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===ATP Bound and Unbound Conformations===
===ATP Bound and Unbound Conformations===
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As an [https://en.wikipedia.org/wiki/ATP-binding_cassette_transporter ABC Transporter], ABCG2 exhibits ATPase activity by using the energy of ATP hydrolysis to facilitate transport. After substrates bind in the TMD, one molecule of <scene name='83/832932/Atp_bound_use2/3'>ATP binds each NBD</scene> (2 molecules of ATP total) causing a conformational change of the overall structure from an <scene name='83/832932/Overall_use_2/3'>inward-facing conformation</scene> to an <scene name='83/832932/Outward_facing_conformation/4'>outward-facing conformation</scene>. ATP coordinates with various residues and a magnesium ion in the <scene name='83/832932/Atp_bound_in_nbd/2'>binding site of each NBD</scene>. One molecule of ATP is hydrolyzed to transport substrates across the cell membrane while the second molecule of ATP is hydrolyzed to reset the transporter to its inward-facing conformation.<ref name="Robey"/>
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As an [https://en.wikipedia.org/wiki/ATP-binding_cassette_transporter ABC Transporter], ABCG2 exhibits ATPase activity by using the energy of ATP hydrolysis to facilitate transport. After substrates bind in the TMD, one molecule of <scene name='83/832932/Atp_bound_use2/3'>ATP binds each NBD</scene> (2 molecules of ATP total) causing a conformational change of the overall structure from an <scene name='83/832932/Overall_use_2/3'>inward-facing conformation</scene> to an <scene name='83/832932/Outward_facing_conformation/4'>outward-facing conformation</scene>. ATP coordinates with various residues and a magnesium ion in the <scene name='83/832932/Atp_bound_in_nbd/2'>binding site of each NBD</scene> which is bordered with [https://en.wikipedia.org/wiki/Walker_motifs Walker A and B motifs]. One molecule of ATP is hydrolyzed to transport substrates across the cell membrane while the second molecule of ATP is hydrolyzed to reset the transporter to its inward-facing conformation.<ref name="Robey"/>
When ATP binds, α-helices in the NBD <scene name='83/832932/Atp_bound_nbd/3'>rotate</scene> approximately 35° relative to the <scene name='83/832932/Overall_structure_nbd_unbound/5'>inward-facing conformation of NBD</scene>. This shift in the NBD causes slight shifts of α-helices in the TMD; these helices are <scene name='83/832932/Atp_bound_use_tmd/4'>pushed toward each other</scene> relative to the <scene name='83/832932/Overall_structure_tmd_unbound/4'>inward-facing conformation of TMD</scene>. The overall shift from inward-facing to outward-facing promotes the transport of substrates through the transporter.<ref name="Manolaridis"/>
When ATP binds, α-helices in the NBD <scene name='83/832932/Atp_bound_nbd/3'>rotate</scene> approximately 35° relative to the <scene name='83/832932/Overall_structure_nbd_unbound/5'>inward-facing conformation of NBD</scene>. This shift in the NBD causes slight shifts of α-helices in the TMD; these helices are <scene name='83/832932/Atp_bound_use_tmd/4'>pushed toward each other</scene> relative to the <scene name='83/832932/Overall_structure_tmd_unbound/4'>inward-facing conformation of TMD</scene>. The overall shift from inward-facing to outward-facing promotes the transport of substrates through the transporter.<ref name="Manolaridis"/>
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===Cavities and Leucine Plug===
===Cavities and Leucine Plug===
[[Image:Cavities_and_leucine_plug_abcg2.png|250 px|right|thumb|Figure 2. Locations of Cavities 1 and 2 and the Leucine Plug in ABCG2. The protein is in the inward-facing conformation with Cavity 1 open to the cytosol for substrate recruitment, the Leucine Plug is intact, and Cavity 2 is completely occluded. [https://www.rcsb.org/structure/5NJ3 (5NJ3)]]]
[[Image:Cavities_and_leucine_plug_abcg2.png|250 px|right|thumb|Figure 2. Locations of Cavities 1 and 2 and the Leucine Plug in ABCG2. The protein is in the inward-facing conformation with Cavity 1 open to the cytosol for substrate recruitment, the Leucine Plug is intact, and Cavity 2 is completely occluded. [https://www.rcsb.org/structure/5NJ3 (5NJ3)]]]
