2.A.6 The Resistance-Nodulation-Cell Division (RND) Superfamily
Characterized members of the RND superfamily all probably catalyze substrate efflux via an H+ antiport mechanism.These proteins are found ubiquitously in bacteria, archaea and eukaryotes (see Nikaido 2018 for a review of RND transporters). They fall into ten recognized phylogenetic families, three primary phylogenetic families that are restricted largely to Gram-negative bacteria (families 1-3, see below), the SecDF family (family 4) that is represented in both Gram-negative and Gram-positive bacteria as well as archaea, the HAE2 family (family 5) that is restricted to Gram-positive bacteria, one very diverse eukaryotic family (family 6), one archaeal plus spirochete family (family 7) (Tseng et al., 1999), a recently identified family that includes a probable pigment exporter in Gram-negative bacteria (TC #2.A.6.8.1; Goel et al., 2002), Dispatched (family 9), an exporter of the amino terminal portion (19 kDa) of Hedgehog which has a C-terminal cholesterol covanlent linkage that may be recognized by Dispatched (Nikaido 2018), and the uncharacterized family 10 from Actinobacteria.
Clustering patterns in the Gram-negative bacterial families of the RND superfamily correlate with substrate specificity with family 1 catalyzing export of heavy metals, family 2 catalyzing export of multiple drugs, cluster 3 probably catalyzing export of lipooligosaccharides concerned with plant nodulation for the purpose of symbiotic nitrogen fixation, cluster 4 corresponding to the SecDF proteins, and cluster 8 catalyzing pigment export. Within family 2, MdtABC, consisting of an MFP (TC #8.A.1) and two RND family proteins (MdtB [TC #2.A.6.2.12] and MdtC [TC #2.A.6.2.14]) may form a complex exhibiting broader specificity than either MdtAB or MdtAC (Baranova and Nikaido, 2002; Nagakubo et al., 2002). The ActII3 protein, one of the two partially characterized member of family 5, has been implicated in drug resistance. The MmpL7 protein, also of this family, catalyzes export of an outer membrane lipid, phthiocerol dimycocerosate (PDIM) in M. tuberculosis. The SecDF proteins (family 4) function as nonessential constituents of the IISP protein secretory system (TC #3.A.5). They seem to allow coupling of substrate protein translocation to the proton motive force by facilitating deinsertion of the SecA component of the IISP system, thus rendering this system partially ATP-independent.
Nies (2003) subdivied the HME-RND proteins (2.A.6.1) into subgroups according to substrate specificity. HME1 (Zn2+, Co2+, Cd2+), HME2 (Co2+, Ni2+), HME3a (divalent cations), HME3b (monovalent cations) HME4 (Cu+ or Ag+) and HME5 (Ni2+). Kim et al. (2011) have proposed two models for the extrusion of heavy metals (2.A.6.1) from the periplasm to the extracellular medium, the 'switch' and the 'funnel' mechanisms. The funnel model involves the shuttling of periplasmic substrate from the membrane fusion protein to the RND transporter and further on through the outer membrane factor to the extracellular space. Conversely, the switch model requires substrate binding to the membrane fusion protein, inducing a conformational change and creating an open-access state of the tripartite protein complex. They favor the switch mechanism.
Some or all of the eukaryotic proteins (family 6) may function in cholesterol/lipid/steroid hormone transport, reception, regulation or catalysis. One such protein complex includes the RND family disease protein, Niemann-Pick C1, which may function in the export of cholesterol and lipids from lysosomes in conjunction with a soluble lysosomal protein with cholesterol binding properties, NPC2 (TC #2.A.6.6.1; Sleat et al., 2004). The disorder is typified by inhibited egress of cholesterol and glycosphingolipids from endosomal and lysosomal compartments. In the majority of NPC patients, mutations in the NPC1 gene can be identified, but about 5% of patients show mutations in the NPC2 gene. Many different mutations can cause NPC disease, and multiple variants not associated with the disease are known in both genes. There is an NPC disease gene variation database (NPC-db; http://npc.fzk.de). Non-transporter homologues possess the sterol recognition domain and do not exhibit the typical RND family internal duplication (see below). The functions of the archaeal and spirochete proteins of family 7 have not been investigated.
Water-soluble Niemann-Pick C2 (NPC2) and membrane-bound NPC1 are cholesterol-binding lysosomal proteins required for export of lipoprotein-derived cholesterol from lysosomes. The binding site in NPC1 is located in its N-terminal domain (NTD), which projects into the lysosomal lumen. Transfer of cholesterol from NPC2 to NPC1 requires three residues that form a patch on the surface of NPC2. Wang et al. (2010) previously identified a patch of residues on the surface of NPC1(NTD) that is required for transfer. They presented a model in which these two surface patches on NPC2 and NPC1(NTD) interact, thereby opening an entry pore on NPC1(NTD) and allowing cholesterol to transfer without passing through the water phase. They referred to this transfer as a hydrophobic handoff and hypothesized that this handoff is essential for cholesterol export from lysosomes (Wang et al., 2010).
Most of the RND superfamily transport systems consist of large polypeptide chains (700-1300 amino acyl residues long). These proteins possess a single transmembrane spanner (TMS) at their N-termini followed by a large extracytoplasmic domain, then six additional TMSs, a second large extracytoplasmic domain, and five final C-terminal TMSs. In the case of one system (NolGHI) the system may consist of three distinct polypeptide chains, and most of the SecDF homologues consist of two polypeptide chains. Most others probably consist of a single polypeptide chain. The first halves of RND family proteins are homologous to the second halves, and the proteins therefore probably arose as a result of an intragenic tandem duplication event that occurred in the primordial system prior to divergence of the family members. One protein homologue from Methanococcus jannaschii is of half size and has no internal duplication. It can be postulated to function as a homo- or heterodimer in the membrane. The same is true of the eukaryotic RND family homologues that do not appear to function in transport. Some of the eukaryotic proteins have hydrophilic C-terminal domains.
Crystal structures of the RND drug exporter of E. coli, AcrB (TC #2.A.6.2.2), have been solved at 3.5 Å and 2.8 Å resolution (Murakami et al., 2002, 2006). Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer consists of a 50 Å thick transmembrane domain and a 70 Å headpiece, protruding from the external membrane surface. The top of the headpiece opens like a funnel, and this may be a site of interaction with the MFP, AcrA (TC #8.A.1.6.1) and the OMF, TolC (TC #1.B.17.1.1). A pore formed by the three α-helices connects the funnel with a central cavity at the bottom of the headpiece. The 12 TMSs in the membrane domain are visible. Substrates are presumably successively transported through the channels of AcrB and TolC (Murakami et al., 2002). An MFP such as MexF of P. aeruginosa facilitates proper assembly of the RND permease as well as stabilization of the OMF such as OprN (Maseda et al., 2002). Vestibules are part of substrate path in AcrB multidrug efflux transporter of Escherichia coli (Husain et al., 2011). Pagès et al. (2011) have described several classes of efflux pump inhibitors that counteract MDR.
The large external cavity is of 5000 cubic angstroms. Several different hydrophobic and amphipathic ligands can bind in different positions within the cavity simultaneously. Binding involves hydrophobic forces, aromatic (π) stacking and van der Waals interactions (Yu et al., 2003). Crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. The external large cleft in the periplasmic domain of AcrB appears to be closed in the crystal structure of one of the three protomers. Conformational changes, including the closure of the external cleft in the periplasmic domain, are apparently required for drug transport by AcrB (Takatsuka and Nikaido, 2007; Takatsuka et al., 2010).
Murakami et al. (2006) have described crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of three protomers, each of which has a different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures indicate that drugs are exported by a three-step functionally rotating mechanism in which substrates undergo ordered binding change. A crystal structure at 2.9 Å resolution of trimeric AcrB was reported by Seeger et al. (2006) and shows asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.
