Editing ABC transporter (section)

== ABC exporters ==
Prokaryotic ABC exporters are abundant and have close homologues in eukaryotes. This class of transporters is studied based on the type of substrate that is transported. One class is involved in the protein (e.g. [[toxins]], [[hydrolase|hydrolytic enzymes]], S-layer proteins, [[lantibiotics]], [[bacteriocins]], and competence factors) export and the other in drug efflux. ABC transporters have gained extensive attention because they contribute to the resistance of cells to [[antibiotics]] and [[anticancer agents]] by pumping drugs out of the cells.<ref name=":0" /><ref name=":1">{{Cite journal|last1=Gilson|first1=L.|last2=Mahanty|first2=H. K.|last3=Kolter|first3=R.|date=December 1990|title=Genetic analysis of an MDR-like export system: the secretion of colicin V|journal=The EMBO Journal|volume=9|issue=12|pages=3875–3884|issn=0261-4189|pmid=2249654|doi=10.1002/j.1460-2075.1990.tb07606.x|pmc=552155}}</ref><ref name=davidson/> A common mechanism is the overexpression of ABC exporters like [[P-glycoprotein]] (P-gp/ABCB1), multidrug resistance-associated protein 1 ([[MRP1]]/[[ABCC1]]), and [[breast cancer resistance protein]] (BCRP/ABCG2) in cancer cells that limit the exposure to anticancer drugs.<ref>{{Cite journal|last1=Choi|first1=Young Hee|last2=Yu|first2=Ai-Ming|date=2014|title=ABC Transporters in Multidrug Resistance and Pharmacokinetics, and Strategies for Drug Development|journal=Current Pharmaceutical Design|volume=20|issue=5|pages=793–807|doi=10.2174/138161282005140214165212|issn=1381-6128|pmc=6341993|pmid=23688078}}</ref>

In gram-negative organisms, ABC transporters mediate secretion of protein substrates across inner and outer membranes simultaneously without passing through the periplasm. This type of secretion is referred to as ''type I secretion'', which involves three components that function in concert: an ''ABC exporter'', a ''[[membrane fusion protein]] (MFP)'', and an ''outer membrane factor (OMF)''. An example is the secretion of [[hemolysin]] (HlyA) from ''E. coli'' where the inner membrane ABC transporter HlyB interacts with an inner membrane fusion protein HlyD and an outer membrane facilitator TolC. TolC allows hemolysin to be transported across the two membranes, bypassing the periplasm.<ref name=":0" /><ref name=":1" /><ref name=davidsonchen/>

Bacterial drug resistance has become an increasingly major health problem. One of the mechanisms for drug resistance is associated with an increase in antibiotic efflux from the bacterial cell. Drug resistance associated with drug efflux, mediated by [[P-glycoprotein]], was originally reported in mammalian cells. In bacteria, Levy and colleagues presented the first evidence that antibiotic resistance was caused by active efflux of a drug.<ref name="pmid7001450">{{cite journal | vauthors = McMurry L, Petrucci RE, Levy SB | title = Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 77 | issue = 7 | pages = 3974–7 | date = Jul 1980 | pmid = 7001450 | pmc = 349750 | doi = 10.1073/pnas.77.7.3974 | bibcode = 1980PNAS...77.3974M | doi-access = free }}</ref> P-glycoprotein is the best-studied efflux pump and as such has offered important insights into the mechanism of bacterial pumps.<ref name=davidson/> Although some exporters transport a specific type of substrate, most transporters extrude a diverse class of drugs with varying structure.<ref name=pohl/>  These transporters are commonly called [[multi-drug resistant]] (MDR) ABC transporters and sometimes referred to as "hydrophobic vacuum cleaners".<ref name=msbachang2003/>