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Substrates are transported through ABCG2 via two cavities separated by a leucine plug (Figure 2). <scene name='83/832932/Cavity_1_zoom_out/2'>Cavity 1</scene> acts as a multidrug binding pocket and is formed by helices at the interface of the monomers in the TMD. When ATP is not bound to the NBDs, Cavity 1 is <scene name='83/832932/Overall_structure_cavity_1hel/3'>open to the cytosol</scene> in order to recruit substrates for transport. Cavity 1 is <scene name='83/832932/Cavity_1_narrow_surface/4'>narrow</scene> and full of nonpolar, hydrophobic residues and, as a result, prefers nonpolar, hydrophobic substrates, particularly flat, polycyclic molecules. Substrates, such as estrone sulfate, <scene name='83/832932/Cavity_1_-_use2/4'>form hydrogen bonds and stacking interactions</scene> with residues from each subunit in Cavity 1.<ref name="Taylor"/>
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Substrates are transported through ABCG2 via two cavities separated by a leucine plug (Figure 2). <scene name='83/832932/Highlight_cavity_1/3'>Cavity 1</scene> acts as a multidrug binding pocket and is formed by helices at the interface of the monomers in the TMD. When ATP is not bound to the NBDs, Cavity 1 is <scene name='83/832932/Overall_structure_cavity_1hel/4'>open to the cytosol</scene> in order to recruit substrates for transport. Cavity 1 is <scene name='83/832932/Cavity_1_narrow_surface/4'>narrow</scene> and full of nonpolar, hydrophobic residues and, as a result, prefers nonpolar, hydrophobic substrates, particularly flat, polycyclic molecules. Substrates, such as estrone sulfate, <scene name='83/832932/Cavity_1_-_use2/4'>form hydrogen bonds and stacking interactions</scene> with residues from each subunit in Cavity 1.<ref name="Taylor"/>
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After substrates bind in Cavity 1, ATP binds each NBD leading to the transporter shifting from inward-facing to outward-facing. The outward-facing conformation results in the <scene name='83/832932/Atp_bound_cavity_2/3'>collapse of Cavity 1</scene> in the TMD in which the cavity is no longer <scene name='83/832932/Overall_structure_cavity_1hel/3'>open to the cytosol</scene>. This collapse forces the substrate to move forward to Cavity 2 as there is no longer room in Cavity 1 to accommodate substrates.<ref name="Manolaridis"/> <scene name='83/832932/Atp_bound_use_cav_2/2'>Cavity 2</scene>, which is occluded when the protein in is the inward-facing conformation, is now open to the extracellular space and able to release the substrate. Cavity 2 contains a less hydrophobic environment and, as a result, substrates are released due to hydrophobic mismatch.<ref name="Taylor"/> <scene name='83/832932/Atp_bound_use_el_disulfides/3'>Disulfide bonds</scene> in the external loops near the exit of Cavity 2 also help promote substrate release.<ref name="Manolaridis"/> Once Cavity 2 is empty, the protein reverts to the inward-facing conformation via hydrolysis of ATP.
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After substrates bind in Cavity 1, ATP binds each NBD leading to the transporter shifting from inward-facing to outward-facing. The outward-facing conformation results in the <scene name='83/832932/Atp_bound_cavity_2/3'>collapse of Cavity 1</scene> in the TMD in which the cavity is no longer <scene name='83/832932/Overall_structure_cavity_1hel/4'>open to the cytosol</scene>. This collapse forces the substrate to move forward to Cavity 2 as there is no longer room in Cavity 1 to accommodate substrates.<ref name="Manolaridis"/> <scene name='83/832932/Atp_bound_use_cav_2/3'>Cavity 2</scene>, which is occluded when the protein in is the inward-facing conformation, is now open to the extracellular space and able to release the substrate. Cavity 2 contains a less hydrophobic environment and, as a result, substrates are released due to hydrophobic mismatch.<ref name="Taylor"/> <scene name='83/832932/Atp_bound_use_el_disulfides/3'>Disulfide bonds</scene> in the external loops near the exit of Cavity 2 also help promote substrate release.<ref name="Manolaridis"/> Once Cavity 2 is empty, the protein reverts to the inward-facing conformation via hydrolysis of ATP.