The RND members of families 1-3 function in conjunction with a 'membrane fusion protein' (MFP; TC #8.A.1) and an 'outer membrane factor' (OMF; TC #1.B.17) to effect efflux across both membranes of the Gram-negative bacterial cell envelope in a single energy-coupled step. They may also pump hydrophobic substances from the cytoplasmic membrane, and toxic hydrophilic substances (i.e., heavy metals) from the periplasm to the external medium. The large periplasmic domains of RND pumps are involved in substrate recognition and form a cavity that can accommodate multiple drugs simultaneously (Mao et al., 2002). A comprehensive review of the classes of efflux pump inhibitors from various sources, highlighting their structure-activity relationships, which can be useful for medicinal chemists in the pursuit of novel efflux pump inhibitors, has appeared (Durães et al. 2018).
Symmons et al., 2009 showed that the adaptor termini assemble a beta-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous beta-roll, beta-barrel, and lipoyl domains of the adaptor. The flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter-adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. AcrA(3)-AcrB(3)-TolC(3) is a 610 KDa, 270-A-long efflux pump crossing the entire bacterial cell envelope (Symmons et al., 2009).
RND transporters such as AcrD of E. coli can capture drugs such as aminoglycosides, from the periplasm and maybe from the cytoplasm (Aires and Nikaido, 2005). The latter process has been referred to as periplasmic vacuuming where, in this case, AcrD is the periplasmic vacuum cleaner (Lomovskaya and Totrov, 2005). This allows Gram-negative bacteria to protect themselves against cell wall biosynthetic inhibitors (drugs) that act in the periplasm. It also explains why HAE1 family members are largely restricted to Gram-negative bacteria. They are rarely found in Gram-positive bacteria or archaea.
A novel member of the RND superfamily, very distantly related to other established members of the superfamily, was shown to be a pigment (xanthomonadin) exporter in Xanthomonas oryzae (Goel et al., 2002). This protein (TC #2.A.6.8.1) has close homologues in various species of Xanthomonas as well as Xylella, Ralstonia and E. coli (AAG58596). These proteins comprise the eighth recognized family in the RND superfamily.
Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by the proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has been shown to function in late stages of protein secretion and membrane protein biogenesis. Tsukazaki et al. (2011) determined the crystal structure of Thermus thermophilus SecDF TC# 2.A.6.4.3) at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and the proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, Tsukazaki et al. (2011) proposed that SecDF functions as a membrane-integrated chaperone, powered by the proton motive force, to achieve ATP-independent protein translocation. Furukawa et al. 2018 reported the crystal structure of SecDF in a form named the super-membrane-facing form, demonstrating a beta-barrel architecture instead of the previously reported beta-sheet structure. A remote coupling model was proposed in which a structural change of the transmembrane region drives a functional, extracytoplasmic conformational transition.
AcrB actively exports a wide variety of noxious compounds using the proton-motive force for energy. AcrB adopts an asymmetric structure of three protomers with different conformations that are sequentially converted during drug export; these cyclic conformational changes during drug export are referred to as functional rotation. Using different protonation states for the titratable residues in the middle of the transmembrane domain, simulations revealed a correlation between the specific protonation states and the side-chain configurations. Changing the protonation state for Asp408 induced a spontaneous structural transition, which suggests that the proton translocation stoichiometry may be one proton per functional rotation cycle. Simulations also demonstrate that alternating the protonation states in the transmembrane domain induces functional rotation in the porter domain, which is coupled to drug transport (Yamane et al. 2013). A mechanism involving two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 A apart, has been proposed (Eicher et al. 2014). Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system.
The generalized transport reaction catalyzed by functionally characterized RND proteins is:
Substrates (in) + nH+ (out) → Substrates (out) + nH+ (in).
Substrates: (a) heavy metals, (e.g., Co2+, Zn2+, Cd2+, Ni2+, Cu+ and Ag+; family 1); (b) multiple drugs (e.g., tetracycline, chloramphenicol, fluoroquinolones, β-lactams, etc.; family 2); (c) lipooligosaccharides (nodulation factors; family 3); (d) unfolded proteins as for the SecDF-mediated translocation of substrate proteins (Family 4), (e) lipids and possibly antibiotic drugs in Gram positive bacteria (e.g., outer membrane mycolic acid-containing lipids in actinobacteria and actinorhodin; family 5), (f) possibly sterols in eukaryotes (family 6), (g) fused pentacyclic ring compounds such a hopanoids in bacteria (family 7), (h) pigments (family 8), and (i) cholesterol-modified peptides such as 'hedgehog', a sterol sensor in animals (family 9).
Heavy metal (Ni2+ and Co2+) efflux pump, CnrA. Functions with CnrB (TC# 8.A.1.2.1) and CnrC (TC# 1.B.17.2.1) (Grass et al. 2000; Tibazarwa et al. 2000).
CnrA of Cupriavidus (Ralstonia; Alcaligenes) metallidurans (eutrophus or eutropha) (P37972)
Putative Cu2+ exporter, Czc2ABC. (induced by Cu2+ and other heavy metal ions; Moraleda-Muñoz et al., 2010)
Czc2AB of Myxococcus xanthus
Czc2A (RND) (Q1D665)
Czc2B (MFP) (Q1D664)
Putative metal ion exporter (induced by starvation; Moraleda-Muñoz et al., 2010)
Czc3ABC of Myxococcus xanthus
Czc3A (RND) (Q1CVN2)
Czc3B (MFP) (Q1CVN1)
Czc3C (OMF) (Q1CVN0)
NccABC Ni2+, Co2+, Cd2+ resistance efflux pump (Schmidt and Schlegel, 1994).
NccABC of Alcaligenes xylosoxidans
NccA (RND) (Q44586)
NccB (MFP) (Q44585)
NccC (OMF) (Q44584)
CzrABC Cd2+, Zn2+ resistance efflux pump (Hassan et al., 1999).
CzrABC of Pseudomonas aeruginosa
CzrA (RND) (Q9RLI8)
CzrB (MFP) (Q9RLI9)
CzrC (OMF) (Q9RLJ0)
CznABC Cd2+, Zn2+, Ni2+ resistance efflux pump. Required for urea modulation and gastric colonization (Stähler et al., 2006).
CznABC of Helicobacter pylori
CznA (RND) (O25622)
CznB (MFP) (O25623)
CznC (OMF) (O25624)
The CzrCBA operon is induced by Cd2+ and Zn2+. CzrCBA transports Cd2+, Zn2+, and Co2+ but not Ni2+ (Valencia et al., 2013, in press).
CzrCBA of Caulobacter crescentus NA1000
CzrA (RND) (B8H146)
CzrB (MFP) (B8H144)
CzrC (OMF) (B8H143)
The nczCBA operon (also called the czc operon) is induced maximally by Ni2+ and Co2+, moderately by Zn2+ but not by Cd2+. NczCBA transports Ni2+ Co2+and probably Zn2+ and Cd2+(Valencia et al. 2013).
NczCBA of Caulobacter crescentus NA1000
NczA (RND) (B8GZE9)
NczB (MFP) (B8GZE8)
NczC (OMF) (B8GZE7)
Zn2 exporter, ZniA. Functions with an MFP and an OMF (Nies,2013, in Microbial Efflux Pumps, EW Yu, Q Zhang and MH Brown, editors, Caister Acadmic Press, 2013).
ZniA of Cupriavidus metallidurans
Ni2 , Co2 exporter, NimA. Functions with an MFP and an OMF (Nies, 2013, in Microbial Efflux Pumps, EW Yu, Q Zhang and MH Brown, editors, Caister Acadmic Press, 2013).
NimA of Cupriavidus metallidurans
Heavy metal (Co2+, Zn2+, Cd2+) efflux pump, CzcAB. Functions with CzcC (P13509; 1.B.17.2.2).