=== Human ABCB1/MDR1 P-glycoprotein ===
{{main|P-glycoprotein}}

P-glycoprotein (3.A.1.201.1) is a well-studied protein associated with multi-drug resistance. It belongs to the human ''ABCB (MDR/TAP)'' family and is also known as ''ABCB1'' or ''MDR1 Pgp''. MDR1 consists of a functional monomer with two transmembrane domains (TMD) and two nucleotide-binding domains (NBD). This protein can transport mainly cationic or electrically neutral substrates as well as a broad spectrum of amphiphilic substrates. The structure of the full-size ABCB1 monomer was obtained in the presence and absence of nucleotide using [[electron crystallography|electron cryo crystallography]]. Without the nucleotide, the TMDs are approximately parallel and form a barrel surrounding a central pore, with the opening facing towards the extracellular side of the membrane and closed at the intracellular face. In the presence of the nonhydrolyzable ATP analog, AMP-PNP, the TMDs have a substantial reorganization with three clearly segregated domains. A central pore, which is enclosed between the TMDs, is slightly open towards the intracellular face with a gap between two domains allowing access of substrate from the lipid phase. Substantial repacking and possible rotation of the TM helices upon nucleotide binding suggests a helix rotation model for the transport mechanism.<ref name=pohl/>

=== Plant transporters ===
The genome of the model plant ''Arabidopsis thaliana'' is capable of encoding 120 ABC proteins compared to 50-70 ABC proteins that are encoded by the human genome and fruit flies (''[[Drosophila melanogaster]]'').  Plant ABC proteins are categorized in 13 subfamilies on the basis of size (full, half or quarter), orientation, and overall amino acid sequence similarity.<ref name="Rea_2007">{{cite journal | vauthors = Rea PA | title = Plant ATP-binding cassette transporters | journal = Annual Review of Plant Biology | volume = 58 | pages = 347–75 | year = 2007 | issue = 1 | pmid = 17263663 | doi = 10.1146/annurev.arplant.57.032905.105406 | bibcode = 2007AnRPB..58..347R }}</ref> Multidrug resistant (MDR) homologs, also known as P-glycoproteins, represent the largest subfamily in plants with 22 members and the second largest overall ABC subfamily. The B subfamily of plant ABC transporters (ABCBs) are characterized by their localization to the plasma membrane.<ref name="Bailly_Yang_2012">{{cite journal | vauthors = Bailly A, Yang H, Martinoia E, Geisler M, Murphy AS | title = Plant Lessons: Exploring ABCB Functionality Through Structural Modeling | journal = Frontiers in Plant Science | volume = 2 | pages = 108 | year = 2011 | pmid = 22639627 | pmc = 3355715 | doi = 10.3389/fpls.2011.00108 | doi-access = free }}</ref> Plant ABCB transporters are characterized by heterologously expressing them in ''Escherichia coli'', ''[[Saccharomyces cerevisiae]]'', ''[[Schizosaccharomyces pombe]]'' (fission yeast), and [[HeLa]] cells to determine substrate specificity. 
Plant ABCB transporters have shown to transport the phytohormone indole-3-acetic acid ( IAA),<ref name="Geisler_Murphy_2006">{{cite journal | vauthors = Geisler M, Murphy AS | title = The ABC of auxin transport: the role of p-glycoproteins in plant development | journal = FEBS Letters | volume = 580 | issue = 4 | pages = 1094–102 | date = Feb 2006 | pmid = 16359667 | doi = 10.1016/j.febslet.2005.11.054 | s2cid = 23368914 | doi-access =  | bibcode = 2006FEBSL.580.1094G }}</ref> also known as [[auxin]], the essential regulator for plant growth and development.<ref name="Yang_Murphy_2009">{{cite journal | vauthors = Yang H, Murphy AS | title = Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe | journal = The Plant Journal | volume = 59 | issue = 1 | pages = 179–91 | date = Jul 2009 | pmid = 19309458 | doi = 10.1111/j.1365-313X.2009.03856.x | doi-access = free }}</ref><ref name="Blakeslee_Peer_2005">{{cite journal | vauthors = Blakeslee JJ, Peer WA, Murphy AS | title = Auxin transport | journal = Current Opinion in Plant Biology | volume = 8 | issue = 5 | pages = 494–500 | date = Oct 2005 | pmid = 16054428 | doi = 10.1016/j.pbi.2005.07.014 | bibcode = 2005COPB....8..494B }}</ref> The directional polar transport of auxin mediates plant environmental responses through processes such as phototropism and gravitropism.<ref name="Kretzschmar_Burla_2011">{{cite journal | vauthors = Kretzschmar T, Burla B, Lee Y, Martinoia E, Nagy R | title = Functions of ABC transporters in plants | journal = Essays in Biochemistry | volume = 50 | issue = 1 | pages = 145–60 | date = Sep 2011 | pmid = 21967056 | doi = 10.1042/bse0500145 | url = https://www.zora.uzh.ch/id/eprint/53838/1/Kretzschmar_EssaysBiochem_2011.pdf }}</ref> Two of the best studied auxin transporters, ABCB1 and ABCB19, have been characterized to be primary auxin exporters<ref name="Yang_Murphy_2009"/> Other ABCB transporters such as ABCB4 participate in both the export and import of auxin<ref name="Yang_Murphy_2009"/> At low intracellular auxin concentrations ABCB4 imports auxin until it reaches a certain threshold which then reverses function to only export auxin.<ref name="Yang_Murphy_2009"/><ref name="KubešYang2012">{{cite journal | vauthors = Kubeš M, Yang H, Richter GL, Cheng Y, Młodzińska E, Wang X, Blakeslee JJ, Carraro N, Petrášek J, Zažímalová E, Hoyerová K, Peer WA, Murphy AS | title = The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis | journal = The Plant Journal | volume = 69 | issue = 4 | pages = 640–54 | date = Feb 2012 | pmid = 21992190 | doi = 10.1111/j.1365-313X.2011.04818.x }}</ref>