Cavities 1 and 2 are separated by a <scene name='83/832932/Leucine_plug_open_con/7'>leucine plug</scene> which likely acts as a substrate check-point during transport; changes to either of these leucine residues have exhibited an increase in transport and a decrease in substrate specificity.<ref name="Manolaridis"/> After the substrate binds Cavity 1 and ATP molecules bind each NBD, the <scene name='83/832932/Atp_bound_cavsleu/5'>leucine plug opens</scene> to allow the substrate to enter Cavity 2. Once the substrate enters Cavity 2, the plug is able to reform and promote substrate release and conversion to the inward-facing conformation.
Cavities 1 and 2 are separated by a <scene name='83/832932/Leucine_plug_open_con/7'>leucine plug</scene> which likely acts as a substrate check-point during transport; changes to either of these leucine residues have exhibited an increase in transport and a decrease in substrate specificity.<ref name="Manolaridis"/> After the substrate binds Cavity 1 and ATP molecules bind each NBD, the <scene name='83/832932/Atp_bound_cavsleu/5'>leucine plug opens</scene> to allow the substrate to enter Cavity 2. Once the substrate enters Cavity 2, the plug is able to reform and promote substrate release and conversion to the inward-facing conformation.
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Due to the potential for ABCG2 inhibition to aid in cancer treatment, efforts have been made to develop specific inhibitors of ABCG2 and other ABC transporters. The ABC transporter ABCB1, also known as multidrug resistance 1 (MDR1), was a therapeutic target in previous studies which produced three generations of MDR1 inhibitors, such as [https://en.wikipedia.org/wiki/Verapamil verapamil], [https://en.wikipedia.org/wiki/Valspodar valspodar], and [https://en.wikipedia.org/wiki/Zosuquidar zosuquidar]; however, many of these inhibitors had neurotoxic effects that discouraged their use in cancer treatment.<ref name="Leonard"/><ref name="Binkhathlan"/><ref name="Witherspoon"/>
Due to the potential for ABCG2 inhibition to aid in cancer treatment, efforts have been made to develop specific inhibitors of ABCG2 and other ABC transporters. The ABC transporter ABCB1, also known as multidrug resistance 1 (MDR1), was a therapeutic target in previous studies which produced three generations of MDR1 inhibitors, such as [https://en.wikipedia.org/wiki/Verapamil verapamil], [https://en.wikipedia.org/wiki/Valspodar valspodar], and [https://en.wikipedia.org/wiki/Zosuquidar zosuquidar]; however, many of these inhibitors had neurotoxic effects that discouraged their use in cancer treatment.<ref name="Leonard"/><ref name="Binkhathlan"/><ref name="Witherspoon"/>
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While ABC transporter inhibition was dismissed after failed clinical trials, interest in revisiting ABC inhibition has reemerged due to new developments made in recent years.<ref name="Robey"/> Kol143 (Figure 3) is a compound derived from [https://en.wikipedia.org/wiki/Fumitremorgin fungal toxin fumitremorgin C] (FTC), a selective inhibitor of ABCG2 which exhibits undesirable neurotoxic effects.<ref name="Allen"/> Kol143 was found to be less toxic and more potent than FTC; however, this inhibitor is nonselective toward ABCG2.<ref name="Weidner"/> Various inhibitors were derived from Kol143 with changes made at positions 1 and 2 in Figure 3, which are carbons 3 and 9 respectively. Modifications at these positions prove to affect the inhibitory capacities of compounds. A promising compound which has shown a high degree of potency is the inhibitor MZ29 (Figure 3).<ref name="Jackson"/>