CzcA/CzcB of Cupriavidus (Ralstonia; Alcaligenes) metallidurans (eutriphus or eutropha)
Cu+ /Ag+ efflux pump, CusABCF (may pump ions from the periplasm to the external medium); CusF is a periplasmic Cu+ /Ag+ binding receptor essential for full resistance (Franke et al., 2003). Bagai et al. (2007) reported that CusB (MFP) binds one molecule of Ag+ or Cu+ via four conserved methionines and induces a substrate-linked conformational change (Bagai et al., 2007). The crystal structures of CusB are available (Su et al., 2009). The crystal structure of the CusAB complex has been solved (PDB# 3K07) (Su et al., 2011a). CusC is listed under TC# 1.B.17.3.5. The metal-binding methionines play a role in restricting the substrates to monovalent heavy metals (Conroy et al., 2010). It has been reported to export L-cysteine (Yamada et al., 2006). Crystal structures of the CusA efflux pump suggested that methionine residues in a 3-methionine cluster, bind the metal as a transport intermediate (Long et al., 2010). Four methionine pairs in the transmembrane region, and one in the periplasmic domain may comprise the channel. Cu+ is exported from the cytoplasm to the periplasmic chaparone, CusF in the extracellular space (Padilla-Benavides et al. 2014). The Cus efflux system removes Cu+ and Ag+ from both the cell cytoplasm and the periplasm (Su et al., 2011b; Delmar et al. 2014). Metal-bound CusB is required for activation of Cu+ transfer from CusF directly to a site in the CusA antiporter (Chacón et al. 2014). Metal transfer occurs between CusF and apo-CusB, and when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm. The ratio of CusA (RND):CusB (MFP):CusC (OMF) is 3:6:3 (Delmar et al. 2013). Intermediates in metal transfer reactions have been measured (Chacón et al. 2018).
CusCFBA of E. coli:
CusC (OMF) (see 1.B.17.3.5)
The Zn2+-specific exporter, ZneABC. The ZneB MFP plays an active role in substrate efflux through metal binding and release. Its 2.8 Å structure is available (De Angelis et al., 2010). 3.0 Å intermediate conformational structures of ZneA have been determined, revealing two Zn2+ binding sites separated by a channel, and the protein has been shown to catalyze electrogenic Zn2+:H+ antiport (Pak et al. 2013).
ZneABC of Cupriavidus (Ralstonia) metallidurans (eutrophus or eutropha)
ZneC (DMF) (Q1LCD9)
ZneA (RND) (Q1LCD8)
ZneB (MFP) (Q1LCD7)
Putative Zn2+ exporter, Cus1ABC (induced by Zn2+; Moraleda-Muñoz et al., 2010)
Cus1ABC of Myxococcus xanthus
Cus1A (RND) (Q1DDM9)
Cus1B (MFP) (Q1DDM8)
Cus1C (OMF) (Q1DDM7)
Putative Cu2+ exporter, Cus2ABC (induced by Cu2+; Moraleda-Muñoz et al., 2010)
Cus2ABC of Myxococcus xanthus
Cus2A (RND) (Q1DDM4)
Cus2B (MFP) (Q1DDM3)
Cus2C (OMF) (Q1DDM2)
Putative heavy metal (Me2+) exporter, Czc1ABC (induced by heavy metals, but not Cu2+; Moraleda-Muñoz et al., 2010)
Czc1ABC of Myxococcus xanthus
Czc1A (RND) (Q1D6S7)
Czc1B (MFP) (Q1D6S8)
Czc1C (OMF) (Q1D6S9)
Multidrug (acriflavin, doxorubicin, ethidium, rhodamine 6G, SDS, deoxycholate) resistance pump [required for normal chromosomal condensation and segregation as well as cell division] (Lau and Zgurskaya, 2005). Exports L-cysteine (Yamada et al., 2006).
AcrEF (EnvCD) of E. coli
AcrE (MFP) (P24180)
AcrF (EnvD) (RND) (P24181)
TtgGHI of Pseudomonas putida
Heteromeric multidrug/detergent resistance protein YegM/YegN/YegO (MdtA/MdtB/MdtC) (Nishino and Yamaguchi 2001). Exports nalidixic acid, norfloxacin, cloxicillin, enoxacin, kanamycin, benzalkonium, bile salts, SDS and deoxycholate. It forms a complex with MdtA (YegM) (an MFP, TC# 8.A.1.6.2). Drug resistance depends on the simultaneous presence of all three proteins (Baranova and Nikaido, 2002). (Also contributes to copper and zinc resistance; regulation is mediated by BaeSR, and indole, Cu2+ and Zn2+ induce (Nishino et al., 2007)). MdtB:C stoichiometry = 2:1; MdtB and MdtC may play different roles (Kim et al., 2010), MdtB transporting the proton and MdtC transporting the drug (Kim and Nikaido 2012). MdtBC is reported to export bile salts without MdtA (Nagakubo et al. 2002), but this conclusion seems questionable.
MdtB/MdtC of E. coli
MdtB (YegN) (P76398)
MdtC (YegO) (P76399)
Multidrug/dye/detergent resistance protein, YhiU/YhiV or MdtE/MdtF (Nishino and Yamaguchi 2001) MdtE (YhiU) is listed under TC# 8.A.1.6.3. The system exports erythromycin, doxorubicin, crystal violet, ethidium, rhodamine 6G, TPP, benzalkonium, SDS, deoxycholate and growth inhibitory steroid hormones (estradiol and progesterone) (Elkins and Mullis, 2006).
YhiUV or MdtEF of E. coli
SmeVWX MDR efflux pump. Drugs include chloramphenicol, quinolones, tetracyclines and aminoglycosides, but not β-lactams and erythromycin (Chen et al., 2011).
SmeVWX of Stenotrophomonas maltophilia
SmeV (MFP) (B2FLY3)
SmeW (RND) (B2FLY4)
SmeX (OMF) (B2FLY6)
Multidrug efflux pump, MexD (exports β-lactams, fluoroquinolones, tetracycline, macrolides, chloramphenicol, biocides, including levofloxacin, carbenicillin, aztreonam, ceftazidime, cefepime, cefoperazone, piperacillin, erythromycin, azithromycin, chloramphenicol, etc.; Mao et al., 2002). Functions with MexC (MFP) and OprJ (OMF) (Mao et al., 2002; Poole, 2008).
MexD of Pseudomonas aeruginosa
Multidrug efflux pump, MexF (exports fluoroquinolones, chloramphenicol, biocides, xenobiotics and chloramphenicol; functions with MexE (MFP) and OprN (OMF)) (Kohler et al., 1997; Poole, 2008). The P. putida orthologue also exports solvents such as farnesyl hexanoate (Dunlop et al. 2011).
MexF of Pseudomonas aeruginosa (AAG05882)
Multidrug efflux pump, MexK (exports fluoroquinolones, macrolides, chloramphenicol; biocides, and triclosan [with MexJ but without OprM] as well as tetracycline, erythromycin [requiring both MexJ and OprM]; Chuanchuen et al., 2002). Can function with OpmH (BAC24099) instead of OprM (Poole, 2008).
