complex TAP, returned a Z-score of 7.7.²⁴ While intriguing and appealing in its simplicity, such systematics do not automatically suggest that the MacB/FtsX family is a basal group, as it could be a product of degenerative evolution.

More recently, Murakami et al.⁵⁸⁰ also extended their DALI analysis to the type V family members ABCG2 (PDB ID 5NJG)⁶³² and ABCA1 (PDB ID 5XJY)⁶³³ that returned high Z-score values of 7.6 and 7.5, respectively. While the overall score is lower, these transporters allow better superposition, covering all four TMs of MacB, and importantly, they provide a dimer interface matching that of the MacB.

Our initial analysis of MacB-structures has also suggested a connection of the MacB/FtsX with type V floppases/exporters such as Wzm-WztN and the ABCG/ABCA family but also with the yet-to-be classified Mla-system members due to the overall topology of TMDs, the presence of the amphipathic connecting helices (CoH), linking the NBDs to the TM helices, and the close association of the NBDs with the membrane. In Figure 26 (panel B), we have expanded the topological classification discussed above,⁶³⁴ incorporating the data from the recently solved structures of the yet-to-be classified MlaEF-ABC transporter system,⁴³⁷⁻⁴³⁹ the structure of the permease subunit of which, MlaE displays a number features linking it with the MacB and LptB₂FG families, plus a set of unique elements, that in our view justify classifying it as a separate group of ABC transporters, which we tentatively assign to type VIII for clarity of presentation below. Notably, the MlaE topology can be readily derived from a MacB/FtsX family basal topology by addition of C-terminal helix 5 (Figure 26, panel B). Furthermore, the TM5-helices of MlaE pack analogously to the similarly derived helix 5 in the LptFG complex, and crucially, the packing is dominated by close bundling of TM2 which is only observed in the MacB family, setting these two groups into a close sister grouping within the ABC-superfamily, reaching similar conclusions from the independent topological analysis by Coudray et al.⁴³⁹ Helical packing diagrams (Figure 26, panel A) provide further evidence of common patterns across the ABC-groups V–VII.

### 5.7.8. TMD and Energy Coupling.
As mentioned earlier, the analysis of the topologies of the available ABC transporter structures revealed that there are at least seven structurally distinct ABC transporter folds,⁴²⁸ of which MacB is the most-recently determined representative. MacB can therefore be considered the “holotype” for the whole of the type VII ABC transporter superfamily⁴²⁸,⁴³¹ and has become the first structurally characterized member of the ABC3 superfamily of the ABC transporters as defined by Wang et al.⁴²⁷ Its unique architecture suggests a mechanistically distinct functional cycling mechanism.

The *EcMacB* has been revealed in a nucleotide free form (PDB ID 5NIK), with the NBDs apart and unable to bind ATP.²⁷² However, an ATP-bound (PDB ID 5LJ7) and nonhydrolyzable ATP-analogue bound form (PDB ID 5LJ6, 5LOL)⁴³¹ have also been reported. For *AaMacB* and *AbMacB*, an ADP-analogue bound form (e.g., adenosine-5'-(*β*-thio)-diphosphate (PDB ID 5WS4)) and a nucleotide-free form
(PDB ID 5GKO) have been obtained, respectively.²⁴ In addition, the structure of the Streptococcal MacB-like transporter Spr0694–0695 (PDB ID 5XU1)⁵⁷¹ was revealed in a nucleotide-free state and the comparative analysis of these structures has allowed us to reconstruct a plausible energy-coupling cycle of the MacB/FtsX-like transporters.

In none of the structures does the tightly packed dimer interface of MacB present any credible space for a trans-membrane cargo to be accommodated. However, the nucleotide-free, as well as the ADP-bound A. baumannii structures clearly resemble each other with the extra-membrane helical protrusions of the TM1 and TM2 being bent away from the central axis of symmetry, creating a “Y-shaped” crevice, which appears accessible to potential periplasmic cargoes.²⁴,²⁷² The PCDs in the nucleotide-free forms and in the ADP-bound form appear to be separated. In the presence of ATP, the NBDs are brought together, which causes the major coupling helix CH1 to push up the TM2, causing contraction of the interhelical gap, which results in a transformation of the loose helical arrangement into a rigid four-helical bundle,⁴³¹ and this “zipping-up” of the interhelical interface causes a marked contraction of the PCDs, which are squeezed together. Such long-range communication from the NBDs through the TM-stalk helices all the way to the periplasmic domain has been dubbed “mechanotransmission”, as the stroke from the ATP-binding is not used to transport the substrate across the membrane in which the ABC transporter resides but to cause conformational changes into the periplasm, or extracellular space, respectively. Consistent with such an interpretation, disulfide cross-linking of the stalk-helices has been demonstrated to severely impact MacB function *in vivo*⁴³¹ (see Figure 27).

Thus, in MacB, the tight coupling between the TMD and the NBDs via CH1 and CH2 is likely used not solely for conformational changes of TM helices but is also translated into long-range communication with the PCDs, which is in turn converted into substrate transport via the PAP- and OMF-interactions.

Under the currently prevailing paradigm, ATP-binding and hydrolysis on the inside of the membrane cause large, transmembrane conformational changes in the MacB structure, leading to “zipping-up” of the central stalk-helices, and this has been suggested to drive the PCD-domain rearrangement which leads to cargo expulsion, although the exact binding sites and extrusion mechanism are not fully clear. It is clear, however, that rather than transporting substrates across the inner membrane, MacB-family transporters remove their substrates from the periplasm, utilizing long-distance conformational coupling of the NBD dimerization, in a mechanism which was dubbed “mechanotransmission”. Members of the MacB/FtsX family participating in tripartite complex formation may use the mechanotransmissive conformational coupling of the stalk helices to the PCDs to efflux substrates across the outer membrane via OMFs ducts, while the MacB/FtsX homologues that function alone can utilize such motions for exporting across the peptidoglycan layer in the Gram-positive organisms, as well as for a related function in lipoprotein trafficking and transmembrane signaling.

**5.7.9. Substrate Binding Site.** In the MacB/FtsX family, the extra-membrane extensions of TM1 and TM2 protrude out of the membrane forming a kinked, “Y-shaped” conformation with a small semioccluded cavity, that is accessible from the periplasm. In the cryo-EM structure of MacB (PDB ID 5NIL),

<figure><img src="image_38.png" /><figcaption>**Figure 27.** Mechanotransmission mechanism of MacB. Crystal structures suggest a possible cycling mechanism for MacB. In the nucleotide-free structures, the NDBs of the transporter are separated and appear unable to bind ATP. This coincides with the separation of the PCDs and lack of helical-bundle formation in the stalk helices. To the contrary, the ATP-bound forms show significant rearrangement of the PCDs, which also coincides with a helical bundle formation in the TM-domain, suggesting that the ATP-binding is communicated long-range from the NBDs across the membrane to the periplasmic domain through the TM-stalk helices. This has been dubbed “mechanotransmission”, as the stroke from ATP-binding is not used to transport the substrate across the membrane in which the ABC transporter resides but rather to cause conformational changes in the periplasm. (Modified, based on the model by Crow et al.⁴³¹)</figcaption></figure>

an unidentified electron density, occluding this region between the periplasmic extensions of TM1 and TM2, has been reported.²⁷² Although, the low resolution of the associated cryo-EM map (at 5.3 Å) did not permit a clear interpretation, it has been speculated that it might represent a putative substrate binding site.²⁷² While there is no direct structural data on substrate binding, structure-guided mutagenesis was used to narrow down the potential substrate binding residues,⁴³¹ showing a plausible erythromycin binding site, covering predominantly bulky hydrophobic residues (including Y315, F320, F320, F503, and M507), mapping mostly to the PD-subdomain facing the interdomain interface. Their impact on the activity of the pump was also validated using bacitracin and colistin. Given their distribution across the PCD, it appears plausible that the PD subdomain is primarily engaged with cargo processing, while the SABRE domain, which shows significant conformational transitions and has been implicated in protein–protein interactions, is central to coupling to the PAP and the transmission of the conformational changes outward to the OMF, although future research is still needed to validate this hypothesis.

As mentioned above, the structure of the related Gram-positive pump Spr0694-0695, despite overall similarity with MacB, reveals a departure from its blueprint in the TMD portion of the protein.⁵⁷¹ Specifically, there is divergence in the SABRE domain, where there is a short extra helical insertion (helix 6 of the PCD or rather in that case the extra-cellular domain or ECD) with the extra-membrane protrusions of the TM helices 1–3. Several charged residues are seen on this helix (e.g., K207-R208, K210), and it has been suggested that these may form a “guard helix” protecting a putative substrate-entrance tunnel. Mutation of the charged residues of the helix
reduced the ATPase activity, suggesting it may also play a role
in energy coupling.

In *Lactococcus lactis*, the *hrtB* gene (UniProt A0A2N5WEC6) codes for the permease, and the *hrtA* gene (A0A0H1RJ19) codes for the NBDs of a MacB-like heme transporter, which was initially suggested to remove it from the cytoplasm or the membrane-solubilized pool,⁶³⁶,⁶³⁷ although the exact mechanism remains unclear. Newer work suggests that the heme may instead be removed from the periplasmic side of the membrane in *S. aureus*⁶³⁸ and consistent with that mutation of residues in the HrtB of *L. lactis* mapping to the SABRE domain of the PCD (Y168) and the upper part of stalk-helix 2 (Y231), speculated to coordinate heme-ligands, impacted function,⁶³⁹ suggesting this region may be involved in ligand binding. Despite this circumstantial evidence, the detailed characterization of the binding sites of MacB-like transporters must await another day.

In summary, the unique structural organization of the
MacB/FtsX family suggests a distinct mode of operation for
the binary PAP transporter pairs and for the tripartite
assemblies in which they participate. We will discuss that in
the context of the structure of the complete tripartite
complexes with their participation below.

# 6. OUTER MEMBRANE FACTORS (OMFS): TOLC AND OTHERS

## 6.1. Introduction to the OMFs

Ubiquitous in Gram-negative bacteria, proteins from the TolC family were initially characterized based upon their pleiotropic roles: their ability to induce tolerance to colicin E1,¹⁹⁶ a role of the surface receptor for bacteriophages,¹⁹⁷ resistance to antibacterial agents,⁶⁴⁰ or secretion of virulence factors.⁶¹ Several structural descriptions of TolC⁶⁴¹ and its homologues have emerged over the last 20 years, notably OprM,²⁶⁹,⁶⁴²,⁶⁴³ OprN, and OprJ from *P. aeruginosa*,⁶⁴⁴,⁶⁴⁵ MtrE from *Neisseria gonorrhoeae*,²⁹⁷ VceC from *Vibrio cholerae*,⁶⁴⁶ CmeC from *Campylobacter jejuni*,⁶⁴⁷ and CusC from *E. coli*.⁶⁴⁸ These structures build upon an important number of (rational or random) side-directed mutagenesis, biochemical and biophysical studies that have shed light on the way OMFs open and assemble with their partners. The purpose of the following paragraphs is to try to confront structural and functional data obtained over the years and highlight the breakthroughs and paradoxes that have emerged.

## 6.2. Overall Organization of OMFs

OMF structures share a very similar architecture although their percentage of sequence identity is relatively low. OMFs are trimeric proteins, which form a long tubular channel one end of which is inserted in the outer membrane with the other end extending into the periplasmic space and interacting with the peptidoglycan layer.

They share a characteristic channel-like organization.⁶⁴¹
TolC from *E. coli* is a protein consisting of 471 residues whose
protomers assemble into a homotrimer. This homotrimer of
approximately 150 kDa forms a 140 Å long channel organized
in two main domains: a transmembrane domain that adopts a
porin-like fold consisting of 12 β-strands organized in a β-
barrel of approximately 40 Å inserted into the outer membrane
and a periplasmic domain of about 100 Å long. This second
domain presents a unique “α-barrel” architecture that consists
of 12 α-helices organized into three subdomains: the α-
domain, the equatorial domain, and the coiled-coil domain.

Each protomer is formed by the repetition of a conserved motif made of two transmembrane β-strands, of a long helix (H3 in repeat one; H7 in repeat two), and finally of two helices aligned along their axis (H2/H4; and H6/H8, respectively), with each having one of their ends at the equatorial domain, thus forming a pseudohelix of the same length as the long helix. These pseudocontinuous helices give the trimer extra flexibility. The equatorial domain (or mixed α/β domain) consists of a mixture of secondary structures (α-helices and β-strands) that forms a belt around the medial part of the α-barrel (see Figure 28).

<figure><img src="image_39.png" /><figcaption>**Figure 28.** General organization of the typical OMF channel on the example of OprM. (A) Topological diagram. Principal helices (H) and β-sheets (β) are denoted. The chain is colored rainbow from the N-terminus to the C-terminus. Pseudosymmetry within the protomer provided by the structural repeats, arising from a gene duplication event, is evident. (B) 3D structure based on the palmitoylated OprM structure (PDB ID 4Y1K).⁶⁴³ See text for more details.</figcaption></figure>

OprM in *P. aeruginosa* is a large, slightly conical cylinder about 140 Å long, 40 Å wide at the top, and 70 Å wide at the bottom.²⁶⁹,⁶⁴² OprM protomer consists of four transmembrane antiparallel strands (β1, β2, β3, and β4) and eight periplasmic helices (H1 to H8) (Figure 28). The β-strands are composed of a dozen residues which combine to form an antiparallel β single sheet curving at a remarkable angle of ~60°. Each β-strand is inclined by about 45° relative to the axis of symmetry of the trimer. The H1 helix consists of 13 residues, H2/H6 of 23 residues, H4/H8 of 36 residues, and H3/H7 of 67 residues. The smaller helices join together with the larger ones to form a large, curved sheet of antiparallel helices. Each helix bundle tilts approximately 20° from the axis of symmetry of the trimer. The three protomers come together to form a transmembrane β-barrel about 40 Å long and wide which extends toward the periplasm by a large β-barrel 100 Å long and 70 Å wide. The structure of OprM can be separated into three domains: the transmembrane domain which forms a β-barrel, the periplasmic domain which forms an α-barrel, and the equatorial domain located at the level of the middle part of the α-domain. The latter is a mixed structure consisting of two α-helices (H1 and H5), loops, and 3₁₀ helices. The equatorial domain forms a ring around the α-barrel.

Although the structures are very similar, there are some
differences between TolC and OprM. First, the N-terminal
extremity of OprM is anchored to the outer membrane via a
<figure><img src="image_40.png" /><figcaption>**Figure 29.** (A) Highlight of the conserved amino acids among 5 OMFs. The protomeric structures of TolC (1EK9, blue), OprM (3DSK, purple), VceC (1YC9, ochre), CmeC (4MT4, green), and CusC (3PIK, red) are shown in the upper part of the panel. The side chains of the preserved residues are represented as spheres of different colors according to their intrinsic location in the protein: orange for the β-barrel part, green for the equatorial part, and blue for the periplasmic part. The position of each conserved amino acid within the primary sequence is shown in the table in the lower part of the panel. (B) Visualization of the β-barrel domains within the quaternary structures of 5 OMFs. Each porin retains the same structural color as in panel A. Two views are shown: a side view first and a top view second.</figcaption></figure>

palmitoyl moiety that originates from the equatorial domain.⁶⁴²,⁶⁴³,⁶⁴⁵ The palmitate anchor provides a second attachment point of the protein to the outer membrane.