=== Sav1866 ===
The first high-resolution structure reported for an ABC exporter was that of Sav1866 (3.A.1.106.2) from ''Staphylococcus aureus''.<ref name=pohl/><ref>{{cite journal | vauthors = Dawson RJ, Locher KP | title = Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP | journal = FEBS Letters | volume = 581 | issue = 5 | pages = 935–8 | date = Mar 2007 | pmid = 17303126 | doi = 10.1016/j.febslet.2007.01.073 | s2cid = 19960736 | url = https://www.dora.lib4ri.ch/psi/islandora/object/psi%3A17836 | doi-access =  | bibcode = 2007FEBSL.581..935D }}</ref> Sav1866 is a homolog of multidrug ABC transporters. It shows significant sequence similarity to human ABC transporters of subfamily B that includes MDR1 and TAP1/TAP2. The ATPase activity of Sav1866 is known to be stimulated by cancer drugs such as [[doxorubicin]], [[vinblastine]] and others,<ref name=velamakanni2008>{{cite journal | vauthors = Velamakanni S, Yao Y, Gutmann DA, van Veen HW | title = Multidrug transport by the ABC transporter Sav1866 from Staphylococcus aureus | journal = Biochemistry | volume = 47 | issue = 35 | pages = 9300–8 | date = Sep 2008 | pmid = 18690712 | doi = 10.1021/bi8006737 }}</ref> which suggests similar substrate specificity to P-glycoprotein and therefore a possible common mechanism of substrate translocation. Sav1866 is a homodimer of half transporters, and each subunit contains an N-terminal TMD with six helices and a C-terminal NBD. The NBDs are similar in structure to those of other ABC transporters, in which the two ATP binding sites are formed at the dimer interface between the Walker A motif of one NBD and the LSGGQ motif of the other. The ADP-bound structure of Sav1866 shows the NBDs in a closed dimer and the TM helices split into two "wings" oriented towards the periplasm, forming the outward-facing conformation. Each wing consists of helices TM1-2 from one subunit and TM3-6 from the other subunit. It contains long intracellular loops (ICLs or ICD) connecting the TMDs that extend beyond the lipid bilayer into the cytoplasm and interacts with the 8=D. Whereas the importers contain a short coupling helix that contact a single NBD, Sav1866 has two intracellular coupling helices, one (ICL1) contacting the NBDs of both subunits and the other (ICL2) interacting with only the opposite NBD subunit.<ref name=rees/><ref name=sav1866/><ref name=oldham2008/>