MexK of Pseudomonas aeruginosa
The polycyclic aromatic hydrocarbon (phenanthrene; anthacene; fluoranthene)/drug (chloramphenicol; nalidixic acid) exporter, EmhABC (Hearn et al., 2003; 2006)
EmhABC of Pseudomonas fluorescens
Multidrug/dye/detergent/bile salt/organic solvent resistance pump (substrates include: chloramphenicol, tetracycline, erythromycin, nalidixic acid, fusidic acid, fluoroquinolones, lipophilic β-lactams, norfloxacin, doxorubicin, novobiocin, rifampin, trimethoprim, acriflavin, crystal violet, ethidium, disinfectants, rhodamine-6G, TPP, benzalkonium, SDS, Triton X-100, deoxycholate/bile salts/organic solvents (alkanes), growth inhibitory steroid hormones (estradiola and progesterone), and phospholipids) (Elkins and Mullis, 2006). Lateral entry of substrates from the lipid bilayer into AcrB and its homologues has been proposed (Yu et al., 2003a; 2003b). [An asymmetric trimeric structure is established with AcrA having a hexameric structure, and TolC having a trimeric structure (Seeger et al., 2006]. A structure of a complex with YajC is also known (Törnroth-Horsefield et al., 2007). A covalently linked trimer of AcrB provides evidence for a peristaltic pump, alternative access, rotation mechanism (Takatsuka and Nikaido, 2009;Nikaido and Takatsuka, 2009; Pos, 2009) Further evidence for a rotatory mechanisms stems from kinetic analyses for cephalosporin efflux which can exhibit positive cooperativity (Nagano and Nikaido, 2009). May also export signaling molecules for cell-cell communication (Yang et al., 2006). The substrates may be captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate, and finally to the AcrA funnel that connects AcrB to TolC (Husain & Nikaido et al., 2010). AcrB has been converted into a light-driven proton pump using delta-rhodopsin (dR) linked to AcrB via a glycophorin A transmembrane domain. This created a solar powered protein capable of selectively capturing antibiotics from bulk solutions (Kapoor and Wendell 2013). The trimeric structure is essential for activity (Ye et al. 2014). Association with AcrZ (TC# 8.A.50), a small 1 TMS protein (49 aas) that modifies the substrate specificity of AcrAB, has been demonstrated (Hobbs et al. 2012). In a similar way, the binding of YajC to AcrB stimulates the export of ampicillin (Törnroth-Horsefield et al. 2007). AcrZ binds to AcrB in a concave surface of the transmembrane domain (Du et al. 2015). Substrate binding accelerates conformational transitions and substrate dissociation, demonstrating cooperativity (Wang et al. 2015). The overall structure of AcrAB-TolC exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the alpha-barrel tip region of TolC. Direct interaction between AcrB and TolC is not allowed (Kim et al. 2015). TMS2 in AcrB is required for lipophilic carboxylate binding. A groove shaped by the interface between TMS1 and TMS2 specifically binds fusidic acid and other lipophilic carboxylated drugs (Oswald et al. 2016). After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion (Su et al. 2006). His978 is probably on the H+ pathway (Takatsuka and Nikaido 2006). AcrAB-TolC segregates to the old pole following cell division, causing the two daughter cells to exhibit different drug resistances (Bergmiller et al. 2017). The hoisting-loop is a highly flexible hinge that enables conformational energy transmission (Zwama et al. 2017). AcrB exhibits three distinct conformational states in the transport cycle, substrate access, binding, and extrusion, or loose (L), tight (T), and open (O) states, respectively (Yue et al. 2017). Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Release of a proton from Asp408 in the O state results in large conformational changes. Simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB (Yue et al. 2017). The three-dimensional structures of the homo-trimer complexes of AcrB-like transporters, and a three-step functional rotation helps to explain the mechanism of transport, but a more comprehensive model has been proposed (Zhang et al. 2017). Preparation of the trimeric complex (AcrAB/TolC) for cryo EM has been described (Du et al. 2018). The structural and energetic basis behind coupling functional rotation to proton translocation has been presented (Matsunaga et al. 2018). Protonation of transmembrane Asp408 in the drug-bound protomer drives rotation. The conformational pathway identifies vertical shear motions among several transmembrane helices, which regulate alternate access of water as well as peristaltic motions that pump drugs into the periplasm (Matsunaga et al. 2018). CryoEM of detergent-free AcrB preserves lipid-protein interactions for visualization and reveals how the lipids pack against the protein (Qiu et al. 2018). In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action (Nolivos et al. 2019).
AcrABZ of E. coli
AcrA (MFP) (P31223)
AcrB (RND) (P31224)
AcrZ of 49 aas (P0AAW9)
The multidrug (aminoglycosides, β-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, erythromycin, ofloxacin, etc.) efflux pump, MexXY-OprM (Jeannot et al., 2005). The 3-d structurre of OprM (also called OprK) is known (1WP1). RND-type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu (Li et al. 2006).
MexXY-OprM of Pseudomonas aeruginosa
The conjugated and unconjugated bile (bile-inducible)/multidrug (ethidium, ciprofloxacin, norfloxacin, tetracycline, cefotaxime, rifampicin, erythromycin, chloramphenicol, salicylate; drug-noninducible) efflux pump, CmeABC (Lin et al., 2005). The 3-d structure of the OMF, CmeC, has been determined (Su et al. 2014). The system is involved in biofilm production (Teh et al. 2017).
CmeABC of Campylobacter jejuni
CmeA (MFP) (AAL74244)
CmeB (RND) (AAL74245)
CmeC (OMF) (AAL74246)
The multidrug (β-lactams, aminoglycerides (gentamycin and streptomycin) macrolides (erythromycin) and dye (acriflavin)) efflux pump, BpeAB-OprB (Chan et al., 2004; Chan and Chua, 2005). It also exports acyl homoserine lactones including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone (Chan et al., 2007). Q9HWH6 is a DoxX family member (see 9.B.214.2).
BpeAB-OprB of Burkholderia pseudomallei
BpeA (MFP) (AAQ94109)
BpeB (RND) (AAQ94110)
OprB (OMF) (AAQ94111)
The gold (Au2+) resistance efflux pump, GesABC (induced by GolS in the presence of Au2+; also mediates drug resistance when induced by Au2+ (Pontel et al., 2007). Also exports a variety of organic chemicals including chloramphenicol (Conroy et al., 2010).
GesABC of Salmonella enterica
GesA (MFP) (Q8ZRG8)
GesB (RND) (Q8ZRG9)
GesC (OMF) (Q8ZRH0)
The multidrug efflux pump, VmeAB-VpoC (Matsuo et al., 2007). There are 11 RND-type efflux transporters in Vibrio parahaemolyticus, and several (VmeCD, VmeEF and VmeYZ) contribute not only to intrinsic drug resistance but also to virulence (Matsuo et al. 2013).
VmeAB-VpoC of Vibrio parahaemolyticus:
VmeA (MFP) (Q2AAU4)
VmeB (RND) (Q2AAU3)
VpoC (OMF) (Q87SJ8)
Multidrug efflux pump, AcrAB (Bina et al. 2008).
AcrAB of Francisella tularensis
The AdeIJK MDR pump (contributes to resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolines, fusidic acid, tigecycline, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, triclosan and sodium dodecyl sulfate) (Damier-Piolle et al., 2008; Fernando et al. 2014)
AdeIJK of Acinetobacter baumannii
AdeI (MFP) (Q2FD95)
AdeJ (RND) (Q24LT7)
AdeK (OMF) (Q24LT6)
Isoflavonoid efflux pump, IfeB
IfeB of Agrobacterium tumefaciens
Multidrug efflux pump, MexHI OpmD (exports fluoroquinolones; Poole, 2008). The encoding genes are part of the SoxR regulon (Naseer et al. 2014). These genes are preceded by a gene encoding PA4205, a 148 aas 4 TMS protein, MexG, a member of the DoxX family (TC# 9.B.214) of unknown function, but possibly a component of this ABC transporter (Naseer et al. 2014).
MexHI OpmD of Pseudomonas aeruginosa
MexH (MFP) (Q9HWH5)
MexI (RND) (Q9HWH4)
OpmD (OMF) (Q9HWH3)
MexG (4 TMS protein (Q9HWH6)
Multidrug efflux pump, MexVW OmpM (exports fluoroquinolones, macrolides, chloramphenicol, and tetracycline) (Poole, 2008).