The equatorial domain also presents significant variations, particularly in the C-terminal, probably reflecting interaction with structurally diverse PAPs in the tripartite efflux pump. As will be discussed in more detail below, proposed models of assembly between TolC and AcrA show that this loop is involved in the recruitment and interaction of the OMF porin with its PAP.⁶⁴⁹,⁶⁵⁰ The importance of the equatorial domain has been shown by loss-of-function mutants of TolC.⁶⁵¹⁻⁶⁵³ It was shown that TolC requires a leucine residue at position 412, located in an equatorial domain that protrudes from the main body of TolC into the periplasm, to be functional.⁶⁵¹ Similarly, residues located at the base of carboxy-terminal elongation in the equatorial domain of AatA, homologous to *E. coli* TolC in enteroaggregative *Escherichia coli*, are required for the export of its substrate, Aap.⁶⁵⁴ Finally, the inability for TolC to form a functional complex with CusAB and to confer resistance to copper and silver ions *in vivo* was also suggested

to be caused by the seemingly different equatorial domains of CusC and TolC.⁶⁴⁸ In VceC, part of the loop is not visible in the crystallographic structure probably because of its great flexibility.

The C-terminal tail also seems to be of importance for OprM as it is involved in its interaction with MexAB.⁶⁵⁵ The role of the C-terminal tail of OprM has also been studied for the hybrid complementation between OprM and VceAB in order to highlight its role in the specificity of recruitment in the assembly. OprM can no longer function with VceAB when its 20 C-terminal residues are truncated,⁶⁵⁶ but when a modified VceA with mutation in the hairpin domain is used, the function with OprM-Δ20Cter is restored.⁶⁵⁷ As will be discussed below, this region has long been thought to be involved in the interaction with the partner PAP. Overall, the equatorial domain takes on a unique shape for each porin, which is not surprising since it is one of the least conserved regions. Accordingly, major structural differences between OprM, OprN, and OprJ also lie at the equatorial domain and at the periplasmic end of the β-barrel.⁶⁴⁵ However, the H1/H5
helices and the large L1 loop appear to share redundant patterns. In TolC, it was shown that exchanging the H4–H5 loop of TolC with that of other OMFs decreases susceptibility to novobiocin, suggesting that this loop affects the transport activity of AcrAB–TolC; hence, part of loop L1 (N188–K214) is essential for the function of the channel.⁶⁵⁸ Compared to its homologues, TolC is the only one to have an additional β-strand in this domain. Unlike the α- and β-domains, the equatorial domain does not appear to be a duplication of the same structural pattern.

Despite the strong structural analogy of OprM (PDB code: 3D5K) with its counterparts (TolC, PDB code: 1EK9; VceC, PDB code: 1YC9; CmeC, PDB code: 4MT4; CusC, PDB code: 3PIK), the identity of their primary sequence is rather low (19% for TolC and 23% for VceC). A multiple alignment of OprM and OMPs from different species highlights the residues conserved during evolution (Figure 29A). These residues, which probably play a key role in the structure or function of OMPs of the TolC family, have a preferential distribution at the ends of the α-helices and at the level of the connections between the α-domain and the β-domain. Furthermore, the majority of the conserved residues are located on the C-terminal motif which could be an evolutionarily ancestral sequence.

Gram-negative bacteria encode multiple RND transporters which pair with a number of PAPs and OMFs forming a variety of efflux systems with different substrate profiles (Table 1). TolC from *E. coli* can be associated with several export machineries located in the inner membrane including the α-hemolysin HlyBD-TolC secretion system⁶¹ and antibiotic efflux pumps (e.g. AcrAB-TolC, MacAB-TolC, EmrAB-TolC). CusC is an exception in the versatility of association among efflux pumps as it is specific for the CusBA CusC metal efflux pump.¹⁰ OprM from *P. aeruginosa* also shows some promiscuity as it is the common OMF for MexAB,⁶⁵⁹ MexXY,⁶⁶⁰ and MexJK.⁶⁶¹ In addition, it can replace OprJ and form a complex with MexCD.⁶⁶² However, OprN is only able to interact with MexEF.⁸²,⁶⁶³ Electrostatic distribution at the periplasmic entrance regions may be important for formation of functional tripartite efflux pumps. OprM is fully functional in complex with MexAB, MexCD, and MexEF, and OprJ is partially functional in complex with MexAB, whereas OprN is not.⁶⁵⁹,⁶⁶²,⁶⁶⁴ Crystal structures of the OMFs allowed the suggestion that the electrostatic distribution at the periplasmic entrance regions may be important for formation of functional tripartite efflux pumps containing MexAB. Indeed, electrostatic distribution of OprM at the interprotomer and intraprotomer grooves of the periplasmic entrance of the α-barrel domain is more similar to that of OprJ than that of OprN.⁶⁴⁵ The substrate profile also seems to be a determinant of assembly as it was shown that MexY forms a complex with OprA when it effluxes β-lactams, carbenicillin, or sulbenicillin and with the OprM when aminoglycosides are transported.⁶⁶⁵

Apart from direct involvement in efflux, OMFs play additional roles, notably as adhesion molecules and phage receptors. In *Burkholderia*, the gene for the outer membrane collagen-like protein 8 (BucL8)⁶⁶⁶ colocalizes with the downstream fusCDE genes encoding fusaric acid resistance leading to the suggestion that it forms a tetrapartite efflux pump.⁶⁶⁷ The recent characterization of the BucL8 is an unexpected unique architecture, where the canonical TolC-like N-terminal portion of the protein is followed by two C-terminal regions, one of which is a collagen-like (CL) region

composed of an uncommon repeating (Gly-Ala-Ser)ₙ sequence followed by a noncollagen low-complexity C-terminal tail (Ct). The CL-region has been shown to form a triple helix, whereas functional assays screening for BucL8 ligands identified binding to fibrinogen.⁶⁶⁷ While little is known about this protein, it significantly expands the structural gallery of OMF proteins and suggests additional adhesion and host-invasion-associated functions.

### 6.3. Gates within the OMFs

OMFs of the TolC family display a mean internal diameter of 20 Å (reaching 26 Å at the widest) along the channel. When not engaged with the cognate transporter-PAP pair, both ends of the channel are suggested to be occluded, to prevent uncontrolled diffusion of solutes, despite the fact that some crystal structures (see e.g. CusC, pdb 3PIK, Figure 29B) have been found to have external loops open. We discuss the molecular explanations for the channel gating and the locks that exist along the channel path and how recruitment of protein partners allows for a long-range, allosteric, active, and controlled opening.

#### 6.3.1. Gates within the OMFs: Outer Membrane Loops.
Similar to other OMF family members known to date, the transmembrane domain of OprM is a β-barrel formed by the association of 12 antiparallel strands.⁶⁶⁸ In transverse view, the β-barrel of OprM appears to be rather triangular, unlike that of TolC, VceC, or CusC, which are cylindrical (Figure 29B). As pointed out by Wong et al.,⁶⁶⁹ the β-barrel of OMPs is delimited by two rings of aromatic residues, on either side of the membrane, which ensures the stabilization and the vertical positioning of the channel within the phospholipid bilayer. The aromatic residues involved in the stability of the β-barrel are predominantly phenylalanines. They are mainly located at the level of the lower crown which also corresponds to the junction zone with the α-domain. Furthermore, the hydroxyl of the tyrosine residues logically points to the polar groups of the phospholipids. The nature and number of these residues is very variable from one porin to another. Unlike OprM and VceC, aromatic residue crowns are not well-defined in TolC. In the case of VceC, the side chain of residues F340, Y131, and Y143 is localized inside the porin and therefore does not interact with the cell membrane. It is possible that these residues reorient outward to stabilize the β-barrel when the canal is opened.

The cavity of porins is generally capped by a large loop which regulates the flow. In the case of TolC and its counterparts, there is no such loop, and the interior of the channel is therefore completely free. However, extracellular loops, topping their upper end, were postulated for the closed state observed in crystallographic structures. Short molecular dynamics simulations initially suggested that large motion of the extra cellular loops indeed plays a gate function over the β-barrel like a lid.⁶⁷⁰ However, simulation performed in the presence of NaCl ruled out this outer gate hypothesis and concluded that TolC is locked at the periplasmic side only.⁶⁷¹ Differences most probably arose because it was shown that at physiological values of ions, cations of the solvent are seemingly able to constitute ionic bonds in place of the removed salt bridges, which inhibited the opening of the aperture in simulations.⁶⁷² Similar studies performed for OprM showed that extracellular loops open and close freely in the simulations, suggesting that in the conditions of the simulation
<figure><img src="image_41.png" /><figcaption>**Figure 30.** "Knobs into holes" packing analysis of the helix-helix interfaces of TolC. (A) The structure of TolC (PDB ID: 1EK9) is visualized as a ribbon, with residues forming canonical coiled-coils (yellow and orange spheres) and residues forming noncanonical coiled coils (red and blue spheres). (B and C) A closer look into the knobs-into-holes packing patterns at the interfaces between α-helices, which are also depicted in "ladder" representation. Helical IDs are listed at the top, and amino acid residues are specified by the one-letter code. Heavy lines and bold helix identification numbers correspond to α-helices viewed from the exterior, while dotted lines and light identification correspond to α-helices viewed from the interior. (B) The layout of the four interfaces between α-helices participating in the construction of the 12-helix tube forming the α-cylinder. (C) The layout of the two interfaces between α-helices in the construction of the four-helix bundle forming the canonical coiled-coil restricting the periplasmic extremity of the TolC channel.</figcaption></figure>

(isolated protein, absence of peptidoglycan and LPS), there is no gating mechanism on the extracellular side.⁶⁷³

Studying the influence of the composition of the outer membrane on porin is a difficult experimental challenge, and examples of reliable *in vitro* models of envelopes of Gram-negative bacteria are scarce in the literature.⁶⁷⁴,⁶⁷⁵ Remarkably, major advances in the field of molecular dynamics now allow for more and more accurate models of very complex lipid environments.⁶⁷⁶⁻⁶⁷⁸ In the latter study, a fully assembled OM bilayer of *P. aeruginosa* was built *in silico* in order to understand the molecular determinants contributing to the rigidity and stiffness of LPS-containing bilayers, e.g the low permeability of cell envelopes of Gram-negative bacteria. In addition, OprM was inserted, and its conductivity was calculated in a model OM by comparison to a pure 1-palmitoyl-oleoyl-*sn*-glycero-phosphocholine (POPC) bilayer. The authors conclude that the presence of LPS modulates the conductivity of the channel, increasing its conductance (from 600 pS to 890 pS) and increasing the selectivity toward anions in an LPS-embedded OM bilayer by comparison to what is found in pure POPC bilayers. Accordingly, LPS was found to interact with the residues of the extracellular loop of the protein through the interaction of a network of hydrogen bonds. Opening of the outer gate was not affected by the presence of LPS *per se* but had a more pronounced interaction with the loops which is in good agreement with a direct effect on the function of the porin, as already shown for other porins.⁶⁷⁹⁻⁶⁸¹ A cryo-ET structure of AcrAB-TolC in *E. coli* intact cells⁶⁸² suggests a critical role of the loop in keeping the outer cavity open: according to this structure, upon interaction with LPS at the

vicinity, the outer diameter is kept 10 Å wide, which is enough to expel substrates.

**6.3.2. Gates within the OMFs: Knob-into-Holes and Bistable Switching between Opening and Closing of the α-Helical Bundle.** Behind the structural similarities of the α-barrel domain of OMFs of the TolC family is hidden interhelical interaction geometries that differ according to the porin, in particular at the level of the upper α-cylinder. They have been described through geometrical principles derived from the definition of coiled coils by Francis Crick.⁶⁸³

In TolC, the α-barrel is separated into two subdomains, on either side of the equatorial domain. In its lower part, H3 and H7 initiate supercoiling with H4 and H8, respectively, hence forming two canonical coiled coils which, in turn, result in a curvature of the α-helices of about 20° with respect to the axis of the channel curl and constrict to close the lower end of the porin. Formed with a periodicity of seven residues over two helical turns and a supercoil in the opposite direction of the helices, these structures are stabilized by the interlocking of side chains, most often nonpolar (dubbed "knobs"), in small concavities (dubbed "holes") formed by four residues of the opposite helix. The packing cutoff is the distance between the "knobs" residue and those which form the "holes", which is classically 7 Å.⁶⁸⁴ The periodicity of the lattice line that contains the hydrophobic residues determines the "helix crossing angle", e.g. the hand of the supercoiling.⁶⁸⁵ (Figure 30).

The upper part corresponds to an organization called the α-cylinder,⁶⁸⁶ that can be imagined to be like an assembly of coiled-coils. An α-domain, corresponding to a large barrel formed from hydro soluble α-helices encompassing half of the
A

<figure><img src="image_42.png" /></figure>

B

<figure><img src="image_43.png" /></figure>

C

<figure><img src="image_44.png" /></figure>

D

<figure><img src="image_45.png" /><figcaption>**Figure 31.** View of the periplasmic tip of representative OMFs: TolC (1EK9), OprM (3D5K), CusC (3PIK), and BesC. (A) TolC. (B) OprM. (C) CusC. (D) BesC. Structures are shown as cartoons, and residues involved in the channel gating are shown as ball and sticks representation, colored depending on the type of interactions between them. In the resting state of the channels, the trajectories of the H7/H8 helices are bent and they are prevented from adopting a relaxed superhelical trajectory by anchoring interactions with the H3/H4 helices, known as "primary gates". These are suggested to be disrupted upon interaction with the helical-hairpins of their PAP-binding partners. Intraprotomer interactions within primary gates are colored red; interprotomer interactions are shown in orange; van der Waals interactions forming the hydrophobic gates in, e.g., OprM and CusC are colored blue. The aspartate ring forming the "secondary" gate is shown in gray. The predicted primary gates of BesC (homology model), from the *B. burgdorferi* pump BesABC, which operates with the PAP BesA, that lacks the helical hairpin domain, are significantly less stable.</figcaption></figure>
periplasmic space, is the signature of TolC-like proteins. The interacting helices do not coil or adopt the canonical “knobs-into holes” configuration because they have to adjust along the curved surface of the cylinder to maintain a perfectly uniform diameter. Two distinct deformations are needed: the bending of the helices in a curve and the untwisting along the axis so that they arrange like a sheet with the α-cylinder formed by the association of three curved leaflets. By contrast to helices found in classical two-stranded coiled-coils, the axis of each helix is now tilted and translated, and α-barrel helices untwist and distort so as to make their knobs approach closely one end of the complementary holes. In addition, each helix packs laterally with two neighbors to form parallel or antiparallel sheets. As a consequence, this arrangement must fulfill geometrical constraints on two separate interfaces simultaneously, on either side of the helix. The periodicity of amino-acid side chains is crucial because of the mechanical requirements for interfacial compression and expansion on either side.⁶⁸⁵

**6.3.3. Gates within the OMFs: Primary and Secondary Periplasmic Gates.** The periplasmic end is a key region for channel opening⁶⁴¹,⁶⁶⁸,⁶⁸⁷ as the constriction at the periplasmic end has to allow the passage of cargoes as large as α-hemolysin (160 kDa). The coiled-coil-derived “hydrophobic core” does not have a sufficient stabilizing effect for the tight maintenance of the bundle, and additional specific salt bridges and/or hydrogen-bonding links between adjacent α-helices are necessary to stiffen the interactions between the internal and external helices or between protomers. Reciprocally, the positioning of an intertwined coiled-coil on top of the α-cylinder leads to an intrinsically tensed edifice, so that opening movement can spontaneously arise upon the mere breaking of the periplasmic gate(s): removal of the hydrogen bonds and the salt bridges between these residues causes a reorientation of the internal helices (H4 and H8) and the external helices (H3, H7) of the trimer linked to the relaxation of the torsional stress and to a widening of the periplasmic pore. An additional constriction, located higher in the channel, is constituted by an anionic ring formed by the carboxylate groups of the side chains of the Aspartate residues present in this region. This ring forms a cation-selective filter. These two obstacles do not allow the efflux of substrates and need to be removed before a productive efflux event.