=== MsbA ===
MsbA (3.A.1.106.1) is a multi-drug resistant (MDR) ABC transporter and possibly a lipid [[flippase]]. It is an [[ATPase]] that transports [[lipid A]], the hydrophobic moiety of [[lipopolysaccharide]] (LPS), a glucosamine-based saccharolipid that makes up the outer monolayer of the outer membranes of most gram-negative bacteria. Lipid A is an [[endotoxin]] and so loss of MsbA from the cell membrane or [[mutations]] that disrupt transport results in the accumulation of lipid A in the inner cell membrane resulting to cell death. It is a close bacterial homolog of P-glycoprotein (Pgp) by protein sequence homology and has overlapping substrate specificities with the MDR-ABC transporter LmrA from ''Lactococcus lactis''.<ref name=msbareuter2003>{{cite journal | vauthors = Reuter G, Janvilisri T, Venter H, Shahi S, Balakrishnan L, van Veen HW | title = The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities | journal = The Journal of Biological Chemistry | volume = 278 | issue = 37 | pages = 35193–8 | date = Sep 2003 | pmid = 12842882 | doi = 10.1074/jbc.M306226200 | doi-access = free }}</ref> MsbA from ''E. coli'' is 36% identical to the NH<sub>2</sub>-terminal half of human MDR1, suggesting a common mechanism for transport of amphiphatic and hydrophobic substrates. The MsbA gene encodes a half transporter that contains a transmembrane domain (TMD) fused with a nucleotide-binding domain (NBD). It is assembled as a homodimer with a total molecular mass of 129.2 kD. MsbA contains 6 TMDs on the periplasmic side, an NBD located on the cytoplasmic side of the cell membrane, and an intracellular domain (ICD), bridging the TMD and NBD. This conserved helix extending from the TMD segments into or near the active site of the NBD is largely responsible for crosstalk between TMD and NBD. In particular, ICD1 serves as a conserved pivot about which the NBD can rotate, therefore allowing the NBD to disassociate and dimerize during ATP binding and hydrolysis.<ref name=davidson/><ref name=davidsonchen/><ref name=pohl/><ref name=rees/><ref name=msbareyes2006/><ref name=oldham2008/><ref name=msbachang2003/><ref name="pmid17362200">{{cite journal | vauthors = Raetz CR, Reynolds CM, Trent MS, Bishop RE | title = Lipid A modification systems in gram-negative bacteria | journal = Annual Review of Biochemistry | volume = 76 | pages = 295–329 | year = 2007 | pmid = 17362200 | pmc = 2569861 | doi = 10.1146/annurev.biochem.76.010307.145803 }}</ref> 
[[Image:Msba.jpg|thumb|Structures of MsbA depicting the three conformational states: open apo ({{PDB|3b5w}}), closed apo ({{PDB|3b5x}}), and nucleotide-bound ({{PDB|3b60}})]]

