dataesr/scientific-paragraphs-categorization huggingface dataset:
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 cargoprotein. 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.
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 usB 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.
] [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 imilar 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 Zn 2+ ion coordinated by H220, H284, E328, and a water molecule at the interface between the β-barrel and the MPD domain. 46 he 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 we will discuss in the following paragraphs for each of the main systems.
726 The bacterial flagellar motor consists of a number of conserved components which include the innermembrane 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),  772  which associates with the outer membrane H-and T-rings in Vibrio, is comprised of a six-stranded β-barrel, nearly identical to the Nterminal domain of the β-subunit of F 1 -ATPase (e. g. , bovine mitochondrial F1-ATPase PDB ID 1BMF),  773  the catalytic subunit of the ATP-synthase complex. 772 his 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 β1strand and β2-strand forming the core of the barrel.
726 onsistent with that, despite lacking discernible sequence homology, FlgT-C superposes on the Cα-backbone of the βbarrel 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 f lagellar 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, homohexameric 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 he 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 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.
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 proteins  287, 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 lipids  270, 304, 306  or even resolved by in situ by cryoelectron 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 5NG5). 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 nonequivalent interfaces on the surface of the transporter and are 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 RNDprotomers 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. 305 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. 305 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.
307 The tip-totip model initially came to existence from the observation of the crystallographic arrangement of the PAP MacA from the Actinobacillus actinomycetemcomitans (PDB ID 3FPP),  737  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. 687 Furthermore, similar tip-totip assembly is formed by the OMF in isolation (PDB ID 2VDD),  689  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 "adapterbridging" model,  737, 750  something that has been further supported by cryo-microscopy of engineered MacA-TolC chimeric proteins.
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 αhairpin domain of MacA which was able to engage with MacA,  746  while a MacA-MexA chimeric protein, containing the P. aeruginosa MexA α-helical domain (PDB ID 4DK1)  751  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.
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.
803 Such interpretation is compatible with the position of the PAP2 observed in the "zero-degree" structures of the OMF (PDB ID 5NG5, 305 6IOK, 306 6TA6 270 ). 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. 302 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.
This system can elegantly explain the lack of cooperativity or directionality observed in individual transporters. 299 n respect to the PAP-OMF interface our model allows for the first time to tentatively assign a specific role to each PAP conformer within the cycle. As described above in the closed, resting state of the OMF the coiled-coil α-helical hairpins at its tip are stabilized by a network of predominantly ionic interactions, aka "primary gates", that keep the inner coiledcoil helices (H7/H8) in a tensed supercoiled state relative to the relaxed superhelical trajectories of the outer coiled-coil helices (H3/H4). 8] [689]  This interaction is induced by the PAP-OMF interaction and does not require energy input from the transporter. 720 Analysis of the positions of the PAP1 and PAP2 in the zero-degree structures of MexAB-OprM (PDB ID 6IOK, 306 6TA6)  270  and the corresponding AcrAB-TolC  305  suggests that PAP2 is the main interacting conformer with the OMF-R2 (H7/H8) motif, which is consistent with it being the final recipient (and stabilizer) of its relaxed helical trajectory, the release of which could plausibly be deduced to be triggered by an initial docking of the PAP1.
Due to the differing helical trajectories of the R1 (H3/H4-associated) and R2 (H7/H8associated) motifs of the OMF, the primary interaction of PAP1 (based on the TriA data) is suggested to be the R1motif, although in single-PAP assemblies such as MexAB and AcrAB the interface is quasi-6-fold symmetrized, and 60°r otation of the OMF may possibly produce functional complexes as suggested by the existence of the alternative structures (e. g. , PDB ID 6IOL, 306 6TA5 270 ). In the case of a binary AcrAB complex which is transitioning from an LLL state, the drug binding and conversion on L to T would serve as an allosteric signal from PAP1 toward the OMF enabling its attachment. The initial docking of the PAP1 to the H3/H4 helices of the OMF, which serves as an upward communication signal, triggers at least partial release of the H7/H8-helices, possibly by attacking the interprotomer H8-H4 attachment gate, the full relaxation of which is achieved by breakage of the intraprotomer H7-H4 gate (containing a conserved Y-residue, e.