The residues involved in the formation of the salt bridges and intra- and interprotomer hydrogen bonds at the extremities of the periplasmic helices differ between OMFs (Figure 31). In TolC, the key interaction providing the obstruction of the OMF periplasmic end is the intraprotomer bond Y362-R367 that tethers the inner coiled-coil H7/H8 to the outer H3/H4 coiled-coils. There, two major locks were described: the D153-Y362 intraprotomer hydrogen bond between helices H4 and H7 and the D153-R367 interprotomer salt bridge between helices H4 and H8. In addition, T152 also provides coordination to R367.

Mutational inactivation of the charged interactions that anchor the H7/H8 to the H3/H4 helices results in spontaneous relaxation of the superhelical trajectories of the former, leading to a “leakiness of the channel” that could be detected by electrophysiology essay,⁶⁸⁷ and the resulting relaxation was also trapped in a crystal structures of the TolC channel.⁶⁸⁸ That led to the suggestion that these residues are targeted during the formation of the tripartite complex not the partner proteins, and they were dubbed “primary gates”.⁶⁸⁹ The exact mechanics of these interactions are discussed in the

following sections, but it is also notable that in the TolC D371 and D374,⁶⁹⁰ a double concentric ring is formed above the primary gates, which also prevents the passage of any cargo beyond cations, which is often referred to as “the secondary gate”, as it has to be disrupted to allow passage of the cargo.

In order to validate the importance of these residues, site-directed mutagenesis and electrophysiology experiments were carried out. On TolC, by electrophysiology measurements carried out on the wild-type form and on the D371A-D374A mutant, it was shown that porin was a selective channel with respect to cations and this via a ring of negative charges formed by D371 and D374.⁶⁸⁷,⁶⁹⁰,⁶⁹¹ Breakdown of three inter- or intramolecular bonds (D153A–Y362 between H4 and H7 intraprotomer, R367S–D153A and R367S–T152 V between H4 and H8 interprotomers) induces a 6-fold increase in conductance (500 pS instead of 80 pS) within the channel compared to the wild-type TolC protein, reflecting a greater opening. Combining mutations R367S and Y362F even made it possible to increase the conductance further. Inactivation of the ionic bond network at the R367S and Y362F mutants relaxes the protein accompanied by an increase in conductance at 205–370 and 800–1000 pS, leading to an approximately 16 Å opening at the periplasmic end.⁶⁸⁷ A reverse experiment, aiming at blocking the movement of the helices thanks to the introduction of cysteines, confirmed, both by electrophysiology and complementation in a strain of *E. coli*, the importance of the opening of the helices for the efflux of antibiotics. Indeed, if a disulfide bridge between the H4 and H7 interprotomer helices (A159C–S350C) reduces the export of hemolysin by a factor of 5, it is completely abolished when the H4 and H8 helices or Asp374C is cross-linked.⁶⁹² However, these mutants retain the ability to interact with their partners, which suggests that the movement of the helices is essential for the efflux of molecules. The importance of residues D374 as well as D371 was later confirmed by a biochemical and structural study of the porin in the presence of hexaamminecobalt which has a 20 nM affinity for TolC and forms a plug at the entrance to the periplasmic pore upon interacting with aspartate D374.⁶⁹³ The TolC periplasmic end is tightly sealed, and several laboratories have provided insights into how the association of the channel with its inner membrane partners may cause its opening,⁶⁹⁴–⁶⁹⁸ as will be discussed with more details below.

In OprM, the equivalent residues are not all conserved (Figure 31B). Surprisingly, only one aspartate ring is present at D416 (corresponding to D374 in TolC), contributing to the closure by creating three interprotomer salt bridges with R419. Other salt bridges were also described as important for the opening mechanism: between D205–Y404 (equivalent to D153–Y362 in TolC) and D209–R403 (D153–R367 in TolC). In addition, L412 adds additional “glue” at the smallest constriction of the tunnel via van der Waals interactions. Note that X-ray crystallography under xenon gas pressure located xenon trapped in a sandwich between the three symmetry-related corresponding leucines in OprN (L405) forming a tight hydrophobic constriction of the channel. Xenon was further coordinated by the three-symmetry related D409 (corresponding to D416 in OprM), part of a salt bridge with R412 (R419 in OprM).⁶⁴⁴ Recent cryo-EM structures of the complete MexAB–OprM pump²⁷⁰,³⁰⁶ have allowed for a detailed reconstruction of the trajectories of the gating helices, which are discussed in section 8.2 (Figure 42).

Electrophysiology recordings were similarly used for the characterization of OprM⁶⁶⁹,⁶⁹⁹ and EefC from *Enterococcus*
aerogenes.⁷⁰⁰ Similarly, D371 and D374 are conserved in TolC of *E. aerogenes* (D365 and D368) but are replaced by a serine (S401) and a threonine (T404) in EefC. Both TolC from *E. coli* and *E. aerogenes* have similar ion selectivity. EefC was found approximately 10-fold lower than that of *E. aerogenes* TolC. Interestingly, single channel conductance studies performed at acidic pH showed that conductance decreases when pH decreases,⁶⁹¹,⁷⁰⁰ probably because protonation of D371 and D374 reduces the cation selectivity and the like charge repulsions that help to expand the tunnel entrance. This hypothesis was supported by systematic molecular dynamics simulations of WT versus mutated TolC that showed that the closed state is favored at low pH.⁷⁰¹ Along similar lines, MD simulations suggest that cations of the solvent may form ionic bonds when salt bridges are removed, thereby inhibiting the opening of the aperture.⁶⁷² More specifically, TolC seems to be locked in a sodium-dependent manner: the inner periplasmic bottleneck region at D374 is kept closed unless all NaCl is removed from the simulation.⁶⁷¹

The resolution of the X-ray structure of VceC⁶⁴⁶ suggested that opening occurs by changes at the interfaces between helix H4 of one protomer and the helical pair H7–H8 of the adjacent protomer concomitant to changes between helices H3 and H7–H8 of the same protomers. VceC is structurally closer to OprM than to TolC, and comparison of the structures revealed that hydrogen bonds and salt bridges are not conserved between VceC and TolC (Figure 31C). Although we do not have structures of the open VceC channel, it has been supposed that it is stabilized in its closed conformation by a different set of interactions than those present in TolC. The two intraprotomer links D153–Y362 and Q136–E359 from TolC are not conserved in VceC. The TolC D153 corresponds to S233, and the serine side chain interacts with a glutamine rather than the corresponding tyrosine.

TolC Q136 and D359 correspond in VceC to T216 and T430, respectively. The side chains of these threonines are too distant to form a hydrogen bond. None of the TolC interprotomer salt bridges (D153–R367 and R367–T152) are possible in VceC where the D153, R367, and T152 are respectively replaced by S233, A438, and G232. In CusC, the surface of the periplasmic end is relatively hydrophobic. The channel is gated by van der Waals contacts between L403 residues from the three protomers. An adjacent ring of negatively charged residues (D407) provides an additional layer of closure.⁶⁴⁸ TolC is not able to functionally replace CusC in the CusABC-mediated resistance to copper and silver,³⁵⁷ probably because the α-helical subdomain of CusB is very different from that of all other known PAPs.⁷⁰² For the sake of systematic comparison, we have computed a model of BesC from *Borrelia burgdorferi*⁷⁰³ based on the structure of VceC. It shows a gating merely located on H8, through interprotomer salt bridges between K391 and S390 (Figure 31D). This rather simplistic lock is consistent with the fact that its corresponding PAP has no actual hairpin and may in fact not be involved in maintaining the OMF open.

Opening of the channel is necessary during the translocation process, and it was long thought that the other two partners were needed to open. Deletion of efflux pump genes gives rise to various sensitivity profiles,⁵⁶ and use of drug sensitive mutants to monitor the effect of pump deletion, sequence insertion, and site-directed mutagenesis to select suppressor mutants has long been a method of reference.³¹¹ to depict what could be the residues involved in the opening of

the OMFs. In the following we describe how this strategy that allowed understanding of the reversible assembly of the pump acts as a switch for opening of the periplasmic gate. The role of the PAP in the bridging and in the stabilization of a tripartite pump has been well documented. For instance, Mokhonov et al.⁷⁰⁴ showed that there is tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to copurify MexB or OprM. The same conclusion was raised from *in vitro* experiments where MexAB OprM was reconstituted in respective liposomes and subjected to pull-down *in vitro*.⁷⁰⁵ But the PAP is certainly much more than a simple connector and could play an active role in channel opening by transmitting the work liberated at the level of the RND transporter, in the inner membrane. The interplay within the tripartite assembly is of definitive complexity. The pump is described as a highly allosteric system in which conformational changes associated with ligand binding to the RND are communicated to the OMF³⁰⁵ and, reciprocally, OMF induces activation of the RND via its PAP (see below), indicating that a mutual interplay exists between the respective partners of the pump.

For instance, it was shown that the PAP is mandatory for the activity of the pump.³¹¹,⁷⁰⁶⁻⁷⁰⁸ Mutual functional interactions have been thoroughly investigated using genetic screens for AcrAB-TolC.⁶⁸⁹,⁶⁹⁵,⁷⁰⁹⁻⁷¹² Lower ion bridges can be destabilized by direct interaction with transporters with large periplasmic domains, such as the RND family.⁷¹¹ However, the Asp-rings were believed to be too far up the channel to be directly affected by the transporter and likely “unlocked” via interaction with the tip of the PAP. Successful unlocking of these bridges designated “primary” and “secondary gates” respectively, is a requirement for productive transport.⁶⁸⁹ The open structure of TolC presents extensive interaction interfaces between the periplasmic and outer membrane proteins, defined by the helices H3–H4 and helices H7–H8 from TolC. Site directed mutagenesis and docking simulation of the coiled-coil domain of AcrA in these grooves were performed and suggested that a functional salt bridge could form in the groove and the equatorial domain, accounting for effects of mutations in this exact region on activity,⁷⁰⁹ in line with mutagenesis,⁶⁵²,⁷¹¹,⁷¹³⁻⁷¹⁵ cross-linking studies,⁶⁵⁰,⁷¹⁶ and mapping of the gain-of-function suppressor alteration of an inactive TolC mutant⁷¹⁰ or mutations in the α-helical region of AcrA allowing cross reactivity within the nonfunctional heterologous TolC-AcrA-MexB pump.⁷¹⁷ It has been proposed that PAP-OMF interaction can also be dependent on the substrate.⁸⁴,⁶⁶¹ For the first time, substrate specificity was also found within AcrAB-TolC after analyzing the effects of site-directed mutations targeting the α-barrel of TolC on substrate transport substrate specificity.⁷¹⁸ Marshall and Bavro⁷¹⁸ showed that those residues affecting the electrostatic properties of the channel caused hypersusceptibility to all efflux-tested substrates and affected cells’ ability to grow even in the absence of antibiotics. Interestingly, mutations were shown to be substrate specific.

Understanding the energy requirements for the assembly has also been the subject of a wide range of converging studies. For instance, the energization of the transition from the closed to open state of MtrE from the efflux pump MtrCDE of *N. gonorrhoeae* was evaluated by assessing the sensibility of the cell to vancomycin. This antibiotic is a very large molecule that does not penetrate the Gram-negative membrane unless the OMF is sufficiently open to passively diffuse into the cell.⁶⁸⁹
<figure><img src="image_46.png" /><figcaption>**Figure 32.** Periplasmic view of TolC in its resting, closed conformation (PDB ID 1EK9) and after full dilation of its aperture upon engagement with the PAP protein as seen in the assembled tripartite complex PDB ID 5NG5. Structures are shown as cartoon view, and aspartate D374 is shown as ball and sticks. Distances between hydroxyl groups of the carboxylic acid of D374 are measured.</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>**Figure 33.** Diversity of PAPs suggesting promiscuity of assembly. (A) In *Salmonella*, at least eight different tripartite assemblies are formed with the participation of the OMF TolC. (B) In *Anabaena* sp. PCC 7120,⁵⁵³ the OMF HgdD partners with at least eight different PAP-transporter pairs. Notably, while the tripartite assemblies in *Anabaena* are dominated by MacB and ABC transporters, the RND-based assemblies are predominantly represented in *Salmonella*. The established roles of the ABC transporters in the lower panel are indicated.</figcaption></figure>

Cells expressing the wild-type MtrE or a mutant (E434 K) which opens more easily than the wild-type remain resistant to vancomycin, but if MtrC is now expressed, cells become more sensitive. This means that MtrC is required by MtrE to open.⁷¹⁹,⁷²⁰ In addition, the full-length PAP is required to open MtrE since a truncated version of the PAP, consisting of the α-helical hairpin domain only, is not able to open the OMF. When the RND, MtrD, is expressed with MtrCE, cells are

resistant to vancomycin whereas when expressed with MtrCEE434K cells are susceptible to vancomycin.

The energy dependency of the pump assembly was investigated, again using nonfunctional mutants of MtrD, mutated in one or more residues involved in the proton-transducing pathway (D405, D406, and K948). If these mutants are expressed with MtrCE or MtrCEE434K, cells remain insensitive to vancomycin. As discussed above, the
TolC<sup>YFRE</sup> variant had significantly lower affinity to AcrB,<sup>698</sup> suggesting that opening of the channel could lead to destabilization of interactions, disengagement of the complex, and channel closing. Thus, the integrity of the assembled tripartite pump seems to be transient, with a proton-independent assembly and a dissociation that is probably performed upon closing of the OMF, which is allosterically coupled with the active transport of proton across the RND transporter. This claim was later confirmed *in vitro* upon reconstitution of the various partners of the MexAB OprM pump into proteoliposomes.<sup>705</sup> This is also in accordance with what was shown for the hemolysin secretion system, in which energy consumption is not necessary for the assembly of the type 1 secretion system<sup>721</sup> and *in vivo* cross-linking copurification and ITC experiments showed that the AcrAB-TolC does not require the proton motive force to assemble.<sup>694,696</sup> Ultimately, structural determinations allowed for a detailed description of the periplasmic opening (Figure 32).<sup>688,689,693</sup> Opening of the periplasmic end is believed to occur in a comparable manner to a photographic diaphragm where the “internal coiled-coils” undergo twisting to align with the “external coiled-coils” H3/H4.<sup>690</sup> Structural and molecular dynamics studies<sup>641,642,689</sup> later confirmed the photographic diaphragm-style opening mechanism to untwist the helices at the level of the coiled-coil domains.

## 7. PERIPLASMIC ADAPTOR PROTEINS (PAPS)/MEMBRANE FUSION PROTEINS (MFPS)

The periplasmic adaptor proteins (PAPs), or as they were previously referred to, “membrane fusion proteins” (MFPs),<sup>722</sup> are a wide and diverse family of transporter-associated proteins the primary function of which is to allow pairing of a limited set of outer membrane factor (OMF) proteins to a wide range of inner membrane transporters ensuring high-fidelity and selectivity of assembly, thus contributing to the combinatorial diversity of the tripartite efflux pumps and greater plasticity of the response to xenobiotic stress.<sup>106,723</sup> The unique feature of PAP proteins that makes them central to the assembly of the trans-envelope pump complex is their ability to interact simultaneously with both the inner-membrane transporter and the OMF using distinct protein-protein interfaces. In this way PAPs allow for “promiscuity” of the OMF relative to the inner membrane transporter, allowing the docking of a number of different transporter types to a single OMF. In *Salmonella* at least seven tripartite complexes are formed, including the five RND complexes (AcrAB, AcrAD, AcrEF, MdsAB, and MdtABC), MFS EmrAB, as well as the ABC transporter bases MacAB that include the TolC as an OMF,<sup>724</sup> which is made possible by the range of PAPs (Figure 33A). Similarly, at least eight separate PAPs appear to dock to the OMF HgdD in *Anabaena* sp. PCC 7120<sup>553</sup> (Figure 33B).