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While ABC transporter inhibition was dismissed after failed clinical trials, interest in revisiting ABC inhibition has reemerged due to new developments made in recent years.<ref name="Robey"/> For instance, Kol143 (Figure 3) is a compound derived from fungal toxin [https://en.wikipedia.org/wiki/Fumitremorgin fumitremorgin C] (FTC), a selective inhibitor of ABCG2 which exhibits undesirable neurotoxic effects.<ref name="Allen"/> Kol143 was found to be less toxic and more potent than FTC; however, this inhibitor is nonselective toward ABCG2.<ref name="Weidner"/> Various inhibitors were derived from Kol143 with changes made at positions 1 and 2 in Figure 3, which are carbons 3 and 9 respectively. Modifications at these positions prove to affect the inhibitory capacities of compounds. A promising compound which has shown a high degree of potency is the inhibitor MZ29 (Figure 3).<ref name="Jackson"/>
ABCG2 inhibitors, <scene name='83/832932/Inhibitor_bound_cavity_1/2'>such as MZ29</scene>, bind Cavity 1 and act as competitive inhibitors against ABCG2 substrates and show a higher affinity toward the transporter. Depending on the size of the inhibitor, one or two molecules can accommodate binding to the cavity and form <scene name='83/832932/Inhibitor_interactions_cavity1/3'>hydrogen bonds, van der Waals, and stacking interactions</scene> within the binding site.<ref name="Jackson"/> Many inhibitors are too big to be transported via the leucine plug resulting in the "clogging" of the transporter. With inhibitors acting as wedges, ABCG2 is locked in the inward-facing conformation and unable to transport molecules out of the cell.<ref name="Manolaridis"/>
ABCG2 inhibitors, <scene name='83/832932/Inhibitor_bound_cavity_1/2'>such as MZ29</scene>, bind Cavity 1 and act as competitive inhibitors against ABCG2 substrates and show a higher affinity toward the transporter. Depending on the size of the inhibitor, one or two molecules can accommodate binding to the cavity and form <scene name='83/832932/Inhibitor_interactions_cavity1/3'>hydrogen bonds, van der Waals, and stacking interactions</scene> within the binding site.<ref name="Jackson"/> Many inhibitors are too big to be transported via the leucine plug resulting in the "clogging" of the transporter. With inhibitors acting as wedges, ABCG2 is locked in the inward-facing conformation and unable to transport molecules out of the cell.<ref name="Manolaridis"/>

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This Sandbox is Reserved from Jan 13 through September 1, 2020 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1598 through Sandbox Reserved 1627.
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ABCG2 Multidrug Transporter

ABCG2 Multidrug Transporter. Green represents residues in monomer A; Purple represents residues in monomer B. Blue is used to highlight areas of interest in select scenes. (PDB Codes: 5NJ3 6HBU 6HCO 6FFC)

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References

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

  1. ↑ 1.0 1.1 1.2 1.3 1.4 Taylor NMI, Manolaridis I, Jackson SM, Kowal J, Stahlberg H, Locher KP. Structure of the human multidrug transporter ABCG2. Nature. 2017 Jun 22;546(7659):504-509. doi: 10.1038/nature22345. Epub 2017 May, 29. PMID:28554189 doi:http://dx.doi.org/10.1038/nature22345
  2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Manolaridis I, Jackson SM, Taylor NMI, Kowal J, Stahlberg H, Locher KP. Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states. Nature. 2018 Nov;563(7731):426-430. doi: 10.1038/s41586-018-0680-3. Epub 2018 Nov, 7. PMID:30405239 doi:http://dx.doi.org/10.1038/s41586-018-0680-3