g. , Y404 OprM or Y362 TolC) upon subsequent PAP2 docking (see Figure  42D , E and Figure  46 ). The relaxed trajectory of H7/H8 is then stabilized by the PAP2 which ends up being attached to the tip of H7-H8 (R2 HTH-motif) with its own RLS-motif, as seen in the recently assigned 0-degree OMF-structures (PDB ID 6IOK, 306 6TA6 270 ) (see Figure  42B  and Figure  46 ). Indeed, the analysis of the tip-to-tip interactions and the possible starting and finishing trajectories of the H7/H8 helices as seen in the 0-degree structures suggests that these are the only arrangements which are compatible with the cross-linking data presented by Ntreh et al. ,  803  strongly indicating that TriB binds in a compatible fashion to the one presented by PAP2.
Concomitantly, the allosteric transitions associated with the L-to-T transition upon the substrate binding are transmitted to PAP1, which initiates OMFrecruitment (Figure  47 ). The binding of the substrate opens the proton access channel in the T-conformer, and the occupation of the proton relay causes the T to C conversion (as demonstrated by a recent MD study). 808 This in turn recruits the PAP2 MPdomain to the interprotomer groove, which by acting as a "door stop" or "latch" stabilizes the C-state, preventing backsliding to the T-conformation, and enables the C-to-O transition. While the occupation of the proton relay facilitates this C-to-O transition in vivo, it can be achieved in the absence of PMF in the isolated transporters. Indeed, the energy barrier of the T-to-C transition appears to be relatively small, as additional binding energy provided by the interaction of the MBX3132 inhibitor over native substrate appears to be sufficient to overcome it and stabilize the pump in a quasisymmetric TTT-state, as seen in the PDB ID 5NG5.
Crystal structures of the binary CusBA complex  359  revealed a hexameric assembly of the PAP forming the typical β-barrel and lipoyl domain gaskets on the top of the transporter; however, CusB forms a clearly delineated trimer of dimers, with each pair being relatively loosely associated with each other. Notably, PAP2 is fully engaged and resolved in the structure, and the electron density is particularly good around the N-terminal segment of PAP2 which appears to be in contact with PAP1 (PDB ID 4DNT, 610 3NE5  359  ). The positions of the different PAP protomers, and especially their respective MPDs, are diverging from the one seen in the cryo-EM structures of the HAE-1 assemblies. The orientation of the MPD domain of PAP2 in the CusBA complex is notably different from that in PAP2 seen in HAE-1 assemblies, where it is firmly associated with the interprotomer crevasse and PN2/ DN domains of the RND protomer.
The PAP2 features an extended N-terminal tail (in PDB ID 4DNT it is resolved from residue 79 to 96), making extensive contact with the surface of the RND transporter, engaging with the PC1 and PN2 domain of the protomer in an unfolded state. Notably this N-terminal tail of PAP2 also contacts PAP1 directly, making specific contact with the equivalent N-terminal tail of PAP1, which however presents a strikingly different configuration, specifically in the contact range (residues 84-93), where if it forms an α-helix, that contrasts the unfolded region of PAP1. Importantly, the helical trajectories of the α-hairpins of CusB are pointing away from the center of the pump axis, without participating in helical-bundle tubular formation (PBD ID 3T53). 610 Along with a loose association of the lipoyl domains, such a hairpin orientation of the PAP is inconsistent with sealing of the OMF-channel.
743  Notably, the lipoyl domains of the PAPs in these assemblies form tight hexameric gaskets, similar to the ones formed by the PAPs in isolation  737  with an extended flexible loop donated from each protomer creating a "gating ring",  272, 571  which is suggested to prevent backflow, favoring unidirectional substrate efflux in an outward direction (Figure  35C ). In the hexameric structure of E. coli MacA, the lipoyl domain loops (G56-L72; T199-I214) protrude into the cavity delineated by the assembled hexamer, providing a constriction of the lumen, which is narrowest at residue N209, which in the stand-alone structure (PDB ID 3FPP)  737  is effectively sealed (Figure  35C  in section 7. 3). Similarly, in the PAP Spr0693 (PDB ID 5XU0), which pairs with the Gram-positive MacB-homologue Spr0694-0695, the hexameric α-helical barrel has an inner diameter of ∼31 Å.