The existence of a wide range of PAPs that are able to interact with a single OMF indicates that the primacy of the determinants of substrate specificity resides with the specific PAP-transporter pair, although the OMF may provide some limited substrate differentiation.<sup>718</sup> Despite their central importance for the transport process, the role and function of PAPs within the assembly remains least-well understood. In the pages below we will try to summarize the recent advances in our understanding of their function. Previous systematic reviews from recent years have focused in more detail on the diversity and phylogenetic grouping of PAPs<sup>725</sup> and structural

organization and overall mechanisms of PAP-transporter coupling,<sup>726,727</sup> respectively.

### 7.1. Historical Note

PAPs participate in the formation of tripartite systems with a number of different transporter classes including ABC transporters (HlyD,<sup>728</sup> MacA<sup>60,206</sup>), MFS transporters (FarAB-MtrE<sup>79</sup>), and RND transporters; however, at the time of writing there have not been any reports of tripartite assemblies formed with the PACE, MATE, or SMR systems. Tripartite efflux systems built around the ABC transporters forming the RTX-toxin secretion systems, which later became known as T1SSs, were the first tripartite systems to be identified and provided the blueprint of the organization of the other such systems, including the multidrug efflux pump assemblies. The first PAP genes were cloned in association with the hemolysin A (HlyA) secretion system from *E. coli*<sup>729</sup> and the leukotoxin secretion systems from *Pasteurella haemolytica*.<sup>535</sup> The HlyBCD system built around the ABC transporter HlyB was one of the first secretion systems to be characterized, and its homology to P-glycoprotein allowed some of the first models of efflux function in the context of multidrug resistance to be proposed.<sup>730</sup> The first indication of the periplasmic localization and organization of the PAPs has consequently also come from topological mapping of the HlyD, part of the ABC-based T1SS HlyBCD responsible for hemolysin export.<sup>728,731</sup> It also provided the first insight into the PAP function and linkage to TolC.<sup>732</sup>

Subsequent bioinformatic analysis of the available PAPs suggested that they form a novel, clearly defined group, which was ascribed “membrane fusion” capability, based on the superficial resemblance of hairpin domain architecture to the coiled-coil domains observed in *bona fide* membrane fusion proteins, e.g., paramyxoviral SVS.<sup>722</sup> This inferred fusion function has never been demonstrated in any PAP since; however, the old-name association still persists, and they are sometimes referred to in the literature as “membrane fusion proteins” or MFPs.

### 7.2. Structural Organization of PAPs

Periplasmic adaptor proteins display significant structural diversity; however, they are built around a common blueprint. Topological analysis of the PAP-fold reveals a rather unique nonlinear architecture, with each of the domains being constructed with the participation of both N- and C-terminal halves of the protein, due to the fact that the polypeptide chain is effectively folded in the middle, similar to a “hairpin” or “safety pin” arrangement.<sup>733,734</sup> While both N- and C-termini of the proteins are either associated with, or held close to, the inner membrane, the elongated body of the molecule extends away from it into the periplasmic space, eventually reaching the OMF.<sup>725,726</sup> This body is composed of three to four “core” domains but is often augmented by additional N- and C-terminal modules, which are specific to the different families of PAPs and are dictated by the cognate transporters.

Rapid advances in the structural characterization of the PAP proteins over the past decade have shed light on all of the main group members, although the RND-associated proteins were the first to be solved; for example, MexA (PDB ID 1T5E;<sup>733,734</sup> PBD ID 2V4D<sup>716</sup>) and AcrA (PDB ID 2F1M<sup>735</sup>) remain the best-studied examples due to the plethora of structures of assembled tripartite complexes with their participation.<sup>270,287,305,306</sup> The structures of the heavy-metal efflux (HME) RND pump-associated PAPs have also
been resolved both in isolation, e.g., the copper-efflux associated CusB (PDB ID 3H94 3OOC, 3OPO, 3OW7)⁷⁰² and ZneB from Cupriavidus metallidurans (PDB ID 3LLN),⁶³ and in complex with cognate transporters, e.g. CusBA (PDB ID 4DNR, 3T51, 3T53, 3T56, 3NE5, 4DNT, 4DOP).³⁵⁹,⁶¹⁰

The structures of MacB-associated PAPs from *E. coli* *EcMacA* (PDB ID 3FPP)⁷³⁶ and *Aggregatibacter (Actino-bacillus) actinomycetemcomitans* AaMacA (PDB ID 4DK0)⁷³⁷ and the structure of the full MacAB-TolC pump have been reconstructed from cryo-EM data (PDB ID 5NIL-5NIK).²⁷² The structures of the PAPs associated with the MacB-type Gram-positive efflux systems have also been resolved, including the *Bacillus YknX*⁵⁹⁴ and the Spr0693 from *Streptococcus pneumoniae* R6 (PDB ID 5XU0),⁵⁷¹ both of which assemble into a hexameric pattern very similar to MacA.

The first MFS transporter-associated PAP was EmrA from *Aquifex aeolicus* (PDB ID 4TKO).⁷³⁸ The last group of PAPs to reveal their structure was the PAPs associated with T1SS. The structure of *E. coli* HlyD (PDB ID 5C21, 5C22)⁷³⁹ was followed by the Serratia *marcescens* LipC PAP (PDB ID 5NEN).⁵¹⁴

In addition, the structures of some aberrant PAPs have become available, such as the PAP BesA from the Lyme disease associated spirochete *Borrelia burgdorferi* (PDB ID 4KKS-4KKU),⁷⁰³ which lacks the helical hairpin domain altogether. In addition, some orphan PAP structures could also be found in the database, as a result of structural genomics efforts, e.g., BACEGG_01895) from a putative efflux pump from *Bacteroides eggerthii* DSM 20697 (PDB ID 4L8J); however, its association with HAE-1 or HME pumps remains to be established. Figure 34 presents the organization of a typical PAP on the example of the MexA which functions with the RND transporter MexB and the OMF OprM in *P. aeruginosa*.⁷⁰⁴

In the paragraphs below, we will “walk” along the structure of the typical PAP from the N-terminal to the C-terminal end, describing briefly the central features and the domains involved in building its unique architecture.

Many RND transporter associated PAPs in their mature form, following processing by signal peptidase 2, are modified by an N-terminal cysteine lipidation (e.g., triacylation or in the case of AcrA, S-palmitoylation), which anchors the protein to the inner membrane. The role of this anchorage is not fully clear, as truncated delipidated AcrA carrying a His-tag at the C-terminal is functional and has been used for biochemical studies.⁷⁴⁰

The PAPs associated with ABC and MFS transporters, as well as those involved in T1SSs, are typically anchored to the inner membrane by an N-terminal transmembrane helix and have sizable cytoplasmic domains;⁷²⁶ however, very little is known of their structure at present. These regions, however, are essential for correct substrate recognition and complex assembly.²⁰⁶

### 7.3. Domain Organization of the PAPs Associated with RND and MacB-Assemblies

The core domains, which are found in all RND-associated PAPs, including MexA, present a beads-on-a-string arrangement; starting with the closest to the membrane are the membrane proximal domain (MPD), the β-barrel domain, the lipoyl domain, and the α-hairpin (coiled-coil) domain. Some modules are universal, while others are only shared within a

<figure><img src="image_48.png" /><figcaption>**Figure 34.** Structural organization of a typical PAP based on the structure of the MexA from *Pseudomonas aeruginosa* (PDB ID 6TA5).²⁷⁰ The topological organization of the principal PAP domains is shown on the right. The polypeptide chain of the PAP has a form with a hairpin-like fold with both N- and C-termini being close to the membrane and both the N- and C-terminal parts of the protein contributing to each domain. The N-terminal cys-residue that is lipidated in the RND-associated PAPs is highlighted in yellow.</figcaption></figure>

subset of the family, and this arrangement correlates with the type of cognate transporter with which the PAP functions.⁷²⁶

The closest domain to the membrane, as the name suggests, is the membrane proximal domain, abbreviated from here on as MPD. It is flexibly linked to the core of the protein and is composed of both N-terminal and C-terminal elements. Its N-terminal β-strand provides the direct link to the inner membrane in the TM-containing PAPs, such as those associated with the T1SS, MacB, and MFS-type transporters. The rest of the domain is composed of the C-terminal part of the protein and presents a β-roll domain topology⁷¹⁶ loosely related to the downstream β-barrel domain, from which it may have originated via an early gene duplication event.

Notably MPDs are missing in the PAPs operating with transporters that lack periplasmic domains, suggesting that their function could be associated with supporting the periplasmic assembly and substrate selection and presentation.⁷²⁶ This role has been clearly demonstrated for PAPs associated with HME RND transporters, such as CusB,³⁶⁰,⁷⁴¹ and hinted at by mutagenesis and transporter studies of chimeric HAE-1 transporter assemblies.⁷¹⁷,⁷⁴² Furthermore, MPDs are essential for the activation of ATPase function in the MacB-class transporters,⁵⁷³,⁷⁴³ and they have been associated with substrate binding as will be discussed further in the
<figure><img src="image_49.png" /><figcaption>**Figure 35.** Hexamerization of PAPs and the formation of the helical–hairpin nanotube. RLS-motif of PAPs and its conservation across different groups. (A) General view of the MacA hexamer from PDB ID 5NIK, highlighting principal domains. (B) Close view of the hairpin domains presenting the RLS signature motif. (C) Top-down view of the lipoyl-ring of MacA as seen in PDB ID 3FPP.⁷³⁷ The N209 from the “gating ring” is restricting the channel in the hexameric form. (D) Canonic RLS-motif as seen in RND- and MacB-associating PAPs. (E) Overlay of the tips of the hairpin domains of MacA and AcrA showing close conservation of the RLS-motif. (F) Overlay of the tips of the hairpin domains of MacA and T1SS-associated HlyD shows deviation from the canonic sequence.</figcaption></figure>

context of their tripartite assembly. A possible break from the rule is observed in the specialized MacB-like transporter DevCA and related pumps in *Anabaena*, the PAPs of which (e.g., DevB), appear not to possess MPDs.²⁰⁶

The MP-domain of RND-associated PAPs was shown to be essential for the assembly and function of the AcrAB-TolC in *E. coli*⁷⁴⁴ and more recently, in a range of *Salmonella* PAPs.⁷⁴² In RND-based assemblies, PAPs play a significant role in stimulation of the efflux activity and consumption of the proton gradient as exemplified by the liposome-reconstituted studies of AcrAB⁷⁰⁶ and MexA–MexB studies.⁷⁰⁷ While in both cases the pump activity was increased in the presence of the cognate PAP, the MexA dramatically increased the activity of MexB only when the substrate was also present, suggesting that substrate engagement plays a significant role in driving the assembly.

The next domain up from the membrane is the self-explanatorily named β-barrel domain, which presents a β-barrel consisting of six antiparallel β-strands capped by a single α-helix⁷³³,⁷³⁴ (Figure 34). The β-barrel domain presents similarity to enzymatic ligand-binding domains such as the flavin adenine nucleotide-binding domain of flavodoxin reductase and ribokinase and also to domains with odorant-binding properties. The roles of this domain appear to be predominantly connected with self-association of the PAPs into hexameric assemblies,⁷³⁷ as well as in forming contacts with the periplasmic domains of respective transporters.²⁷²,²⁸⁷,³⁵⁹

Among the MacA-family PAPs the residues equivalent to the *EcMacA* E231, Y276, and T293 residing within the β-barrel domain are strictly conserved. These three residues are involved in the interactions between the β-barrel domains,

forming a closed six-membered ring in head-to-tail fashion.⁷³⁷ Notably equivalent residues are conserved in PAPs from Gram-positive organisms, e.g., the *B. amyloliquefaciens* YknX and Spr0693,⁵⁷¹,⁵⁹⁴ and similar conservation of equivalent residues in RND-associated PAPs is also observed, highlighting the functional importance of this hexamerization.

At least in some subsets of PAPs, e.g., HME-associated ZneB, the β-barrel domain also participates in ligand binding.⁴⁶ We will discuss these functions further in the context of the specific tripartite assemblies in the sections below.

The next domain up from the β-barrel domain is the lipoyl domain, named due to the homology it exhibits to the biotinyl/lipoyl-carrier domains found in dehydrogenase enzymes.⁷³³,⁷⁴⁵ These domains consist of a β-sandwich of two interlocking motifs, each consisting of three β-strands. The role of these domains also appears to be in stabilizing the oligomeric self-assembly of the PAPs, and mutations targeting the domain have a destabilizing effect, often obliterating function altogether.²⁰⁶ However, unlike the β-barrel domains, the lipoyl domains do not appear to provide contacts with the transporters, at least in the studied RND and MacB assemblies.²⁷²,³⁰⁵,³⁵⁹ The lipoyl domains also provide substrate-vetting and, at least in the case of MacB-based systems, the lipoyl-domains of the MacA-family PAPs create a “gating ring” restricting access to the α-helical tunnel formed by their hexamerised hairpin domains, which is suggested to prevent backflow of substrates.²⁷²,⁷³⁷ (Figure 35, panel D).

The most-distant from the inner membrane and facing toward the OMF is the α-hairpin domain, which in its “canonical” form presents an antiparallel coiled-coil, formed of two helical fragments, divided in the middle by a loop. The distal end of this hairpin is suggested to be responsible for the
<figure><img src="image_50.png" /><figcaption>**Figure 36.** Different types of PAPs associated with different efflux systems. Comparative structural gallery of PAPs of known 3D structure, colored by domain. Membrane proximal domain (MPD) in red; β-barrel domain in green; lipoyl in blue; α-helical-hairpin in gold. From left to right: BesA (PDB ID 4KKS); HME-associated CusB (PDB ID 3OW7); HAE-1 associated MexA (PDB ID 6TA6); MacB-associated MacA (PDB ID 5NIK); MFS-associated AaEmrA (PDB ID 4TKO); and T1SS-associated HlyD (PDB ID 5C21). The missing loops and transmembrane fragments of MacA/EmrA and HlyD have been modeled for illustrative purposes. Note that only RND- and MacB-associated PAPs appear to contain MPDs, while the spirochaetal BesA lacks the α-helical–hairpin domain altogether.</figcaption></figure>

interaction with the reciprocal coiled-coils of the OMF; however, the level of this interaction is either extensive, by forming four-helical bundles in the so-called “deep-inter-penetration” or “wrapping-up” model,<sup>649,716</sup> or strictly restricted to the tip of the hairpin and the loop that connects the two helices in the so-called “cogwheel” or “tip-to-tip” model.<sup>737,746,747</sup> Nestled within the lipoyl domain, the α-hairpin domain is an extension from the very loop that contains the lysine that undergoes lipoylation (covalent attachment of lipoamide, via an amide bond) in the corresponding domain in the dehydrogenase subunits.