MexW of Pseudomonas aeruginosa
MexW (RND) (Q9HW27)
VexB of Vibrio cholerae (Q9KVI2)
VexD of Vibrio cholerae (A6P7H1)
VexK of Vibrio cholerae (Q9KRG9)
THe MuxABC-OpmB multidrug (aztreonam, macrolides, novobiocin and tetracycline) resistance efflux pump complex (with two RND-type proteins (MuxB and MuxC)), both required for activity (Mima et al., 2009).
MuxABC-OpmB complex of Pseudomonas aeruginosa
MuxA (MFP) (PA2528) (Q9I0V5)
MuxB (RND) (PA2527) (Q9I0V6)
MuxC (RND) (PA2526) (Q9I0V7)
OpmB (OMF) (Q9I0V8)
Gram negative bacteria
AdeDE of Acinetobacter sp. 4356
MDR pump, AdeABC. Exports chloramphenicol and tetracycline (Hassan et al., 2011). Also confers resistance to meropenem, tigecycline and ceftazidime (Peleg et al. 2007; Provasi Cardoso et al. 2016).
AdeABC of Acinetobacter baumannii
AdeA (MFP) (Q2FD71)
AdeB (RND) (Q2FD70)
AdeC (OMF) (Q2FD69)
SmeABC MDR efflux pump. Drugs include ciprofloxacin (Cho et al., 2012).
SmeABC of Stenotrophomonas maltophilia
SmeA (MFP) (Q9RBY9)
SmeB (RND) (Q9RBY8)
SmeC (OMF) (Q9RBY7)
SmeDEF MDR efflux pump. Mediates resistance to a wide range of drugs including ethidium bromide and norfloxacin (Alonso and Martínez, 2000). Regulated by SmeT and activated by insertion of the transposon, IS1246 (Gould and Avison, 2006).
SmeDEF of Stenotrophomonas maltophilia
SmeD (MFP) (Q9F241)
SmeE (RND) (Q9F240)
SmeF (OMF) (Q9F239)
Multidrug resistance pump, SmeJK. Shown to export teracycline, minocycline, ciprofloxacin and levofloxacin (Gould et al., 2012).
SmeJK of Stenotrophomonas maltophilia D457
Multidrug efflux pump, AdeFGH. Mediates high level resistance to chloramphenicol, clindamycin, fluoroquinolones, and trimethoprim and decreased susceptibility to tetracycline-tigecycline and sulfonamides; susceptibility to β-lactams, erythromycin, aminoglycosides and rifampin was not affected. It also mediates increased resistance to ethidium bromide, safranin O, acridine orange, trimethoprim and sulfamethoxazole (Coyne et al. 2010).
AdeFGH of Acinetobacter baumannii
AdeF (MFP) (Q2FD82)
AdeG (RND) (Q2FD81)
AdeH (OMF) (Q2FD80)
The AcrA/AcrB multidrug resistance pump. Exports various toxic compounds, including antibiotics, phytoalexins, and detergents. Mutants are less virulent on tomato plants than the wild-type strain (Brown et al. 2007).
AcrAB of Ralstonia solanacearum (Pseudomonas solanacearum)
Solvent (such as limonene) efflux pump, TtgABC (Dunlop et al. 2011).
TtgABC of Alcanivorax borkumensis
Multidrug resistance exporter, OqxA (BepF)-OqxB (BepE) (Taherpour and Hashemi 2013).
OqxAB of Klebsiella pneumoniae
Multidrug resistance (MDR) pump, AcrD, AcrF, Env. Catalyzes efflux of various hydrophilic and amphipathic drugs including clotrimazole and luteolin, but not aminoglycosides. Induction of acrD expression occurs in infected apple tissue but not in pear tissues. Regulated by the two component BaeSR sensor kinase/response regulator (Pletzer and Weingart 2014).
ArcD of Erwinia amylovora, the causal agent of fire blight disease.
Multidrug resistance exporter, AcrABZ. Exports tigecycline and many other drugs (Nielsen et al. 2014; Li et al. 2016; Yuhan et al. 2016; He et al. 2015; Bialek-Davenet et al. 2015). AcrA-AcrB-AcrZ-TolC is a drug efflux protein complex with a broad substrate specificity. AcrZ (YbhT) binds to AcrB and is required for efflux of some but not all substrates, suggesting it may influence the specificity of drug export (Hobbs et al. 2012; Du et al. 2015).
AcrABZ of Klebsiella pneumoniae
AcrZ (YbhT) (RND auxiliary protein; see 8.A.50)
Fatty acid, bile salt, gonadal steroid, antibacterial peptide efflux pump, MtrCDE (Kamal et al., 2007). Opening of the outer membrane protein channel, MtrE, in the tripartite efflux pump, MtrCDE, is induced by interaction with the membrane fusion partner, MtrC (Janganan et al., 2011). The crystal structure of the trimeric MtrE forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end in the open conformational state of this channel (Lei et al. 2014).
MtrCDE of Neisseria gonorrhoeae:
MtrC (MFP) (P43505)
MtrD (RND) (Q51073)
MtrE (OMF) (Q51006)
Multiple drug; N-(3-oxododecanoyl)- L-homoserine lactone autoinducer efflux pump, MexB (functions with MexA (an MFP, 8.A.1) and OprM (an OMF, 1.B.17; see 2.A.6.2.21). All three interact with each other. MexA promotes assembly and stability of the complex (Nehme and Poole, 2007)). Exports β-lactams, fluoroquinolones, tetracycline, macrolides, chloramphenicol, biocides, and a toxic indole compound, CBR-4830, that targets the MreB actin (Robertson et al., 2007). Confers tolerance to tea tree oil and its monoterpene components Terpinen-4-ol, 1,8-cineole and α-terpineol (Papadopoulos et al., 2008) as well as the antimicrobial peptide, colistin (Pamp et al., 2008) (Mao et al., 2002; Poole, 2008). The crystal structure has been solved at 3.0Å resolution (Sennhauser et al., 2009). The MexA-OprM complex has an elongated cylindrical appearance (Trépout et al., 2010). Mutations affecting export of antibiotics with cytoplasmic targets have been identified (Ohene-Agyei et al. 2012). RND-type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu (Li et al. 2006).
MexAB of Pseudomonas aeruginosa
Multidrug efflux pump, AcrD (exports aminoglycosides (amikacin, gentamycin, neomycin, kanamycin and tobramycin) as well as anionic detergents (SDS and deoxycholate) and growth inhibitory steroid hormones (estradiol and progesterone)(Elkins and Mullis, 2006)) (exports aminoglycosides from the periplasm as well as the cytoplasm) (Aires and Nikaido, 2005). (Also contributes to copper and zinc resistance; regulation is mediated by BaeSR, and indole, Cu2+ and Zn2+ induce (Nishino et al., 2007)). Exports L-cysteine (Yamada et al., 2006).
AcrD of E. coli (P24177)
Multidrug efflux pump, ArpB (exports tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin, novobiocin, etc.)
ArpB of Pseudomonas putida
Solvent efflux pump, TtgABC (extrudes toluene, styrene, m-xylene, ethylbenzene, acetate, α-pinene and propylbenzene) (Teran et al., 2007; Dunlop et al. 2011).
TtgABC of Pseudomonas putida:
Putative lipooligosaccharide nodulation factor exporter, NolG (1065 aas; previously thought to be 3 ORFs, NolGHI, an artifact due to sequencing errors and consequent frameshifting (Baev et al. 1991; Ardourel et al. 1994).
NolG of Rhizobium meliloti (P25197)
Multidrug resistance pump, CmeDEF. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin (Pumbwe et al. 2005). This system is similar to the Helicobacter pylori MDR pump, HefABC (Huang et al. 2015).