Previously published (and now retracted) X-ray structures of MsbA were inconsistent with the bacterial homolog Sav1866.<ref name=msbachang2001>{{cite journal | vauthors = Chang G, Roth CB | title = Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters | journal = Science | volume = 293 | issue = 5536 | pages = 1793–800 | date = Sep 2001 | pmid = 11546864 | doi = 10.1126/science.293.5536.1793 | bibcode = 2001Sci...293.1793C }}{{Retracted|doi=10.1126/science.314.5807.1875b|pmid=17185584|intentional=yes}} {{Retracted paper|doi=10.1126/science.314.5807.1875b|intentional=yes}}</ref><ref name=msbareyes2005>{{cite journal | vauthors = Reyes CL, Chang G | title = Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide | journal = Science | volume = 308 | issue = 5724 | pages = 1028–31 | date = May 2005 | pmid = 15890884 | doi = 10.1126/science.1107733 | bibcode = 2005Sci...308.1028R | s2cid = 37250061 }}{{Retracted|doi=10.1126/science.314.5807.1875b|pmid=17185584|intentional=yes}}{{Retracted paper|doi=10.1126/science.314.5807.1875b |intentional=yes}}</ref> The structures were reexamined and found to have an error in the assignment of the hand resulting to incorrect models of MsbA. Recently, the errors have been rectified and new structures have been reported.<ref name=msbaward/> The resting state of ''E. coli'' MsbA exhibits an inverted "V" shape with a chamber accessible to the interior of the transporter suggesting an ''open, inward-facing conformation''. The dimer contacts are concentrated between the extracellular loops and while the NBDs are ≈50Å apart, the subunits are facing each other. The distance between the residues in the site of the dimer interface have been verified by [[cross-link]]ing experiments<ref name="pmid15222771">{{cite journal | vauthors = Buchaklian AH, Funk AL, Klug CS | title = Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling | journal = Biochemistry | volume = 43 | issue = 26 | pages = 8600–6 | date = Jul 2004 | pmid = 15222771 | doi = 10.1021/bi0497751 }}</ref> and [[EPR spectroscopy]] studies.<ref name=dong>{{cite journal | vauthors = Dong J, Yang G, McHaourab HS | title = Structural basis of energy transduction in the transport cycle of MsbA | journal = Science | volume = 308 | issue = 5724 | pages = 1023–8 | date = May 2005 | pmid = 15890883 | doi = 10.1126/science.1106592 | bibcode = 2005Sci...308.1023D | s2cid = 1308350 }}</ref> The relatively large chamber allows it to accommodate large head groups such as that present in lipid A. Significant conformational changes are required to move the large sugar head groups across the membrane. The difference between the two nucleotide-free (apo) structures is the ≈30° pivot of TM4/TM5 helices relative to the TM3/TM6 helices. In the closed apo state (from ''V. cholerae'' MsbA), the NBDs are aligned and although closer, have not formed an ATP sandwich, and the P loops of opposing monomers are positioned next to one another. In comparison to the open conformation, the dimer interface of the TMDs in the ''closed, inward-facing conformation'' has extensive contacts. For both apo conformations of MsbA, the chamber opening is facing inward. The structure of MsbA-AMP-PNP (5'-adenylyl-β-γ-imidodiphosphate), obtained from ''S. typhimurium'', is similar to Sav1866. The NBDs in this ''nucleotide-bound, outward-facing conformation'', come together to form a canonical ATP dimer sandwich, that is, the nucleotide is situated in between the P-loop and LSGGQ motif. The conformational transition from MsbA-closed-apo to MsbA-AMP-PNP involves two steps, which are more likely concerted: a ≈10° pivot of TM4/TM5 helices towards TM3/TM6, bringing the NBDs closer but not into alignment followed by tilting of TM4/TM5 helices ≈20° out of plane. The twisting motion results in the separation of TM3/TM6 helices away from TM1/TM2 leading to a change from an inward- to an outward- facing conformation. Thus, changes in both the orientation and spacing of the NBDs dramatically rearrange the packing of transmembrane helices and effectively switch access to the chamber from the inner to the outer leaflet of the membrane.<ref name=msbaward/> The structures determined for MsbA is basis for the tilting model of transport.<ref name=pohl/> The structures described also highlight the dynamic nature of ABC exporters as also suggested by [[fluorescence]] and EPR studies.<ref name=oldham2008/><ref name=dong/><ref>{{cite journal | vauthors = Borbat PP, Surendhran K, Bortolus M, Zou P, Freed JH, Mchaourab HS | title = Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis | journal = PLOS Biology | volume = 5 | issue = 10 | pages = e271 | date = Oct 2007 | pmid = 17927448 | pmc = 2001213 | doi = 10.1371/journal.pbio.0050271 | doi-access = free }}</ref> Recent work has resulted in the discovery of MsbA inhibitors.<ref>{{cite journal |last1=Zhang |first1=Ge |last2=Baidin |first2=Vadim |last3=Pahil |first3=Karanbir S. |last4=Moison |first4=Eileen |last5=Tomasek |first5=David |last6=Ramadoss |first6=Nitya S. |last7=Chatterjee |first7=Arnab K. |last8=McNamara |first8=Case W. |last9=Young |first9=Travis S. |last10=Schultz |first10=Peter G. |last11=Meredith |first11=Timothy C. |last12=Kahne |first12=Daniel |title=Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors |journal=Proceedings of the National Academy of Sciences |volume=115 |issue=26 |date=7 May 2018 |pages=6834–6839 |doi=10.1073/pnas.1804670115 |pmid=29735709 |pmc=6042065 |bibcode=2018PNAS..115.6834Z |doi-access=free }}</ref><ref>{{cite journal |last1=Ho |first1=Hoangdung |last2=Miu |first2=Anh |last3=Alexander |first3=Mary Kate |last4=Garcia |first4=Natalie K. |last5=Oh |first5=Angela |last6=Zilberleyb |first6=Inna |last7=Reichelt |first7=Mike |last8=Austin |first8=Cary D. |last9=Tam |first9=Christine |last10=Shriver |first10=Stephanie |last11=Hu |first11=Huiyong |last12=Labadie |first12=Sharada S. |last13=Liang |first13=Jun |last14=Wang |first14=Lan |last15=Wang |first15=Jian |last16=Lu |first16=Yan |last17=Purkey |first17=Hans E. |last18=Quinn |first18=John |last19=Franke |first19=Yvonne |last20=Clark |first20=Kevin |last21=Beresini |first21=Maureen H. |last22=Tan |first22=Man-Wah |last23=Sellers |first23=Benjamin D. |last24=Maurer |first24=Till |last25=Koehler |first25=Michael F. T. |last26=Wecksler |first26=Aaron T. |last27=Kiefer |first27=James R. |last28=Verma |first28=Vishal |last29=Xu |first29=Yiming |last30=Nishiyama |first30=Mireille |last31=Payandeh |first31=Jian |last32=Koth |first32=Christopher M. |title=Structural basis for dual-mode inhibition of the ABC transporter MsbA |journal=Nature |date=May 2018 |volume=557 |issue=7704 |pages=196–201 |doi=10.1038/s41586-018-0083-5 |pmid=29720648 |bibcode=2018Natur.557..196H |s2cid=13660653 }}</ref>