The α-helical hairpin domains of the “canonical” PAPs associated with RND assemblies, such as AcrA and the MacB-associated MacA, resemble inverted versions of the helical hairpins forming the structural repeats within the OMF.<sup>716</sup> Indeed, the Cα-backbone of the PAP α-helical hairpin domain can be superimposed on both coiled-coils of TolC, when inverted and viewed in an equivalent orientation. The coiled-coils within this group of PAPs are formed upon a classical heptad repeat (HxxHCxC) principle, where H is a hydrophobic and C is a charged residue,<sup>748,749</sup> and they contain a variable number of repeats. The heptad is also usually depicted as abcdefg, with the core of the hairpin being formed by the residues in the a and d-positions, which are conserved throughout the PAP family and contain bulky hydrophobics, while the exposed helical faces, directly opposite the core of the hairpin are also conserved, with hydrophilic serine/glutamate occupying the c-position, and alanine predominantly found in the f-position<sup>733</sup> (Figure 35 and connected figures in section 8.2). The hairpin domains show considerable diversity in organization, differing in length from four heptad repeats per helix in MexA to 11 heptad repeats in EmrA;<sup>738</sup> however, there are some aberrant forms, such as the metal-efflux pump associated CusB,<sup>702</sup> which features a folded hairpin, with only 2 heptad repeats in its longest helices, as well as the spirochetal BesA,<sup>703</sup> which lacks an α-helical extension altogether.

Sequence alignment of the distal part of the hairpin domains of the canonical PAPs reveals high conservation of the tip-

region of α1 and α2-helices, presenting a consensus sequence RxxxLxxxxxxS/T, which became known as the RLS-motif,<sup>737,750–753</sup> which has been implicated in OMF binding, which was subsequently confirmed by the cryo-EM structure of the complete assemblies. The leucine of the RLS-motif is the most conserved residue within PAPs, mediating strong tip-to-tip interactions between PAPs and OMFs. However, the last position of the motif shows higher variability and can be occupied by a serine or a threonine residue (Figure 35).

The role of the PAP hairpin-domain may not be limited to the RLS-based pairing with the OMF, as witnessed by a number of suppressor and compensatory gain-of-function mutations which map to the stalk of the hairpin.<sup>709,711,717,754,755</sup> The structures of the MacAB-like transport complex from Gram-positive bacteria<sup>571</sup> composed of the ABC transporter Spr0694–0695 and the associated PAP Spr0693 (UniProt ID: Q8DQF9) revealed that despite the strikingly similar architecture to that of the MacAB complexes seen in the Gram-negative bacteria and notable preservation of the hexameric tube-like assembly of PAPs creating a tight gasket-like structure over the transporter, the tips α-helical hairpin is mobile and disordered in the structure. Consistent with a lack of OMF-association, the tips of the Gram-positive PAPs examined to date show little conservation and no obvious RLS-like motif.<sup>571,594,725</sup> These findings highlight the functional importance of PAP–hairpin domain oligomerization. However, they also pose a question regarding their role in these organisms, and while the definitive answer to that is still not known, an increasing amount of circumstantial evidence suggests that they are involved in peptidoglycan binding;<sup>756</sup> for example, both AcrA and YknX were found to bind to peptidoglycan.<sup>594,751</sup> Notably, *E. coli*, but not *S. aureus* peptidoglycan was found to facilitate AcrA-TolC interaction.<sup>751</sup> Furthermore, the reconstruction of the assembled AcrAB dimers from the *in situ* cryo-tomography of AcrAB-TolC complexes is also compatible with the hairpin of the PAP being in direct contact with the PG layer.<sup>307</sup>
The α-helical hairpin of PAPs of the EmrA-family<sup>738</sup> and T1SS-associated PAPs such as HlyD<sup>739</sup> features an “extension subdomain”, representing a departure from the classical heptad packing, which can be seen as an additional helical insertion into the proximal part of the hairpin, which is highly similar to the untwisted pairs of α-helices in the OMF α-barrel.<sup>726</sup>

The coiled coils of the α-hairpin domain show significant flexibility and propensity for self-association, which has been linked to their functional role.<sup>735,736,757,758</sup> Apart from soluble dimer and trimer formation that could be detected in the periplasm, a particular subset of PAPs form hexameric tubular structures, which match remarkably well the outer diameter of the OMF channel.<sup>573,737,759</sup> The stability of these oligomeric assemblies differs between different PAP types and generally correlates with the length of the hairpin domain, resulting in a differential affinity to the OMF.<sup>697</sup> These differences in hairpin domain organization appear to be associated with the different modes of engagement of the PAPs with their respective transporters, and we will discuss in further detail the context of specific assemblies, but here we will highlight some specifics of the PAPs participating in T1SS and MFS transporter assemblies.

## 7.4. PAPs Associated with MFS Transporters and T1SS

Unlike the “canonical” group of PAPs discussed above, which function with transporters that feature large periplasmic domains and only shuttle cargoes from the periplasm, the PAPs from this group associate with transporters that transfer cargoes directly from the cytoplasm and lack pronounced periplasmic domains. Correspondingly, these PAPs present a different architecture, as they face dual challenges—on one hand they have to provide extensive sealing of the relatively small transporters to avoid leakage of substrate in the periplasm, and on the other hand, they are required to reach further out into the periplasm to bridge the OMF as they are located much closer to the inner membrane.

Notably, PAPs from this group lack a MPD domain and present a very long helical extension although they have slightly different solutions to the common problem discussed above (Figure 36). The MFS-associated group is exemplified by the structure of the EmrA from the hyperthermophile Aquifex aeolicus (AaEmrA from here on).<sup>738</sup> The lipoyl and β-barrel domains are rather conserved and match those seen in the rest of the family, with the exception of a notable loop (P321-V343) in the β-barrel domain, which is both unique to and highly conserved across the EmrA family, suggesting it may play an important functional role. The periplasmic part of the protein binds the transported drugs, and in addition, the *E. coli* homologue, *EcEmrA*, is anchored in the membrane by an N-terminal helix, which contains a leucine-zipper-like dimerization domain and is involved in oligomerization and transporter association of the PAP.<sup>760</sup>

The crystal structure of the AaEmrA revealed an antiparallel, two-stranded, α-helical coiled-coil domain of 168 residues and 127 Å long with 11 heptad repeats per helix. Thus, the hairpin-domain in AaEmrA is over twice the length of that in RND-type PAPs, e.g., MexA and AcrA (47 and 58 Å, respectively), although in its *E. coli* counterpart *EcEmrA*, that is estimated to be around 80 Å in length<sup>738</sup> (Figure 36). Notably, the coiled-coil in EmrA appears nonideal due to disruption in packing caused by positively charged residues in its hydrophobic core, i.e., K84, K98, R130, R182, and K119 which occupy the normally hydrophobic a and d heptad positions. In addition,

the heptad repeat pattern is disrupted by a four-residue insertion in the N-terminal helix (residues 112–115) and a three-residue insertion in the C-terminal helix (residues 210–212), approximately 70 Å from the tip of the structure and close to the center of the coiled-coil.<sup>738</sup> These insertions disrupt the knobs-into-holes packing, resulting in a weakened interhelical interaction in this region of the coil. Heptad shifts, known as “stutters” and “stammers”, are commonly found in fibrous proteins with an extended coiled-coil structure,<sup>761</sup> and it has been long hypothesized that such regions serve as points of flexibility for extremely long coiled-coils.<sup>762,763</sup>

## 7.5. T1SS Associated PAPs

Similar to the PAPs associated with the MFS assemblies, the structure of the representative T1SS-associated PAP, namely *E. coli* HlyD (referred to as *EcHlyD* henceforth) from the α-hemolysin secretion system, presents an architecture lacking an MPD and is characterized by an oversized α-helical hairpin domain measuring ~115 Å in length, thus being second only to the MFS-associated EmrA, which is almost 130 Å long (PDB ID 5C21-5C2).<sup>739</sup>

Similar to AaEmrA discussed above, the α-helical hairpin domain of *EcHlyD* is divided roughly in the middle, into an N-terminal and C-terminal part, by a loop at its distal tip (L2), which is supposed to contact the OMF. Unlike the MFS-associated PAPs, however, which have two continuous α-helices forming each half of the α-hairpin domain, the HlyD hairpin presents a complex, discontinuous architecture, which deviates from the standard coiled-coils observed in the classical RND-associated transporters and is composed of three helical fragments (α1 to α3), linked by two connecting loops L1 and L2.<sup>739</sup>

While the C-terminal part of the hairpin domain is formed by a continuous α3-helix (residues 251–325), which runs the entire length of the hairpin, the N-terminal part of the hairpin domain is discontinuous and formed by two shorter helices, α1 and α2, connected by the long and flexible loop L2. While the exact role of the long loop L1 is unclear, it provides a hingelike arrangement for the α1- and α2-helices, allowing them to form independent antiparallel-coiled coils with the α3, and such an arrangement may purposefully allow for allosterically controlled destabilization of the PAP α-barrel, providing greater flexibility, and it may facilitate the disassembly of what otherwise may be a too-rigid conduit. Thus, the breakage of helices observed in the T1SS-associated HlyD likely serves the same purpose as the nonheptad insertions in the EmrA, allowing to alleviate structural stress, and furthermore, helical breaks allow the helical trajectories to be corrected, providing both more flexible lateral packing and easier disassembly of the complex. Intriguingly, a similar solution to the cladding of the alpha-barrel problem is presented by the OMF-proteins, the inverted repeats of which follow the same discontinuous hairpin arrangement.

Oligomerization of HlyD appears to be prerequisite for TolC recruitment,<sup>492</sup> and consistent with that forced hexamerization of a HlyD fragment (covering 81–383) fused to an Hfq-hexamer ring was able to recruit a TolC-MacA chimeric protein, while the same HlyD fragment was unstable on its own and was not capable of TolC-MacA binding.<sup>739</sup> Indeed, formation of a helical bundle via lateral oligomerization, similar to those observed for MacA and AcrA, has been suggested based on the crystal structure of the hairpin and lipoyl domain of HlyD,<sup>739</sup> and modeling of hexamer creates a
<figure><img src="image_51.png" /><figcaption>**Figure 37.** Modularity of the assembly of tripartite complexes. A schematic representation of the different tripartite assemblies based on their transporter types reveals striking correlation with the domain composition of the PAP proteins associated with them. PAPs of the MFS- and T1SS ABC transporter-based complexes lack MP domains, which is also correlated to the transport of their cargoes across the inner membrane (IM). On the other hand, the MacB and RND transporter-based assemblies pick their cargoes from the periplasmic side of the membrane associated with PAPs that have MPDs, hinting toward their role in presentation of the cargo to the transporters. Notably, the *β*-barrel domains of the PAPs are universally present and allow adaptation to diverse transporter stoichiometries.</figcaption></figure>

plausible PAP assembly, with an internal diameter of ~40 Å. Providing support to the plausibility of the model, packing of the helices within the model brings into proximity two critically important residues, mutation of which abolishes HlyD function *in vivo*, as established from functional studies (R186 from α2-helix and D309 from α3-helix).⁷⁵² Superposition of the distal parts of the α-helical hairpins of HlyD and MacA provides a good structural match (Figure 35E); however, the RLS-motif is misaligned relative to the earlier sequence-alignment-based predictions, which designated the R186, L190, and T197 as the signature triplet.⁷⁵²

Furthermore, the RLS-motif as positioned at the L2-loop appears to be significantly degenerated relative to the other members of the PAP family which interact with TolC and present a sequence which in the *EcHlyD* is formed of D238, L243, and A250. The lack of RLS conservation suggests higher promiscuity at the tip of the assembly than might be expected by the limited interface that it presents. This unique organization of the hairpin in T1SS-PAPs has also been confirmed from the structure of the *Serratia marcescens* LipC (PDB ID 5NEN), a PAP that forms a T1SS with the ATPase LipB and the OMF LipD in the transport of the lipase cargo-protein.⁵¹⁴ The “RLS” sequence of the LipC is further divergent from the canonical one, with S183 (in place of R128 AcrA/D239 HlyD respectively); L187 (L132/L243 being the only fully conserved residue within the motif); P194 (S139/A250); and E200 (D144/E256) in place.

### 7.6. PAPs Associated with the Heavy Metal Efflux (HME) Family

PAPs associated with the heavy metal efflux (HME) family of RND transporters often contain additional N- and C-terminal domains, e.g., SilB from *Cupriavidus metallidurans* CH34,⁷⁶⁴ the C-terminal domain of which has been shown to provide metal-chaperoning function. These domains have arisen from

gene-fusions with metal-binding proteins and could also be found as stand-alone polypeptides (e.g., CusF of *E. coli*; PBD ID 3E6Z), which display a unique metal-binding *β*-barrel fold.⁷⁶⁵,⁷⁶⁶

CusB protein is part of the CusCBA periplasmic Cu⁺/Ag⁺ efflux system in Gram-negative bacteria,⁷⁶⁷ and it was the first HME-associated PAP whose structure was solved (PDB ID: 3H9I)⁷⁰² and presented several unexpected structural features; notably, its hairpin domain was significantly shorter than the ones of “canonical” HAE-1 associated PAPs and was folded into an antiparallel, three-helix bundle. In addition, CusB was found to directly bind to Cu⁺ ions via an intramolecular relay of conserved methionine residues (e.g., M49, M64, M66, and M311), undergoing a conformational transition, implicated in the transport cycle of the pump.⁷⁶⁸⁻⁷⁷⁰

The structure of ZneB (PDB ID 3LNN), a PAP associating with the second branch of HME transporters, responsible for efflux of divalent cations, has also been solved; it presented an architecture very similar to the canonical HAE-1 PAPs.⁴⁶ Similar to CusB, it also has a specific metal-binding site, which however is much more localized and located in the junction between the *β*-barrel and the MP-domain. While the PAP ZneB presents an architecture very similar to the PAPs associated with the HAE-1 type RND transporters, it also acts as a 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 the MPD domain.⁴⁶

The demonstration of active substrate-binding and presentation function, as exemplified by the metal-pump associated PAPs, showed that the PAP-proteins are more than just passive adaptors and has shifted the attention to their active role in organization of the tripartite assemblies, which
<figure><img src="image_52.png" /><figcaption>**Figure 38.** Structural organization of the PAP *β*-barrel domains links them with *β*-barrel domains in flagellar basal body proteins. (A) Structure of the isolated *β*-barrel domain of the MFS-associated PAP EmrA, based on PDB ID 4TKO. (B) Topological diagram. The lipoyl and *α*-hairpin domains can be seen as inserted between the *β*1- and *β*2-strands of the *β*-barrel. (C) Structure of the C-terminal domain of the flagellar basal body protein FlgT from Vibrio.</figcaption></figure>

we will discuss in the following paragraphs for each of the main systems.

### 7.7. Fellowship of the Rings—The Role of PAP *β*-Barrel Domains in Assembly and Processivity of Tripartite Efflux Pumps

In the preceding sections we described a wide variety of transporter families, PAPs and OMFs, which combine to create a motley crew of tripartite assemblies. This leads to the logical question, as to what the unifying feature of these assemblies is and what brings them under the same umbrella. Linked to the common use of the OMFs, the unifying feature of these assemblies is the use of the PAP-family for bridging the transporters. As noted previously,⁷²⁶ the domain utilization of the PAPs is associated with specific transporter families (Figure 37), with the presence of MP domains being restricted to transporters which have large periplasmic domains, i.e. the MacB- and RND-families, which correspondingly take their cargoes predominantly from the periplasm.