CmeDEF of Campylobacter jejuni
CmeD (OMF, 424 aas)
CmeE (MFP, 246 aas)
CmeF (RND. 1005 aas
RND family protein involved in virulence and resistance to antimicrobial agents, BesABC. BesC forms channels in lipid bilayers (Bunikis et al. 2008).
BesABC of Borrelia burgdorferi
BesA, 317 aas
BesB, 1070 aas
BesC, 428 aas
The multidrug efflux porter, HefABC; HefA is an OMF (TC#1.b.17) of 477 aas and 1 N-terminal TMS; HefB is an MFP (TC# 8.A.1) of 234 aas and 1 N-terminal TMS. HefC is the RND pump of 1028 aas and 12 TMSs (Mehrabadi et al. 2011; Liu et al. 2008).
HefABC of Helicobacter pylori
RND exporter of thiophosphate of 1085 aas and 12 TMSs, functioning with an MFP of 337 aas and 1 N-terminal TMS.
Thiophosphate exporter of Shewanella oneidensis
NolG homologue, Atu4636
NolG of Agrobacterium tumefaciens (A9CGX6)
NolG of Acinetobacter baumanii (E8PBU7)
NolG of Myxococcus xanthus (Q1DEX6)
NolG of Synechococcus sp. PCC7335 (B4WH09)
NolG of Oceanobacillus iheyensis (Q8CX78)
Putative Cu2+ exporter, Cus3ABC. Induced by Cu2+; Moraleda-Muñoz et al., 2010)
Cus3ABC of Myxococcus xanthus
Cus3A (RND) (Q1CZ65)
Cus3B (MFP) (Q1CZ64)
Cus3C (OMF) (Q1CZ66)
Efflux pump for antifungal and antibacterial syringopeptin and syringmycin lipodepsipeptides (see 1.D.35) as well as acriflavin, erythromycin and tetracycline, PseABC (Kang and Gross 2005).
PseABC of Pseudomonas syringae
PseA (OMF) (L8NE56)
PseB (MFP) (L8NGR5)
PseC (RND) (L8NFZ8)
Primary surfactin (a lipodepsipeptide) exporter of 1056 aas and 12 TMSs, YerP (Li et al. 2015).
YerP of Bacillus subtilis
The secretory accessory proteins, SecDF. The first periplasmic domain of SecDF has been crystallized (Echizen et al., 2011) as has the intact SecDF complex (Tsukazaki and Nureki 2011). SecDF has been reported to function as a pmf-driven H+ transporter that facilitates protein translocation (Tsukazaki et al. 2011). It may assume at least two conformations differing by a 120 degrees rotation during polypeptide translocation (Mio et al. 2014). SecDF is proposed to undergo repeated conformational transitions to pull out the precursor protein from the SecYEG channel into the periplasm (Tsukazaki 2018). Once SecDF captures the precursor protein on the periplasmic surface, it can complete protein translocation even if SecA function is inactivated by ATP depletion, implying that SecDF is a protein-translocation motor that works independent of SecA. Structural and functional analyses of SecDF suggested that SecDF utilizes the proton gradient and interacts with precursor proteins in the flexible periplasmic region. The crystal structures of SecDF in different states at more than 3 Å resolution were reported in 2017 and 2018, which further improved our understanding of the dynamic molecular mechanisms of SecDF (Tsukazaki 2018).
SecDF of E. coli; SecD; SecF
SecDF of Bacillus subtilis
Protein translocase subunit SecDF. The 3-dimensional structure is known at 3.3 Å resolution (Tsukazaki et al. 2011). SecDF serves several functions, such as stabilizing other Sec translocon components within the membrane, maintaining the transmembrane (TM) potential, and facilitating the ATP-independent stage of the translocation mechanism. SecDF also undergoes functionally important conformational changes that involve mainly its P1-head domain, and these changes are coupled with the proton motive force (Δpmf). Using all-atom molecular dynamics simulations combined with umbrella sampling, Ficici et al. 2017 studied the P1-head conformational change and how it is coupled to the pmf. They reported potentials of mean force along a root-mean-square-distance-based reaction coordinate obtained in the presence and absence of the TM electrical potential. Their results showed that the interaction of the P1 domain dipole moment with the TM electrical field lowers the free-energy barrier in the direction of the F-form to I-form transition, two conformations that vary by the relative positioning of the P1-head
subdomain—the large periplasmic domain of TtSecDF—which is suggested to
undergo a hinge motion (Ficici et al. 2017).
SecDF of Thermus thermophilus
SecDF of 1254 aas and 12 TMSs
SecDF of Spiroplasma diminutum
Multidrug resistance protein, CmpL1, of 772 aas and 12 TMSs. Mutants are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellular trehalose monocorynomycolates, free corynomycolates, and a previously uncharacterized corynomycolate-containing lipid. It is inferred that this transporter exports one or more of these lipids. Evidence for a pmf-dependent mechanism was obtained (Yang et al. 2014).
CmpL1 of Corynebacterium glutamicum
CmpL4 of 801 aas and 12 TMSs. Multidrug resistance protein of 801 aas and 12 TMSs. Mutants are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellular trehalose monocorynomycolates, free corynomycolates, and a previously uncharacterized corynomycolate-containing lipid. It is inferred that this transporter exports one or more of these lipids. Evidence for a pmf-dependent mechanism was obtained (Yang et al. 2014).
CmpL4 of Corynebacterium glutamicum
MmpL4a of 959 aas and 12 TMSs. A rough morphotype has a Y842H mutation that causes a deficiency in glycopeptidolipid production and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was noted, involving massive production of serpentine cords, abscess formation and rapid larval death. Tyr842 is conserved in several MmpL proteins (Bernut et al. 2016).
MmpL4a of Mycobacterium abscessus subsp. bolletii
Lipid (acyl and diacyl trehalose) exporter of 1002 aas and 12 TMSs, MmpL10 (Bailo et al. 2015). A 12 TMS topology has been determined (Belardinelli and Jackson 2017).
MmpL10 of Mycobacterium tuberculosis
Uncharacterized MmpL-like efflux pump of 843 aas and 12 TMSs
MmpL of Entamoeba histolytica
The phthiocerol dimycocerosate (PDIM) lipid exporter, MmpL7. Also confers high level isoniazid efflux and resistance (Pasca et al., 2005).
MmpL7 of Mycobacterium tuberculosis ( P65370)
The putative glycopeptidolipid exporter, TmtpC (most similar to MmpL of M. leprae; implicated in sliding motility). May function with the MmpS4 protein of Mucobacterium smegmatis (A0QPN7) to form a scaffold for coupled biosynthesis and transport (Deshayes et al., 2010).
TmtpC of Mycobacterium smegmatis
sulfolipid, 2,3-diacyl-α, α'-D-trehalose-2'-sulfate (sulfatide precursor) exporter, MmpL8 (Domenech et al., 2004; Seeliger et al. 2012).
MmpL8 of Mycobacterium tuberculosis (CAB10022)
Mycobacterial heme acquisition system, Rv0202c - Rv0207c. Takes up free heme and heme from hemoglobin as an iron source together with the secreted protein, Rv0203 (O53654) (Owens et al. 2013). May function with Rv0206c (MmpL3; TC#2.A.6.5.6) and Rv0202c (Tullius et al., 2011). However, see description of MmpL3 (2.A.6.5.6). These two proteins are targets of drug action (Owens et al. 2013). A 12 TMS topology has been suggested (Belardinelli and Jackson 2017).