=== Mechanism of transport for exporters ===
[[Image:Abc exporter.jpg|thumb|left|Proposed mechanism of transport for ABC exporters. This model was based on structural and biochemical studies on MsbA.]]

ABC exporters have a transport mechanism that is consistent with both the alternating-access model and ATP-switch model. In the apo states of exporters, the conformation is inward-facing and the TMDs and NBDs are relatively far apart to accommodate amphiphilic or hydrophobic substrates. For MsbA, in particular, the size of the chamber is large enough to accommodate the sugar groups from lipopolysaccharides (LPS). As has been suggested by several groups, binding of substrate initiates the transport cycle. The "power stroke", that is, ATP binding that induces NBD dimerization and formation of the ATP sandwich, drives the conformational changes in the TMDs. In MsbA, the sugar head groups are sequestered within the chamber during the "power stroke". The cavity is lined with charged and polar residues that are likely solvated creating an energetically unfavorable environment for hydrophobic substrates and energetically favorable for polar moieties in amphiphilic compounds or sugar groups from LPS. Since the lipid cannot be stable for a long time in the chamber environment, lipid A and other hydrophobic molecules may "flip" into an energetically more favorable position within the outer membrane leaflet. The "flipping" may also be driven by the rigid-body shearing of the TMDs while the hydrophobic tails of the LPS are dragged through the lipid bilayer. Repacking of the helices switches the conformation into an outward-facing state. ATP hydrolysis may widen the periplasmic opening and push the substrate towards the outer leaflet of the lipid bilayer. Hydrolysis of the second ATP molecule and release of P<sub>i</sub> separates the NBDs followed by restoration of the resting state, opening the chamber towards the cytoplasm for another cycle.<ref name=msbaward/><ref name=msbareyes2006/><ref name=higgins/><ref name=msbachang2003/><ref name=dong/><ref name=gutmann2010>{{cite journal | vauthors = Gutmann DA, Ward A, Urbatsch IL, Chang G, van Veen HW | title = Understanding polyspecificity of multidrug ABC transporters: closing in on the gaps in ABCB1 | journal = Trends in Biochemical Sciences | volume = 35 | issue = 1 | pages = 36–42 | date = Jan 2010 | pmid = 19819701 | doi = 10.1016/j.tibs.2009.07.009 | pmc = 4608440 }}</ref>