The examination of the remaining tripartite complexes suggests that the PAP domains minimally required for forming a functional assembly are the *β*-barrel and lipoyl domains, both of which have been shown to form homooligomeric assemblies. Structures of binary PAP-transporter complexes⁶¹⁰ and fully reconstituted pumps⁹,²⁷²,³⁰⁵ indicate that while lipoyl domains never contact the transporter, the *β*-barrel domains form a critical hexameric ring-seal around the transporter in the periplasm, which is a defining structure of the tripartite assemblies, shared across all transporter types.⁷²⁶ This gasket-like assembly allows seamless adaptation of the different transporter stoichiometries and sizes to the trimeric OMF and has some unique features, which we will discuss below.

The PAP *β*-barrel domain shares the topology of the barrel seen in ribokinase enzymes and lipid-binding proteins,⁷³³ and our previous work linked the *β*-barrel domains of the PAPs to the ribokinase-like domains seen in some flagellar basal-body associated proteins.⁷²⁶ The bacterial flagellar motor consists of a number of conserved components which include the inner-membrane spanning MS-ring; a cytoplasmic C-ring and its associated export apparatus; a periplasm-and-outer membrane spanning rod structure; and associated with the latter, the

periplasmic (P-) and outer-membrane embedded L-rings; plus, the optional H- and T-rings which are present in some organisms such as Vibrio.⁷⁷¹ The C-terminal domain of the flagellar protein FlgT (FlgT-C; residues 256–356),⁷²² which associates with the outer membrane H- and T-rings in Vibrio, is comprised of a six-stranded *β*-barrel, nearly identical to the N-terminal domain of the *β*-subunit of F₁-ATPase (e.g., bovine mitochondrial F1-ATPase PDB ID 1BMF),⁷²³ the catalytic subunit of the ATP-synthase complex.⁷²²

This topology is strikingly similar to that observed in the PAP *β*-barrel domains (Figure 38), which only differ by the lipoyl domain being spliced into the loop connecting the *β*1-strand and *β*2-strand forming the core of the barrel.⁷²⁶ Consistent with that, despite lacking discernible sequence homology, FlgT-C superposes on the *Ca*-backbone of the *β*-barrel domain of EmrA (PDB ID 4TKO), with an RMDS of ~3 Å.⁷²⁶ Our new expanded analysis, provided below, expands this association of PAP *β*-barrel domains across a number of the type III secretion system proteins as well, including the cytoplasmic flagellar assembly export apparatus.

Away from the outer membrane, the cytoplasmic flagellar type III export apparatus used for the secretion of the flagellar components consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI, and FliJ.⁷⁷⁴ There, the FliI is a Walker-type, homohexa-meric rotary-ATPase, oligomerization of which is required for ATP-hydrolysis;⁷⁷⁵ FliJ is the stalk unit, and the FliH is a regulatory/stator subunit.⁷⁷⁶ This system is also evolutionarily connected to the virulence associated "injectisome" apparatus,⁷⁷⁷,⁷⁷⁸ e.g. the one coded by the Salmonella pathogenicity island 1 (SPI-1), where the corresponding components in the so-called "sorting platform", forming the base of the SPI-1 injectisome, are the ATPase InvC (aka SctN under the unified nomenclature),⁷⁷⁹ its stalk subunit InvI (SctO), and the regulatory/stator subunit OrgB (SctL).⁷⁸⁰

The FliI~6~FliJ complex was shown to be structurally similar to the *α*~3~*β*~3*γ* complex of F~1~F~0~-ATPase,⁷⁸¹,⁷⁸² with the N-terminal domains in the FliI-type ATPases being highly homologous to the N-terminal domains of the F1/V1-ATPases (e.g., bovine mitochondrial F1-ATPase (PDB ID 1BMF)⁷⁷³ or the *E. coli* F1F0-ATPase (PDB ID 6OQR)),⁷⁸³ in agreement with earlier
<figure><img src="image_53.png" /><figcaption>**Figure 39.** (A) Structural connections of the β-barrel domains in the rotary ATPases and the T3SS-associated ATPases. (B) The β-barrel domains of PAPs structurally homologous to those found in the rotary ATPases. Secondary structure elements are color coded identically to the topological diagram in Figure 38. (C) Overlay of the C-alpha backbone between EmrA and F1-ATPase β-subunit highlights their close structural homology.</figcaption></figure>

bioinformatic predictions.⁷⁸⁴,⁷⁸⁵ This has been further confirmed by the recent cryo-EM structure of the homohexameric T3SS ATPase EscNin complex with its central stalk subunit EscO,⁷⁸⁶ which revealed a rotary catalytic mechanism analogous to that of the heterohexameric F1/V1-ATPases despite its homohexameric nature and, furthermore, elucidated the N-terminal domain of the EscN (residues 35–101) (PDB ID 6NJO; 6NJP), which was missing in the earlier structures. Comparison of these newly resolved N-terminal domains of the InvC-class ATPases, with those belonging to the FliI-type, shows that they are closely connected to each other ( overlaying with an RMSD of ~1 Å over the Cα backbone) and form a closed β-barrel with Greek-key topology.

Startlingly, these N-terminal β-barrel domains of the F1/V1-(e.g., the β-subunit of F1-ATPase PDB ID 6OQR (residues 1–76))⁷⁸³ and T3SS-associated ATPases (e.g., FliI residues 25–97 (PDB ID 2DPY))⁷⁸¹ are also closely connected to the β-barrel domain of the PAPs, presenting an equivalent topology to the one discussed above for the FlgT but lacking the additional β-hairpin insert between β3 and β4 observed in the latter (Figure 39; color scheme is the same as Figure 38).

Within the ATPase assemblies, the N-terminal β-barrel domains form a head-to-tail hexameric ring assembly, which could be formed by alternating α- and β-subunits, e.g., in the case of F1-ATPases,⁷⁷³,⁷⁸³ or can be presented by a homohexameric ring in the case of T3SS ATPases, e.g.

EscN.⁷⁸⁶ As seen in Figure 40, panel A, these rings share a striking resemblance to the ones observed in RND, MacB, PCAT, and MFS assemblies and are maintained by conserved residues as discussed in the preceding PAP section.⁵⁷¹,⁷³⁷

In the type III SS associated ATPases, the N-terminal domain is essential for stable assembly of higher oligomers and activation of the ATPases;⁷⁷⁶,⁷⁸⁷ it interacts with effector proteins (e.g., the Salmonella negative regulator of the ATPase OrgB)⁷⁸⁸ and has intrinsic phospholipid binding affinity.⁷⁸⁹ Similarly, in the N-terminal β-barrel domains of F₁F₀ ATPases have been shown to play a role in stabilization of the complex⁷⁹⁰ and providing contact of stator subunits b and delta.⁷⁸³

Extending the functional analogy, mutations targeting the β-barrel of PAPs impact complex formation and can be detrimental to assembly of the complex presumably by compromising the stability of the hexameric ring formation in both RND- and MacB-based assemblies.⁵⁷¹,⁵⁹⁴,⁷¹¹,⁷³⁷ Regulation of hexamerization and the modulation of the stability of these hexameric rings in response to substrate binding and OMF-engagement provide flexibility to the efflux and secretion machineries employing PAPs.

Paradoxically, the currently accepted model of the peristaltic cycle of the RND pumps³¹⁸ has been inspired by the rotary mechanism of the F1-ATPase⁷⁷³ and the structural connection of the β-barrel rings of PAP and ATPase assemblies presented
<figure><img src="image_54.png" /><figcaption>**Figure 40.** Connection between β-barrel domains and rotary ATPases. (A) Upper panel. A view of the hexameric assemblies presented by the rotary ATPases and PAP β-barrel domains. (B) Left, side view of a hetero-oliomeric F1F0-ATPase from *E. coli*; Center, type 3 secretion system associated ATPase EscN from *E. coli*. Right, CusBA subcomplex from *E. coli*. The hexameric β-barrel rings are highlighted by the red parallelogram, with individual subunits being red and magenta.</figcaption></figure>

here, which allows us to extend this analogy to actual
functional homology between the components of these
complexes.

8. ASSEMBLY AND FUNCTION OF THE RND-BASED TRIPARTITE COMPLEXES

**8.1. Cryo-EM Structures of Fully Assembled RND Complexes**

PAPs appear to play a crucial role in the assembly of the trans-
envelope complexes. Due to their unusual topology, early
studies have speculated that PAPs are able to bridge not just
the transporters and OMFs but also the opposing inner and
outer Gram-negative membranes directly,⁷⁴⁰ which has
partially led to their rather misguided assignment as
"membrane fusion proteins" or MFPs,²² a designation which
could still be found in some literature sources.

Until relatively recently our understanding of the structural
arrangement of the tripartite pumps came from speculative
models based on molecular docking, *in vivo* cross-linking, and
mutagenesis studies.²⁶⁹,⁶⁴⁹,⁶⁵⁰,⁶⁹³,⁷¹³,⁷¹⁶,⁷⁵⁹,⁷⁹¹ While these
produced some convincing models, even such aspects as the
stoichiometry of the assembly remained debatable in the
absence of an experimental structure of a complete tripartite
complex. A quantum leap in our understanding of these

systems has been achieved by the maturing of the cryo-electron
microscopy (cryo-EM) technology. The first to be addressed
were cryo-EM structures of AcrAB-TolC, which were
generated by coexpressing AcrA-AcrB fusion proteins²⁸⁷,⁷⁹²
or after stabilization by *in vivo* disulfide bridges.³⁰⁵ Later,
reconstituted tripartite pumps could be prepared from purified
proteins without additional stabilization in the presence of
native lipids²⁷⁰,³⁰⁴,³⁰⁶ or even resolved by *in situ* by cryo-
electron tomography (cryo-ET)³⁰⁷

A crucial insight has been provided by the higher resolution
structures obtained by the group of Ben Luisi,³⁰⁵ in the
presence and absence of substrate and inhibitors revealing
different conformational states of the pump. The apo-state
(PDB ID 5V5S) revealed a closed tip of the TolC-channel
while the AcrB adopts a symmetric LLL conformation (resting
state). In the presence of puromycin (PDB ID 5O66), the
aperture of the TolC channel is dilated and while the AcrB is
seen adopting an asymmetric LTO conformation, while the
inhibitor in the presence of pyranopyridine inhibitor
MBX3132,³⁴¹ the pump was found in a pseudosymmetrized
TTT state (PDB ID 5NGS).³⁰⁵ Importantly, the PAP, AcrA,
was found to form a hexamer wrapping around the RND
transporter through the trimerization of two conformationally
distinct protomers. These were found bound to two non-
equivalent interfaces on the surface of the transporter and are
<figure><img src="image_55.png" /><figcaption>**Figure 41.** Helix-turn-helix motifs in PAPs and OMF that are supposed to mediate their interaction. (A) Schematic view of the coiled coil packing within the α-helical hairpin of the PAP showing its antiparallel interface. Conserved hydrophobic residues are interacting within the a and d positions of the respective N- and C-helices (projection view looking down the hairpin helices outward from the lipoyl domain). (B) A view of the α-helical hairpin tip of AcrA (based on PDB ID 2F1M),<sup>735</sup> highlighting the interaction positions a and d; V126 (a) and A146 (d); and Y129 (a) and Y143 (a) form heptad-pairs. (C) The RLS-motif is present at the tip of the HTH motif of the PAP hairpin. Presentation of the classic-RLS-motif as observed in the (predominantly) RND- and MacB-associated assemblies, while MFS-associated PAPs show a deviation in the second half of the motif, and the T1SS-associated PAPs only retain a positively charged residue in the front of the motif, suggesting a different association with the OMF. Consistent with the lack of cognate OMFs, the PAPs in the Gram-positive organisms do not share any recognizable RLS. (D) R1 and R2 HTH-motifs at the tip of the static (H3/H4) and mobile (H7/H8) helices of the OMFs present a loosely conserved VG(L) motif, which is suggested to interact with the connecting loop of the RLS in the PAPs. Alignments in this figure have been visualized with Esprit3.<sup>795</sup></figcaption></figure>

referred to here as PAP1 (with binding fully restricted to one RND-protomer and hence also referred to as intraprotomer-PAP) and PAP2 (which binds between the respective RND-protomers and is referred to as interprotomer-PAP) (see section 8.3 and associated figures below for details). The conformationally distinct RND-PAP interfaces between the protomers are suggested to play a key role in the long-distance allosteric coupling between AcrB and TolC. In the apo-state (PDB ID 5V5S), a gap is present at the interfaces between adjacent PAP dimer pairs, so that the PAP helical-hairpin, lipoyl, and β-barrel domains do not pack tightly to seal the channel from the periplasm.<sup>305</sup> Based on these structures, it was suggested that AcrAB-TolC initially forms a complex in the closed state and that TolC opens upon rearrangement of the AcrA-hexamer induced by a conformational change in AcrB.<sup>305</sup> In turn, the AcrA helical bundle would repack, imposing a contraction of the pump along its long axis by approximately 10 Å, and consequently seal gaps that would otherwise cause the substrate to leak out.

Two structures of the nonstabilized wild-type multidrug efflux pump, MexAB-OprM from *P. aeruginosa*, were also resolved at near-atomic resolution.<sup>270,306</sup> In these structures OprM is found in two different orientations relative to MexB, related by a 60° rotation along the long pump axis. In contrast to the AcrAB-TolC structure described by Wang et al., both structures are found with OprM open. In order to account for the activation from a resting to an activated state, Tsutsumi et al.<sup>306</sup> proposed a model where the resting state corresponds to the tripartite structure where MexB is under an LTO conformation but with a closing of the gating loop in its T protomer. Only in the presence of substrate is the gating loop within MexB suggested to experience a slight conformational change, allowing the substrate to access the distal-binding

pocket.<sup>306</sup> The conclusion is slightly different in the structure described by Glavier et al.<sup>270</sup> for which it is described that transition from the resting to activated state is mediated by the binding of both MexA and OprM, allowing MexB to adopt an activated LTO conformation, hence corroborating *in vitro* functional results. In the sections below we will look at the PAP-OMF and PAP-RND interfaces elucidated by these structures in more detail.

## 8.2. PAP-OMF Interface

Due to their “adaptor” function, the PAP family plays a central role in the assembly of the tripartite complexes, effectively bridging the outer membrane (OMF) and inner membrane (energized transporter components). In the absence of direct structural data, a number of studies pointed out the primacy of the α-helical hairpin domain in engagement of the OMF and provided specificity of interaction between different pump complexes. For instance, swapping of the hairpin domain of MexA for that of MexE makes the MexA-E hybrid compatible with the OprM-MexF pump. Conversely, the chimeric construct is not able to function with OprN-MexF, and MexA bearing the hairpin from MexE does not form a functional complex with its cognate components OprM-MexB.<sup>793</sup> Similarly, swapping the AcrA α-hairpin domain for that of MexA makes the chimeric OprM-AcrAB assembly functional.<sup>713</sup> Cross reactivity among heterologous pumps was also tested, further highlighting the major role of PAPs in the specificity of recognition of assembly. For instance, TolC does not assemble with MexAB in *E. coli*<sup>652</sup> but does interact with and functionally complements both the AcrA-MexB<sup>794</sup> and VceAB.<sup>714</sup> Conversely, while VceC is able to interact with AcrAB in *E. coli* it does not provide functional complementation for the loss of TolC.<sup>714</sup>
<figure><img src="image_56.png" /><figcaption>OMF channel viewed from the periplasm</figcaption></figure>

**Figure 42.** Interaction between the tip-regions of OMFs and PAPs on the example of OprM and MexA. (A) Side view of the MexAB–OprM complex as seen in the cryo-EM structure (PDB ID 6TA6<sup>270</sup>). The zone of PAP-OMF interpenetration is indicated by the rectangle. Helices of the respective OMF protomers are shown in red, green, and blue; while the α-helical hairpins of PAP1 and PAP2 are colored orange and magenta, respectively. (B and C) Zoom on the tip-to-tip interaction between OprM H3/H4 and H7/H8 helical tips (shown in blue and green, respectively) and the docked α-hairpins of MexA (shown in orange (PAP1) and magenta (PAP2)). Principal interacting residues are shown (proposed RLS-motifs are in bold). (D and E) Schematic view of the MexA channel and associated PAP protomers looking up the channel in closed and open (MexA-MexB-engaged) form, respectively. The binding of PAP1 protomer to the interprotomer interface is suggested to result in destabilization (orange dotted arrow in D) of the interprotomer gates (indicated by the cyan arrows), resulting in an outward swing of the mobile helices H7/H8 (black curved arrows), which are still attached to the H4 via intraprotomer interactions (gray arrow). The initial relaxation of the H7/H8 allows the docking of PAP2, which in turn destabilizes the intraprotomer gates (see the pink dotted arrow in E), leading to full dilation of the OMF aperture. While the exact gating residues differ between the specific PAP–OMF pairs, the general mechanism of PAP docking and relaxation of H3/H4 helices is suggested to be conserved. See the text for more details.