Heme uptake system of Mycobacterium tuberculosis
MmpL3 (Rv0206; 944 aas) May function with MmpL11 (TC# 2.A.6.5.5) (Tullius et al., 2011). MmpL3 exports trehalose monomycolate, involved in mycolic acid donation to the cell wall core (Tahlan et al., 2012). SQ109, a 1,2,-diamine related to ethambutol is an inhibitor of MmpL3 (Tahlan et al., 2012). May also transport heme. Inhibitors have been identified (Rayasam 2013; Li et al. 2014). MmpL3 has been shown to be a homotrimer of three 12 TMS subunits, confirming its RND-type structure (Belardinelli et al. 2016). MmpL3 is a flipppase for mycolic acids, transporting them from the cytoplasmic side of the inner membrane to the external side. A 1.5-diarylpyrrole compound, BM212, is a potent inibitor (Xu et al. 2017). Inactivation of the mmpL3 gene in M. neoaurum increased the permeability of the outer membrae and allowed increased uptake of sterols for coversion to other sterols for industrial purposes. One such product is 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), used for the synthesis of various steroids in the industry (Xiong et al. 2017). Sincer iron deprivation decreases expression of the mmpL3 gene, a metal chelation strategy could boost the effectiveness of current anti-TB drug regimes to combat drug resistance TB (Pal et al. 2018). Crystal structures are available for MmpL3 alone and in complex with four TB drug candidates. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear facilitate proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs (Zhang et al. 2019). MmpL3 can be directed inhibited by several antitubercular compounds (Li et al. 2019).
MmpL3 of Mycobacterium tuberculosis (O53657)
Siderophore export transporter, MmpL4 (Wells et al. 2013). Functions with MmpS4 (TC#8.A.35.1.1) which is essential for transport activity. MmpL4/MmpS4 and MmpL5/MmpS5 (TC# 2.A.6.5.8 and TC# 8.A.35.1.2, respectively) are two siderophore exporters that overlap in function (Wells et al. 2013). The M. abscessus, subspecies bolletii orthologue (TC# 2.A.6.5.12), of 959 aas, is 65% identical to M. tuberculosis MmpL4 and affects the rough vs smooth phenotype of the cell envelope (Bernut et al. 2016).
MmpL4 of Mycobacterium tuberculosis
Siderophore exporter, MmpL5. Functions with MmpS5, and both proteins are essential for transport activitiy (Wells et al. 2013). Together with MmpS5, it also pumps drugs out of the cell, and upregulation gives rise to drug resistance (Briffotaux et al. 2017).
MmpL5 of Mycobacterium tuberculosis
MmpL-like protein of Bacillus weihenstephanensis (A9VJD5)
Niemann-Pick C1 and C2 disease proteins, NPC1 and NPC2, together may form a lipid/cholesterol exporter from lysosomes to other cellular sites including the plasma membrane (Sleat et al., 2004; Kennedy et al. 2014). NPC1 or NPC2 deficiency causes lysosomal retention of cholesterol, sphingolipids, phospholipids, and glycolipids as well as neuronal dysfunction and neurodegeneration (Infante et al. 2008a). Increased mitochondrial cholesterol, observed in NPC1 or NPC2 deficiency, causes oxidative stress and increased rates of glycolysis and lactate release (Kennedy et al. 2014). NPC1 binds cholesterol, 25-hydroxycholesterol and various oxysterols (Infante et al. 2008b; Liu et al., 2009 ). Soluble NPC2 binds cholesterol, and then passes it to the N-terminal domain of membranous NPC1 (Abi-Mosleh et al., 2009). Cholesterol trafficking in Niemann-Pick C-deficient cells was reviewed by Peake and Vance (2010). NPC1 is a late-endosomal membrane protein involved in trafficking of LDL- derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection. It is the Ebola virus receptor. It contains 13 TMSs, five of which are thought to represent a """"sterol-sensing domain"""", also present in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling. A crystal structure of a large fragment of human NPC1 at 3.6 Å resolution revealed internal twofold pseudosymmetry along TMSs 2-13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen (Li et al. 2016). NPC1's sterol sensing domain forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; this cavity is large enough to accommodate one cholesterol molecule. A model was proposed for cholesterol sensing and transport (Li et al. 2016). Lysosomal cholesterol activates TORC1 via an SLC38A9-Niemann-Pick C1 signaling complex (Castellano et al. 2017). Gong et al. 2016 presented a 4.4 Å structure of the full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains three distinct lumenal domains A (also designated NTD), C, and I. TMSs 2-13 exhibit a typical RND fold, among which TMSs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through domain C (Gong et al. 2016).
The effects of disease-causing mutations on quality control pathways involving the lysosome and endoplasmic reticulum, and how it functions to clear the most common mutant protein found in Niemann-Pick type C patients have been reviewed (Schultz et al. 2016). In the same review, knowledge concerning the mechanisms that degrade misfolded transmembrane proteins in the endoplasmic reticulum is presented. Cholesterol esters are components of low density lipoprotein (LDL), which is brought into the cells of various tissues by targeted endocytosis. Within the endosomes, cholesterol esters are hydrolyzed, releasing free cholesterol, which is finally exported out of the endosome by NPC1 with assistance from a soluble protein NPC2 (Nikaido 2018). The transmembrane helices in the N-terminal half (the SSD, sterol-sensing domain) of NPC1 are homologous to the sterol-binding domains of HMG-CoA reductase, as well as the regulator of cholesterol-regulated transcription activation, SCAP. The domain that binds cholesterol with the highest affinity, within NPC1, however, is the NTD. In the NPC2-NPC1 complex, the substrate is captured at a location far away from the membrane by NPC2, and then is brought to a location close to the membrane surface (NTD of NPC1), and is finally moved to the intramembranous region of NPC1. Degradation occurs via two pathways, the proteasome following MARCH6-dependent ERAD, and an autophagic pathway called the selective ER autophagy (ER-phagy) (Schultz et al. 2018). NPC1 exports LDL-derived cholesterol from lysosomes by carrying it through the 80 Å glycocalyx and the 40 Å lipid bilayer. Transport begins when cholesterol binds to the N-terminal domain (NTD) of NPC1, which projects to the surface of the glycocalyx. Trinh et al. 2018 reconstituted cholesterol transport by expressing the NTD as a fragment separate from the remaining portion of NPC1. When co-expressed, the two NPC1 fragments reconstitute cholesterol transport and showed that cholesterol can be transferred from the NTD of one full-length NPC1 to another NPC1 molecule that lacks the NTD. The locations of buried amino acids and docking studies have identified putative lipid binding domains that are in close proximity to amino acids that, when mutated, are connected to NPC1 loss-of-function (Elghobashi-Meinhardt 2019).
NPC1 and NPC2 of Homo sapiens
Pacific oyster protein of unknown function
Uncharacterized protein of Crassostrea angulata
3-hydroxy-3-methylglutaryl-coenzyme A reductase of 438 aas and 1 or 0 TMSs.
3-hydroxy-3-methylglutaryl-coenzyme A reductase of Bdellovibrio bacteriovorus
Niemann-Pick C1 protein, NPC1 of 1339 aas and 16 TMSs in a 1 + 3 + 1 +5 + 1 +5 arrangement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis clearly suggest that these proteins play a key role in cholesterol uptake (Bolaños et al. 2016).
NPC1 of Entamoeba histolytica
Patched, PTCH1 or PTCH, of 1447 aas and 12 TMSs. RND transporter-like receptor, regulates the activity of Smoothened of 7 TMSs via the Hedgehog (HH) pathway. The Na+ gradient may provide the energy for Patched activity (Myers et al. 2017). Possibly Patched1, chemiosmotically driven by the transmembrane Na+ gradient, regulates Smoothened. 3-d structures have been solved at ~ 3.5 Å resolution by cryoEM (Qi et al. 2018). This protein seems to have both transport and signalling functions, exporting cholesterol from the inner leaflet of the membrane out to the external environment. It displays a tunnel going through the membrane with a lateral opening, all large enough to accomodate cholesterol (Qi et al. 2018; Sommer and Lemmon 2018). The distrutribution of Smoothened on the primary cillium surface has been studied (Yoon et al. 2019).