  3. ↑ 3.0 3.1 3.2 Robey RW, Pluchino KM, Hall MD, Fojo AT, Bates SE, Gottesman MM. Revisiting the role of ABC transporters in multidrug-resistant cancer. Nat Rev Cancer. 2018 Jul;18(7):452-464. doi: 10.1038/s41568-018-0005-8. PMID:29643473 doi:http://dx.doi.org/10.1038/s41568-018-0005-8
  4. ↑ 4.0 4.1 4.2 4.3 Jackson SM, Manolaridis I, Kowal J, Zechner M, Taylor NMI, Bause M, Bauer S, Bartholomaeus R, Bernhardt G, Koenig B, Buschauer A, Stahlberg H, Altmann KH, Locher KP. Structural basis of small-molecule inhibition of human multidrug transporter ABCG2. Nat Struct Mol Biol. 2018 Apr;25(4):333-340. doi: 10.1038/s41594-018-0049-1. Epub, 2018 Apr 2. PMID:29610494 doi:http://dx.doi.org/10.1038/s41594-018-0049-1
  5. ↑ 5.0 5.1 Marzac C, Garrido E, Tang R, Fava F, Hirsch P, De Benedictis C, Corre E, Lapusan S, Lallemand JY, Marie JP, Jacquet E, Legrand O. ATP Binding Cassette transporters associated with chemoresistance: transcriptional profiling in extreme cohorts and their prognostic impact in a cohort of 281 acute myeloid leukemia patients. Haematologica. 2011 Sep;96(9):1293-301. doi: 10.3324/haematol.2010.031823. Epub, 2011 May 23. PMID:21606172 doi:http://dx.doi.org/10.3324/haematol.2010.031823
  6. ↑ 6.0 6.1 Bartholomae S, Gruhn B, Debatin KM, Zimmermann M, Creutzig U, Reinhardt D, Steinbach D. Coexpression of Multiple ABC-Transporters is Strongly Associated with Treatment Response in Childhood Acute Myeloid Leukemia. Pediatr Blood Cancer. 2016 Feb;63(2):242-7. doi: 10.1002/pbc.25785. Epub 2015 Oct, 29. PMID:26512967 doi:http://dx.doi.org/10.1002/pbc.25785
  7. ↑ 7.0 7.1 Mohelnikova-Duchonova B, Brynychova V, Oliverius M, Honsova E, Kala Z, Muckova K, Soucek P. Differences in transcript levels of ABC transporters between pancreatic adenocarcinoma and nonneoplastic tissues. Pancreas. 2013 May;42(4):707-16. doi: 10.1097/MPA.0b013e318279b861. PMID:23462326 doi:http://dx.doi.org/10.1097/MPA.0b013e318279b861
  8. ↑ 8.0 8.1 Mao Q, Unadkat JD. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update. AAPS J. 2015 Jan;17(1):65-82. doi: 10.1208/s12248-014-9668-6. Epub 2014 Sep 19. PMID:25236865 doi:http://dx.doi.org/10.1208/s12248-014-9668-6
  9. ↑ 9.0 9.1 Leonard GD, Fojo T, Bates SE. The role of ABC transporters in clinical practice. Oncologist. 2003;8(5):411-24. doi: 10.1634/theoncologist.8-5-411. PMID:14530494 doi:http://dx.doi.org/10.1634/theoncologist.8-5-411
  10. ↑ 10.0 10.1 Binkhathlan Z, Lavasanifar A. P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives. Curr Cancer Drug Targets. 2013 Mar;13(3):326-46. doi:, 10.2174/15680096113139990076. PMID:23369096 doi:http://dx.doi.org/10.2174/15680096113139990076
  11. ↑ 11.0 11.1 Witherspoon SM, Emerson DL, Kerr BM, Lloyd TL, Dalton WS, Wissel PS. Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918. Clin Cancer Res. 1996 Jan;2(1):7-12. PMID:9816083
  12. ↑ 12.0 12.1 Allen JD, van Loevezijn A, Lakhai JM, van der Valk M, van Tellingen O, Reid G, Schellens JH, Koomen GJ, Schinkel AH. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther. 2002 Apr;1(6):417-25. PMID:12477054
  13. ↑ 13.0 13.1 Weidner LD, Zoghbi SS, Lu S, Shukla S, Ambudkar SV, Pike VW, Mulder J, Gottesman MM, Innis RB, Hall MD. The Inhibitor Ko143 Is Not Specific for ABCG2. J Pharmacol Exp Ther. 2015 Sep;354(3):384-93. doi: 10.1124/jpet.115.225482. Epub , 2015 Jul 6. PMID:26148857 doi:http://dx.doi.org/10.1124/jpet.115.225482

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