Stabilization of this β-barrel gasket-ring is mostly due to the interprotomer interactions of three residues (which in the EcMacA correspond to T293, Y275, and E231), that are strictly conserved across the MacA-family indicating a common mechanism. 737 he β-barrel domains of the three MacA protomers make nonequivalent contacts with the PCDs of the transporter, which could, in an analogy to the PAP1 and PAP2 discussed in the context of RND transporters, be named PAP1-PAP3, respectively (Figure  50 ). Of these, PAP1, similar to the PAP1 in the RND assembly, makes intraprotomer interactions only relative to the transporter (e. g. , using the chain identifiers from the PDB ID 5NIK). 272 PAP1 could be assigned to chain D, and both its MP-domain and β-barrel domain interact with the PD-base of the PCD domain of the MacB (chain J), while PAP2 (chain F) interacts exclusively with the SABRE domain of the same transporter protomer (chain J), and the last, PAP3 (chain G), appears to occupy an interprotomer position extending over the cleft between the neighboring transporter protomers.
272 n an organization which mirrors that observed in PAPs participating in the RND transporter assemblies, the lipoyl domains of MacA form an additional hexameric ring that stacks above the β-barrel gasket but does not contact MacB. The selfassociation of the "lipoyl-gasket" clearly plays a role in stabilization of the whole PAP hexamer, as can be seen in the example of DevB-DevAC interaction. 206 From the lipoyl domain-gasket up, the hairpin arrangement of the PAPs from the MacA-family and that of the AcrA-family follow similar trajectories and present a unified interface to the OMF, which is further supported by the aforementioned chimeric-protein studies, swapping the hairpins between MexA and MacA (PDB ID 4DK1). 751 Consistent with this the MacA-family also shares the conserved RLS-motif at the tip of their α-hairpin.
87 μM. The EmrA trimers were also found to be the prevalent cross-linked species. Although an experimental structure of EmrB is not available at present, reasonably reliable topological estimates could be obtained from the analysis of the structures of the related MFS pumps such as YajR,  382  EmrD (PDB ID 2GFP),  380  and PepTSo (PDB ID 4UVM). 271 The C-terminal part of EmrB likely presents a helical arrangement equivalent of the TM14 of the PepTSo,  271  and earlier studies suggested that this TM region of the MFS transporter may be involved in a leucinzipper like interaction with the TM-domain of the PAP EmrA, forming a TM-helical bundle. 760 A similar suggestion was extended to the N-terminal portion of the transporter, covering a major part of TM1, with the implication of a 2:1 PAP to transporter subunit interaction,  760  and although current structural data on the organization of TM-helices in the known MFS transporters does not lend itself to a straightforward model of such an assembly, as it requires another PAP-binding site per MFS-protomer, such overall arrangement is appealing as (assuming a dimeric transporter MFS) it gives rise to six PAPs and a 34 TM-helical model (six from PAPs and 28 from the transporter-dimer), which is roughly equivalent to the 36 TM-section observed in the RND-assemblies.
g. , the well-studied heme acquisition protein HasA. 529, 854 A major group of the cargoes secreted by the T1SS present additional Ca 2+ binding motifs known as RTXmotifs or GG-repeatsGGXGXDXUX (where X is any amino acid and U is a hydrophobic amino acid)precede the Cterminal secretion signal in some of the passenger proteins, including HlyA and LipA. 449, 497 These play an important role in folding of the cargo, and X-ray structures reveal that they form a β-roll motif (e. g. , PDB ID 1KAP  500, 501  ). Within the RTX-group, there is significant diversity of cargoes, which can be ranked in the order of organizational complexity. The hemolysin/cytolysin family of pore-forming toxins includes hemolysin A (HlyA), in which the region N-terminal of the glycine-repeats code just one functional domainthe pore forming toxin itself  214  and the CyaA from Bordetella pertussis,  855  which apart from the toxin domain, possesses an adenylate cyclase module which affects the cAMP levels upon entering the target host cell.