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched that suppresses the activity of the 7-TMS protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of Patched revealed striking transmembrane domain similarities to prokaryotic RND transporters in whick a central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting that Patched catalyzes cholesterol transport (Zhang et al. 2018). Cholesterol in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Key proteins in the Hedgehog-signalling pathway dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO or SMOH; TC# 9.A.14.16.4) is released of its inhibition by PTCH1 and accumulates in cilia. PTCH1 inactivation by SHH changes the diffusive motion of PTCH1 (Weiss et al. 2019).
PTCH1 of Homo sapiens
Patched-2 (Ptch2) of 1203 aas and 12 TMSs. It plays a role in epidermal development and may act as a receptor for Sonic hedgehog (SHH). The two principal luminal domains of Ptch1 (TC# 2.a.6.6.13) and Ptch2 are interchangeable; the sterol-sensing domains (SSDs) of Ptch-family members exhibit generic activities while the adjacent cytoplasmic and luminal domains determine their protein-specific activities (Fleet and Hamel 2018).
Ptch2 of Homo sapiens
Patched (Ptc) segmentation polarity protein. Patched is known to be an important component of Hedgehog signaling in organisms from Drosophila to humans. It displays a typical RND folding pattern. Although Hedgehog appears to bind to Patched, it probably does not catalyze its import; thus, Patched may function as a Hedgehog receptor (Nikaido 2018). The two extramembranous loops of Patched are thought to face the exterior, and possibly play roles in the binding of the signal protein. However, Patched may indeed function as a transporter, possibly of small molecules (Taipale et al. 2002).
""Patched"" of Drosophila melanogaster
Yeast sterol transport system consisting of two proteins, NCR1 (YPL006w) and NPC2 (YDL046w), components of the Niemann-Pick Type C transporter. It drives sterol integration into the lysosomal membrane before redistributing them to other cellular membranes. Winkler et al. 2019 presented a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting the NTD to the luminal membrane leaflet, 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel, supporting a proton-driven transport mechanism. Winkler et al. 2019 proposed a model for sterol integration that clarifies the role of these NPC proteins.
NCR1/NPC2 of Saccharomyces cerevisiae
SREBP cleavage-activating protein, Scap of 1279 aas. Cholesterol homeostasis is mediated by Scap, a polytopic ER protein that transports SREBPs from ER to Golgi where SREBPs are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. Evidence suggests that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles (Zhang et al. 2016). When ER cholesterol rises, it binds to Loop1 causeing dissociation from Loop7, abrogating COPII binding. Direct binding of the two loops causes dissociation from the membrane, allowing the soluble complex to be secreted. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion.
SCAP of Cricetulus griseus
Liver/intestinal enterocyte brush border Niemann-Pick C1 like 1 (NPC1L1) protein; responsible for ezetimibe-sensitive absorption of luminal lipids and cholesterol via a transport mechanism (Altmann et al., 2004; Davies et al., 2005; Liscum, 2007, Dixit et al. 2007). NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content (Betters and Yu, 2010; Jia et al., 2011). Dietary cholesterol induces trafficking of the intestinal NPC1L1 from the brush boarder to endosomes (Skov et al. 2011). It distributes on the brush border membranes of enterocytes and the canalicular membranes of hepatocytes. It is the target of ezetimibe, a hypocholesterolemic drug which blocks internalization of NPC1L1 and cholesterol in the mouse small intestine (Wang and Song 2012; Xie et al. 2012). Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homology to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). NPC1L1 may have evolved at two sites (apical membrane of enterocytes and canalicular membrane of hepatocytes) to mediate cholesterol uptake through a clathrin-mediated endocytic process, protecting the body against fecal and biliary loss of cholesterol (Yu 2008). NPC1L1-dependent intestinal cholesterol absorption appears to require ganglioside GM3 in membrane microdomains (Nihei et al. 2018).
NPC1L1 of Homo sapiens (NP_037521)
Niemann-Pick C-type protein (NPC) (1342 aas; 16 putative TMSs in a 1+3+1+5+1+5 arrangement)
NPC of Dictyostelium discoideum (Q9TVK6)
Niemann-Pick C1 protein homologue-1, Ncr1; contains a sterol sensing domain. Catalyzes intracellular cholesterol release from endocytic organelles.
Ncr-1 of Caenorhabditis elegans (Q19127)
Niemann-Pick C1 protein homologue-2, Ncr2; contains a sterol sensing domain. Catalyzes intracellular cholesterol release from endocytic organelles.
Ncr-2 of Caenorhabditis elegans (P34389)
Bacterial HAE3 family member
HAE3 family member of Myxococcus xanthus
Bacterial HAE3 family member
HAE3 family member of Myxococcus xanthus
Hopanoid biosynthesis associated RND transporter like protein, HpnN. Required for hopanoid localization to the outer membrane (Doughty et al. 2011).
Putative hopanoid transporter, HpnN, of Rhodopseudomonas palustris
Putative RND lipid exporter
RND exporter of Rhodopirellula baltica
Putative lipid exporter of 797 aas and 12 TMSs.
Putative exporter of Treponema brennaborense
Hopanoid biosynthesis associated RND transporter like protein, HpnN of 877 aas and 12 TMSs. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Kumar et al. 2017 reported crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicated that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm (Kumar et al. 2017).
HpnN of Burkholderia multivorans
Uncharacterized MMLP family protein of 772 aas and 12 TMSs.
UP of Thermotoga caldifontis
Xanthomonadin (brominated, aryl polyene pigment) exporter (to its outer membrane site), ORF4 of 788 aas and 12 TMSs (Goel et al. 2002). This aryl polyene pigment exporter is unusual in having relatively short external loops (Nikaido 2018).
ORF4 in the pig (pigment) gene locus of Xanthomonas oryzae pv. oryzae
RND transporter. Encoded in a 17 cistron operon that appears in pathogenic proteobacteria including pathogenic E. coli strains, but not non-pathogenic E. coli strains like K12. May be involved in host associations (EE Allen, personal communication)
RND transporter of E. coli
RND superfamily, MMLP family transporter of 1126 aas in a 1 + 5 + 1 +5 + 1 TMS arrangement, followed by a hydrophilic domain, possibly with additional TMSs. This last domain may be a glycerol acyltransferase, based on NCBI annotations, suggesting that the RND transporter could be an exporter for an acylated lipid. This fusion of an RND porter to this domain is common in Bacteroidetes and Flavobacteria.
MMLP family transporter of Flavobacterium johnsoniae
Uncharacterized MMLP family protein of 742 aas and 12 TMSs.
UP of Campylobacter concisus
Dispatched is an exporter of the amino-terminal portion (19 kDa) of the C-terminally cholesterol-modified peptide, hedgehog; sterol sensor protein (Ma et al., 2002). Loss prevents hedgehog signaling. (Nakano et al., 2004; Higgins, 2007). The intracellular movement of this Hedgehog signal is complex, and involves insertion into the apical cell membrane, followed by endocytosis, and then secretion at the basolateral membrane by Dispatched (Nikaido 2018). Dispatched shows a typical RND protein folding pattern, although its overall sequence similarity to Patched or NPC1 is low. However, it contains the typical sterol sensing domain, similar to those found in NPC1 as well as in Patched. Hedgehog anchored by glycosylphophatidylinositol, rather than cholesterol, was not exported by Dispatched, suggesting that the cholesterol anchor is recognized by the transporter. Finally, the secretion of Hedgehog by Dispatched across the membrane requires help by other proteins, including a secreted soluble protein called Scube (Nikaido 2018).
Dispatched of Drosophila melanogaster (AAF_23397)
Protein dispatched homologue 1 (dispA). The human ortholog (Q96F81, 1524 aas) is 83% identical. Plays a role in congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) in humans (Takahashi et al. 2018).
Disp1 of Mus musculus