While the central role of the PAP hairpin domain in OMF recognition and binding was never disputed, the nature of this bridging interaction has been a matter of much debate, with two competing models of assembly being proposed, which can be straightforwardly distinguished by the extent of the respective interaction interface between the OMF and the PAP α-hairpins. The first one, known as the “deep-interpenetration” model of assembly, as the name suggests, predicts an extensive binding interface between the PAP α-helical domain with the OMF helical-hairpin repeats, with stable four-helical bundles being formed on the inter- and intraprotomer grooves of the OMF. This model was the first to emerge historically and was primarily derived from biochemical data based on *in vivo* functional studies and cross-linking, as well as *ab initio* docking taking into account the propensity of coiled-coils to form helical bundles.<sup>649,650,713,716,759,791</sup>

The second model, which has become known as “tip-to-tip” or “cogwheel”<sup>756</sup> is predominantly based on the structural information derived from crystallographic,<sup>737,750</sup> cryo-EM,<sup>270,287,305,306,747</sup> and *in situ* cryo-ET data.<sup>307</sup> The tip-to-tip model initially came to existence from the observation of the crystallographic arrangement of the PAP MacA from the *Actinobacillus actinomycetemcomitans* (PDB ID 3FPP),<sup>737</sup>

which revealed a tight hexameric assembly, within which the α-helical hairpins of the PAP have been organized into a tubular structure, with a diameter strikingly similar to that of the fully dilated OMF channel.<sup>687</sup> Furthermore, similar tip-to-tip assembly is formed by the OMF in isolation (PDB ID 2VDD),<sup>689</sup> where two asymmetric TolC trimers were observed to pack crystallographically in an interpenetrative fashion. This similarity led to the suggestion that the two helical α-barrels of the OMF and the hexameric PAPs could interact in a much more limited way, resulting in the “cogwheel”, aka “adapter-bridging” model,<sup>737,750</sup> something that has been further supported by cryo-microscopy of engineered MacA-TolC chimeric proteins.<sup>746</sup> Consistent with the critical role of the RLS-motif (the canonical form of which is RxxxLxxxxxxS/T) and taking the form of a helix-turn-helix (HTX) motif at the tip of the α-helical hairpin domain, its targeting in different PAPs, including AcrA, MacA, and HlyD, has a detrimental effect on TolC-binding.<sup>750–753</sup> The leucine of the PAP RLS-motif in MFPs is the most conserved residue, while the last position of the motif shows higher variability and can be occupied by a serine or a threonine residue (Figure 41, panels A–C).
Similar to the PAPs, the OMFs belonging to the TolC subfamily present conserved HTH-motifs at the tips of their H3/H4 and H7/H8 α-helical hairpins of the coiled-coil domain, referred to as *R1 and R2 motifs*<sup>751,796</sup> or *VGL-motifs*.<sup>747</sup> These motifs contain a Val-Gly-Leu/Thr consensus sequence, which has been suggested to interact directly with the RLS-motif of the PAPs (Figure 41, panel D).

Strong support to the tip-to-tip association as a main driver of PAP-OMF interactions was provided by studies using chimeric PAPs, e.g. a MacA-TolC chimera, with only the R1 and R2 regions of the TolC transplanted onto the tips of the α-hairpin domain of MacA which was able to engage with MacA,<sup>746</sup> while a MacA-MexA chimeric protein, containing the *P. aeruginosa* MexA α-helical domain (PDB ID 4DK1)<sup>751</sup> was able to bind a similarly engineered MacA carrying the tips of the H3/H4 (Repeat 1 or R1) and H7/H8 (Repeat 2 or R2) helices forming the periplasmic tip of the α-barrel of OprM, showing that these are sufficient determinants for interaction.

One of the expected outcomes of such models is a level of promiscuity and interoperability of the PAP-OMF pairs, and indeed there is some experimental evidence in their support; for example, the *Vibrio vulnificus* TolC orthologues TolCV1 and TolCV2 appear able to interact with the *E. coli* PAP MacA, forming functional complexes<sup>753</sup> despite only having 51.3% and 29.6% identity with the *E. coli* TolC. Song et al.<sup>797</sup> have also investigated the interaction between the putative tip-regions of the PAP MdsA, belonging to the *Salmonella*-specific tripartite transporter MdsABC, and its cognate OMF MdsC, as well as TolC, in the formation of functional MdsAB-mediated efflux pumps. Comparative sequence analysis revealed that despite low overall sequence homology between MdsA and MdsC with the endogenous *Salmonella* cognate PAPs (only 15% identity with MacA and 18.7% identify with AcrA) and OMFs, respectively, there is clear conservation of key residues in the putative tip regions of both the PAPs (MdsA has a clearly conserved RLS-motif consisting of R135, L139, and S146) and the OMFs, that suggests allowing interactions and formation of hybrid tripartite systems. The predicted promiscuity was indeed observed in complex formation, suggesting that the productive complex formation is restricted by a small number of conserved residues and interactions at the respective tips of the hairpin domain of the PAP and the H3/H4 (R1) and H7/H8 (R2) of OMF,<sup>797</sup> further enhancing the RLS-R1/R2 interaction hypothesis (Figure 42).

Based on these observations it has been postulated that the RLS-motif is conserved across PAPs engaged in RND pumps, MacB-family ABC transporters,<sup>746,750</sup> as well as PAPs involved in T1SS such as HlyD.<sup>752</sup> However, the predicted RLS-motif in the HlyD family of PAPs appeared to be significantly degenerated, and later crystallographic studies revealed that the predicted tip of the HlyD is misaligned,<sup>739</sup> with the tip sequence differing dramatically from the canonical RSL signature. Similarly, the structure of the LipC from the lipase-secreting T1SS of *S. marcescens*<sup>514</sup> does not present a recognizable RLS-motif, suggesting that the T1SS systems may employ a slightly different mode of OMF-unlocking and/or stabilization and indicating that the role of the motif in OMF-recognition is not as clear-cut as previously thought. In addition, the hairpins of PAPs from Gram-positive organisms have recently come to light, e.g., Spr0693 from *Streptococcus pneumoniae*,<sup>571</sup> and perhaps unsurprisingly they do not show any significant conservation of the hairpin tip, while

maintaining the α-helical domain, raising further questions to its actual function (Figure 41, panel C).

This predicted PAP-OMF interaction was indeed directly observed in the cryo-EM structures derived from chimeric<sup>747</sup> and covalently fused AcrAB-TolC assemblies,<sup>287,792</sup> as well as the later cysteine-cross-link stabilized AcrABZ-TolC structures.<sup>305</sup> More recently conclusive evidence has been provided by the complexes reconstituted without any additional stabilization,<sup>270,304,306</sup> and thus the tip-to-tip model has become a dominant paradigm of PAP-OMF interaction.<sup>756</sup>

The cryo-EM structures reveal an OMF-PAP interface consistent with tip-to-tip interactions between their α-helical hairpins, with participation of the conserved motifs from the respective proteins discussed above; for example, within the AcrAB-TolC complex,<sup>305</sup> the conserved VGL/T R1-motif of TolC is located at the concave region of the TolC cogwheel in close proximity to the conserved R128 and L132 of the AcrA RLS-motif. There, the G365 from the TolC R1-motif is found to form backbone to backbone interaction with AcrA-K140 and the side chain of AcrA-S139.<sup>287,747</sup> Furthermore, consistent with earlier *in vivo* site-specific cross-linking experiments, N145 and T366 of TolC are found to contact L132 from the RLS-motif of AcrA.<sup>736</sup> Due to the quasi 6-fold symmetry of the TolC interfaces, similar interactions are seen between the R2-motif of the OMF and the second copy of the PAP. Homologous contacts can be seen in the high-resolution structures of MexAB-OprM<sup>270,306</sup> as illustrated in Figure 42, lending further support to the tip-to-tip model of interaction. Molecular dynamics simulations in POPC model-lipid bilayers confirmed that this fully assembled tip-to-tip pump is stable over microseconds. In addition, a sequence covariation analysis confirmed that interfacial contacts do involve residues consistent with those identified from the simulations.<sup>798</sup>

Despite the overwhelming structural biology support for it, there are certain issues with the “tip-to-tip” model, stemming from the limited interaction interfaces and the limited use of strong side-chain specific interactions between the predicted PAP-OMF binding surfaces, which limits the ability of each protein to differentiate their cognate binding partners. Furthermore, some experimental data is difficult to reconcile with such models, including functional complementation of concatenated pumps with enforced 3:3:3 stoichiometries,<sup>799</sup> SPR measurements,<sup>698</sup> and a range of direct *in vivo* cross-linking and mutagenesis studies.<sup>650,652,711,717,718,791</sup> While some of these may be dismissed as artifactual, the existence of PAPs without discernible α-hairpin domains, such as BesA,<sup>32</sup> which are able to form functionally active, channel-forming tripartite assemblies could not be readily explained by a classical tip-to-tip model.

### 8.3. RND-PAP Interface: Structural Data Suggests Asymmetry of the Assembled Tripartite Assembly and Different Roles for the PAPs

While earlier models of tripartite assembly gave preference to the 3:3:3 stoichiometry,<sup>649,716</sup> a number of conflicting data have been accumulated that shifted the consensus toward 3:6:3 assembly. These included the discovery of RND transporters which partnered with two separate PAPs, e.g., TriABC-OpmH,<sup>88</sup> as well as the demonstrable functionality of the tandem-fusion AcrA constructs.<sup>736</sup> Furthermore, *in vivo* cross-linking to both inter- and intraprotomer grooves of the OMF MtrE supported the existence of a 3:6:3 stoichiometry of the OMF-PAP-RND, leading to the suggestion that such an
<figure><img src="image_57.png" /><figcaption>**Figure 43.** Two nonequivalent binding sites on the surface of the RND transporter accommodate two distinct conformers of the PAP, which serve distinct functions during the cycling of the pump. (A) Side view of the MexAB-OprM assembly (PDB ID 6TA6)²⁷⁰ showing the binding of the intraprotomer-PAP subunit (PAP1) (orange) and the interprotomer-PAP subunit (PAP2) (magenta). The different RND subdomains are colored, and neighboring RND protomer is colored gray. (B) Cartoon representation of the same angle of view labeling the different subdomains of the PAPs and RND. (C) Isolated views of the contact areas of the PAP1 and PAP2 relative to the RND; for clarity the lipoyl and hairpin domains which do not contact the transporter have been removed.</figcaption></figure>

arrangement would inevitably result in a nonequivalent binding of the PAPs per protomer.⁷⁵⁹ Subsequent structural studies have provided conclusive evidence of this nonequivalent binding from both HAE and HME pumps.²⁷⁰,²⁸⁷,³⁰⁵,³⁰⁶,³⁵⁹ Correspondingly, the nonequivalent binding sites on the surface of the RND pump create two functionally non-equivalent PAP-conformers, which are referred to as PAP1 and PAP2,²⁷⁰,³⁰⁵,³⁰⁶ as seen in Figure 43. PAP1 is exclusively bound within the limits of a single RND transporter protomer with which it makes extensive interactions and is hence referred to also as an "intraprotomer-PAP" relative to the RND subunit. PAP2, on the other hand, contacts both the RND-subunit to which PAP1 is bound and the following anticlockwise protomer (looking from the top down along the axis of the pump from outer cellular space), straddling the RND-interprotomer gap, and hence in the text below is also referred to as an "interprotomer-PAP."

The PAP1 β-barrel domain docks onto the DN- and DC-subdomains of the RND-porter domain (Figure 5 and Figure 6 RND transporter section and Figure 43, showing the arrangement seen in the MexAB-OprM complex), while its MP domain interacts with the PC1 subdomain, as well as with the linker region between PC2 and DC, and an extended loop of the DN subdomain from the follow-up RND (Figure 43, panels A–C). PAP2 on the other hand forms fewer contacts, which are focused predominantly on the crown of the DN-subdomain, which is contacted by the β-barrel domain of PAP2, plus its MP-domain appears to be relatively loosely

associated with the surface of the RND protomer, corresponding to the PN2-DN linker region (Figure 43A, B, and D). Despite the big discrepancy of the PAP binding sites relative to the transporter and their complex 3D arrangement, a closer analysis of the interacting PAP residues revealed that the contacts provided by both PAP1 and PAP2 are closely overlapping and restricted to several discrete linear sequences, which we have previously dubbed "binding boxes".⁷⁴² These are restricted to the β-barrel (boxes 1–5) and MPD domains (6–9) and are highly conserved within specific PAP families; however, not all of them appear to have a pronounced functional role. Disruption of a few key residues within boxes 1, 4, and 5, mapping to the exposed β-barrel loops, abrogates transport, and hence, these have been suggested to play a role in assembly of functional complexes and the differentiation of cognate PAP-transporter pairs.⁷⁴² Notably, while PAP1 and PAP2 share the same β-barrel binding sites, there are discrepancies in the MPD attachment, and the domain is seen to be undergoing significant reorientation akin to "rolling" between the two protomers, resulting in PAP1 and PAP2-specific binding boxes, highlighting their differential modes of engagement and, likely, function.⁷⁴²

Furthermore, comparisons of the structures of RND transporters in isolation (also referred to as "mono"-structures below) vs their structures within tripartite assemblies in the presence of PAPs revealed by cryo-EM demonstrate significant differences, suggesting allosteric changes are enhanced and perhaps actively mediated by the PAPs,³⁰⁵ e.g., spatial
<figure><img src="image_58.png" /><figcaption>**Figure 44.** Comparison of interprotomer interfaces of RND transporters in isolation and upon PAP binding. (A) Side view of the superposition of the T-protomers of AcrB (as seen in asymmetric LTO and symmetrized TTT structures) and MexB (LTO) as seen in tripartite complex as observed in PBD ID 6TA6.<sup>270</sup> RND-conformers are colored: L, blue; T, yellow; O, red; PAP2 conformation, green. (B) Top-down view of the trimeric assemblies of MexB solo (PBD ID 6T7S),<sup>270</sup> MexB "trio" from the tripartite MexAB-OprM structure (PBD ID 6TA6),<sup>270</sup> and the AcrB (TTT-form stabilized by the presence of the MBX3132 inhibitor) (PBD ID 5NG5).<sup>305</sup></figcaption></figure>

orientation of the DN-domain β-hairpin motifs forming its crown, which have been implicated in the assembly of the pump,<sup>712</sup> was noted to be impacted, and it was suggested that this conformational rearrangement facilitates the interactions between the RND-protomer and the β-barrel domain of PAP2, as well as the short "crown" α-helix of the DC-subdomain from the next-RND protomer.<sup>305</sup> Identical contacts are also seen in the MexAB-interface of the assembled MexAB-OprM pump,<sup>270,306</sup> and similar interaction is revealed by the binary CusBA complex,<sup>359</sup> although the orientation of the β-barrel and lipoyl domain differs from the HAE1 pumps and the PAPs are rather loosely fitted to the crown of the transporter. The PAP1 MP-domain locates to the entry of the proximal binding pocket and thus has been suggested to play a role in substrate presentation and/or sensing of the cargo-load state of the transporter, which is also supported by the location PAP1 MPD observed in the CusBA complex, where such a role has been clearly shown.<sup>359</sup>

A significant new insight into the allosteric effects of the PAP-association came from the recent works on the MexAB-OprM,<sup>270,306</sup> which highlighted the special role of the short helix (formally known as Na2'),<sup>279</sup> which is found on the loop connecting the α2-helix and the β4-strand of the central β-sheet of the PD module of the PN1 subdomain of the transporter (Figure 6 and Figure 43, panel C). This short helix, which has been dubbed "helix gate",<sup>270</sup> participates in formation of the exit gate, which controls the exit of the substrates into the PAP-OMF funnel and in the case of MexB

is formed by the Q124, Q124 belonging to the helix gate plus a critical residue (Y758) from the DC domain of the RND.

Comparison of isolated RND transporters (aka "mono"-structures) vs the assembled RND tripartite complexes available shows that the interprotomer groove between T and O protomers is wider than the one observed at the LL-, LT-, or OL-interfaces. Intriguingly, this interface widens even further in the PAP2-engaged fully assembled "trio" state.<sup>270</sup> A closer inspection of the mono structures of MexB revealed that despite the seeming L-T-O cycle being present and the corresponding transformation of the deep binding pocket to outward-facing conformation, the drug exit gate remains occluded, and thus the drug cannot leave the pocket. This "closed" transition state was dubbed the C-state.<sup>270</sup> The closed (C) intermediate appears to be a state between the T and O states. Only after C converts to O is substrate released. Of note, the process from the apo-state (L) over drug bound state (T) to the release of the drug in the O state is postulated to be PMF-independent.

In the "trio"-structure, the PAP2 MPD appears to act as a wedge, which is slotted into the T-O interprotomer crevice, which is associated with a concerted movement of the PN2 subdomain of the T protomer and the PC2 domain of the neighboring O-protomer sidewise (Figure 44). These domain reorganizations have been suggested to translate to the underlying PN1 domain and its "helix-gate" mentioned above, allowing for the transition of the closed C-form to the
open O-form of the transporter,²⁷⁰ emphasizing the specific
role of the PAP2 in the efflux-vetting process.

There is a general consensus that PAP and the RND
transporter initially form a binary complex, as corroborated by
*in vivo* evidence,⁶⁹⁶,⁸⁰⁰ as well as by biochemical inves-
tigations,⁶⁹⁴,⁶⁹⁸ and that subsequently this binary assembly
recruits the OMF to form a functional tripartite complex. It
was suggested for a long time that PAPs play an active role in
transducing conformational energy from substrate/proton
binding in the RND transporter to the OMF to help in
transitioning its channel from its closed to open state allowing
for the creation of a functional efflux conduit, via mediation of
the helical hairpins of the PAPs.²⁸⁸,³¹⁸,⁶⁴⁹,⁶⁸⁹,⁸⁰¹ Substitutions
in the R1 motif targeting the loop connecting H3 and H4 in
TolC result in antibiotic hypersensitivity despite the fact that
their physical interaction is maintained as judged by *in vivo*
chemical cross-linking. Vancomycin sensitivity assay revealed
that AcrA-mediated suppression of this antibiotic sensitivity
was achieved by dilating the aperture of the TolC channel in an
AcrB-dependent manner,⁷¹¹ allowing suggestion that the
allostery of the overall RND-cycle mechanism substrate
binding by AcrB is linked to proton transport, whereby
conformational movements occurring within AcrB would be
captured by the AcrA MP- and lipoyl domains (which at the
time were believed to be in contact with the RND transporter)
and transferred to TolC via the α-helical hairpin of the PAP.

While later structural studies did not support the direct
contact of the lipoyl domains of the PAP with the transporter
and the deep-interpenetration models of PAP-OMF inter-
action have fallen out of fashion, the idea of the conformational
transitions in the RND being conveyed by allosteric PAP
mediation was proven largely correct. The cryo-EM data of the
AcrAB-TolC complex obtained in the presence or in the
absence of puromycin by the group of Ben Luisi allowed us to
further elaborate on an allosteric, concerted, opening³⁰⁵

There, conformational changes in the periplasmic headpiece
of the RND transporter upon substrate binding are suggested
to instigate repacking of all four of the PAP-domains, which in
turn leads to reorganization of the coiled-coiled domain of the
PAPs, which is implied to be the key step in organization of the
RLS-motifs and opening of the OMF channel,³⁰⁵ which is
engaged in a tip-to-tip fashion. Upon opening of the OMF, the
3-fold symmetry in the apo-complex transitions to a quasi-6-
fold symmetry of the interface between PAP α-hairpin and
open-state OMF. This leads to rearrangement of the helical
bundle, creating a tightly sealed nanotube, which in turn leads
to rearrangements further down, resulting in repacking of the
lipoyl and β-barrel domains of the PAPs and sealing of any the
gaps to the periplasm,³⁰⁵ allowing for productive efflux even to
take place. A similar mechanism has been suggested for the
MexAB OprM pump in the presence of novobiocin.³⁰⁶

There is also a visible reorganization of the MPD domains
between the PAPs in apo- and asymmetric, substrate engaged
state, which is likely associated with their changing functional
role through the cycle;³⁰⁵ however, due to the interoperability
of the PAP1 and PAP2 in such pumps as AcrAB-TolC and
MexAB-OprM, narrowing the specific roles of PAP1 and PAP2
has proven difficult.

8.4. Heterooligomeric Complexes Allow Unambiguous Assignment of PAP1 and PAP2 Highlighting Their Distinct Roles during Assembly and Cycling of the Pump

In *P. aeruginosa*, the tripartite pump TriABC-OpmH, which is associated with triclosan resistance,⁸⁸ has a distinct organization, as its transporter component, unlike most of the RND transporters discussed above, which function with a single PAP, creates a heterooligomeric complex with the participation of two different PAP subunits: TriA and TriB. The two PAPs only share 36% identity and are not interchangeable, suggesting specialization of function, which is consistent with each of them occupying a specific binding interface in respect to both the RND transporter TriC and the OMF protein OpmH.²⁰⁲,²⁰³ Due to these unique characteristics, this system has provided a valuable insight into the distinct roles played by PAP1 and PAP2, allowing assignment of each of the PAPs to a specific functional role. A recent cryo-EM structure of the TriABC subcomplex (PDB ID 6VEJ)³⁰² confirmed the locations of the TriA and TriB relative to the transporter and unambiguously assigned the PAP1 and PAP2 roles to them, respectively. The subcomplex was resolved in its unliganded “drug sweeping” state, which displays high mobility of the PAPs, with the hairpin domains being in disarray. However, the MP-domains could be resolved in their respective positions, and as expected the PAP1 (TriA) was found to have high-contact area and occupy the intraprotomer interface between the PC2 and PC1 subdomains, just above the substrate entry point to the proximal drug binding pocket, while the MPD of PAP2 (TriB) was found bound closer to the interprotomer interface between the RND-protomers. Notably, however, while the PAP1 MPD is fully resolved, the electron density for the PAP2 MPD is patchy, corresponding to low occupancy. Furthermore, intriguingly, in this substrate-free state of the pump, the MPD of PAP2 seems to be primarily localized to the PN2 domain of the RND protomer and is very loosely attached to it,³⁰² in stark difference to the snug-fit observed in the recent MexAB-OprM structures, where it is also bridging the interprotomer gap.²⁷⁰,³⁰⁶

Mutation of the RLS-signature motif within the TriA/TriB
seems to have different effects. While the TriA-R130D mutant
fails to assemble a functional TriABC complex with the OMF,
the alteration of the corresponding R118D in TriB does not
significantly affect the assembly and activity of the complex.⁸⁰²
At the same time, mutation of the conserved MPD residue
G350C (TriA) and G339 (TriB) had the opposite effect, with
the first one being tolerated and the second being detrimental
to pump function,⁸⁰² suggesting that it only plays a significant
role in the PAP2. Intriguingly, the mutation of the
corresponding residue, which maps to “binding box 9”, also
obliterates efflux in the *Salmonella* AcrA/AcrE PAPs.⁷⁴²

Antimicrobial susceptibility, site-directed mutagenesis in-
formed by homology modeling, alongside with *in vivo* disulfide
cross-linking and azithromycin-sensitivity assays allowed
further differentiation of the PAP1 and PAP2 roles relative
to the cognate OMF (OpmH).⁸⁰³ It was further confirmed that
while PAP1 (TriA) is required for OMF recruitment, the
activation of the transporter (which, as mentioned above is
linked to the MPD of PAP2) is coupled to the opening of the
OMF aperture.⁸⁰³ Data compiled from azithromycin sensitivity
studies, which indicate leakiness of the OMF-channel, as it
does not normally penetrate the channel and is not a substrate
of the pump, suggested that TriB is essential for keeping the
channel dilated, which is consistent with it associating with the
mobile H7/H8 helices of the R2 motif.<sup>803</sup> Such interpretation is compatible with the position of the PAP2 observed in the “zero-degree” structures of the OMF (PDB ID 5NG5,<sup>305</sup> 6IOK,<sup>306</sup> 6TA6<sup>270</sup>).

Further, *in vivo* proteolysis suggested that both TriA and TriB are conformationally flexible, causing susceptibility of their α-hairpin domains to digestion. However, while TriA (PAP1) is stabilized by the presence of the OMF and the transporter, the proteolysis of TriB (PAP2) is dependent on the presence of transporter, further suggesting that it may be a more dynamic and loosely associated member of the complex.<sup>302</sup> As the TriA and TriB are noninterchangeable, these studies allow us for the first time to correlate the rotationally symmetrical PAP-OMF interface with the underlying and already established PAP-RND interfaces, giving a unified view of the tripartite assembly and the roles of respective components which we will present in the following sections.

## 8.5. Conformational Cycling of the Protomers within the AcrB/MexB Trimer

Before we proceed with a unified model of the assembly, we would like to recapitulate a few key elements of the conformational cycling of the RND transporters. The majority of the current understanding of the RND-transport to date has been derived from the study of the inner-membrane transporters in isolation. The conformational LTO cycling hypothesis suggested an allosteric bisite activation<sup>270,321,335</sup> and is not necessarily suggesting that the three protomer conformations (LTO) are constantly present within the trimer at all times during the cycling. This notion is supported by cross-linking data,<sup>321</sup> suggesting that the LLT, LTT, and TTT trimeric conformations (next to LLL and LTO) are possible trimeric conformations. Structural support for this conformational flexibility came recently by observations via single-particle cryo-EM as this variety of different conformational setups was adopted by trimeric AcrB in the AcrABZ-TolC complex in complex with the inhibitor MBX-3132.<sup>305</sup> Experimental support for bisite activation (allostersy) came from molecular dynamics studies and kinetic β-lactam transport studies in whole cells, which were in dependence of the substrate concentration non-Michaelis–Menten kinetics with Hill-coefficients >1 measured.<sup>346,804–806</sup> All these conformational cycling models did not include a systematic role for the PAPs thus far, despite them being clearly implicated in the initial opening (and closing) of the OMF channel.

In the original LTO cycle,<sup>288,292,318,335</sup> the cycle starts with the symmetric metastable LLL conformation and the efflux substrate is anticipated to bind to the proximal binding pocket of one of the L protomers (to the AP) (Figure 5). Upon conformational change to T (thus forming an LLT trimer), the substrate is suggested to be further transported to the DBP (comprised by the PN2 and PC1 subdomains). In this drug-bound conformation, TM2 transduces the binding energy toward the TMD of the transporter allowing protons to enter the TMD and to bind to the titratable residues (D407 and/or D408 in the case of AcrB),<sup>328</sup> which causes a change in the electrostatics within the TMD. This results in a conformational change, and the transduction of the energy of proton binding is mediated via TM8 to the rigid PN1/PC2 subdomain unit<sup>292</sup> of the periplasmic domain. This results in the closing of the AP and DBP and the opening of a substrate exit gate. However, in

order to anticipate a bisite activation in analogy to the functional rotation cycle of the F₁F₀-ATP-synthase,<sup>320</sup> the neighboring protomer has to be in the T state as well. In the presence of substrates or inhibitor,<sup>305</sup> any L protomer (and T protomer) can accept substrate and reach the conformational transduction state that allows protons to enter from the periplasm to the protonation sites.

Under the current models, the LLL to LTO transition appears to only require substrate binding, which has been corroborated by MD-studies,<sup>806</sup> and indeed, the very existence of the LTO conformation in crystals and EM-resolved complexes reconstituted from purified components suggests that the conformational transitions could be obtained in the absence of PMF energy. However, the pre-existence of the LTO in a resting apo-state has been subject to considerable debate,<sup>338</sup> and recent studies suggest that while LTC transitions could indeed happen in the absence of PAPs, the substrate binding alone may not be sufficient for the transit of the transporter from an LTC to the LTO configuration *in vivo*.

Earlier FRET studies of CmeB transporter reconstituted into liposomes have shown that it could cycle, albeit inefficiently, in the absence of the PAP or OMF components.<sup>299</sup> While individual protomers have been detected undergoing L-T-O transitions, there seems to be little evidence of cooperativity and directionality of cycling. These findings raise the question on whether the role of the PAPs may be associated with both the facilitation of the conformational transitions between the RND protomers and enforcing the directionality of the cycle. In the section below we try to integrate the latest structural advances in understanding of the RND assembly and the roles of the PAPs within it into an integrated model of assembly of the complex, which takes into account both the PAP-OMF and PAP-RND interactions.

## 8.6. Toward the Unified Model of RND Pump Assembly—An Integrated Model of PAP Participation in the Efflux Cycle and Resetting of the RND Assemblies

Corroborating the recent results on the MexAB-OprM<sup>270,306</sup> and TriABC-OpmH systems,<sup>302</sup> with the earlier allosteric model derived from the AcrAB-TolC discussed above,<sup>305</sup> we would like to suggest a modest modification of this model, incorporating a simple mechanism based on varying the affinities of the interprotomer binding sites created by the peristaltic cycling of the RND transporter toward the PAP2.

The key to pump stability and processivity during these cycles and detachments is the continuous association of the hexameric β-barrel gasket with the transporter, which allows for bespoke regulation of the engagement of their MP-domains with the different conformers of the transporter. In essence, we propose that while the intraprotomer (PAP1-binding site) remains high-affinity throughout the pump cycle, the affinity of the PAP2 binding site changes due to the conformational changes of the RND-protomers depending on drug-binding and proton-occupancy, with the L/L, L/T, and L/O presenting low-affinity sites, while the PAP2-binding site formed by the T-protomer is strongest. This applies specifically to the MP-domain, which could possibly detach fully between the different transitions.

The flexibility and the strategic position of the PAP2 straddling the interprotomer interface of the RND make it an ideal candidate for both sensing and communicating the state of the preceding RND-conformer to the next. It seems therefore possible that the PAP2 MPD acts as molecular