Major Photoaffinity Drug Binding Sites in Multidrug Resistance Protein 1 (MRP1) Are within Transmembrane Domains 10–11 and 16–17

MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin orStaphylococcus aureusV8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides (∼6–7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser542-Arg593) and transmembranes 16 and 17 of MSD2 (Cys1205-Glu1253). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.

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Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.022049799 ◽

2002 ◽

Vol 99 (6) ◽

pp. 3511-3516 ◽

Cited By ~ 51

Author(s):

Tip W. Loo ◽

David M. Clarke

Keyword(s):

Multidrug Resistance ◽

Binding Site ◽

Binding Sites ◽

Drug Binding ◽

Atp Binding ◽

Drug Binding Site ◽

P Glycoprotein

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Defining the Drug-binding Site in the Human Multidrug Resistance P-glycoprotein Using a Methanethiosulfonate Analog of Verapamil, MTS-verapamil

Journal of Biological Chemistry ◽

10.1074/jbc.m100407200 ◽

2001 ◽

Vol 276 (18) ◽

pp. 14972-14979 ◽

Cited By ~ 132

Author(s):

Tip W. Loo ◽

David M. Clarke

Keyword(s):

Multidrug Resistance ◽

Binding Site ◽

Drug Binding ◽

Drug Binding Site ◽

P Glycoprotein

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The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression

International Journal of Molecular Sciences ◽

10.3390/ijms22189710 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9710

Author(s):

Gwenaëlle Conseil ◽

Susan P. C. Cole

Keyword(s):

Amino Acids ◽

Multidrug Resistance ◽

Core Structure ◽

Multidrug Resistance Protein ◽

Cytoplasmic Loop ◽

Multidrug Resistance Protein 1 ◽

Resistance Protein ◽

Membrane Spanning ◽

Α Helix ◽

Membrane Spanning Domains

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly392, Tyr404, Arg405), the perpendicular coupling helix (Asn412, Arg415, Lys416), and the ascending α-helix (Glu422, Phe434) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu422 and Arg615 could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.

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Role of the NH2-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e04-12-1113 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2483-2492 ◽

Cited By ~ 52

Author(s):

Christopher J. Westlake ◽

Susan P.C. Cole ◽

Roger G. Deeley

Keyword(s):

Multidrug Resistance ◽

Terminal Region ◽

Multidrug Resistance Protein ◽

Multidrug Resistance Protein 1 ◽

Recycling Endosomes ◽

Protein Fragments ◽

Resistance Protein ◽

Membrane Spanning ◽

Membrane Spanning Domains

Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that independent of cell type, the truncated protein accumulates in early/recycling endosomes. Using a real-time internalization assay, we demonstrate that MSD0 is important for MRP1 retention in, or recycling to, the plasma membrane. We also show that MSD0 traffics independently to the cell surface and promotes membrane localization of the core-region of MRP1 when the two protein fragments are coexpressed. Finally, we demonstrate that MSD0 becomes essential for trafficking of MRP1 when the COOH-terminal region of the protein is mutated. These studies demonstrate that MSD0 and the COOH-terminal region contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated. These data explain apparent differences in the trafficking requirement for MSD0 and the COOH-terminal region of MRP1 compared with other ABCC proteins.

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Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082072 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1793-C1793

Author(s):

Paul Rowland ◽

Onkar SINGH ◽

Leila Ross ◽

Francisco Gamo ◽

Maria Lafuente-Monasterio ◽

...

Keyword(s):

Drug Resistance ◽

Binding Site ◽

Crystal Structures ◽

Point Mutations ◽

Crystal Form ◽

Drug Binding ◽

Binding Modes ◽

Resistance Mutations ◽

Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

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All-Atomic Molecular Dynamic Studies of Human and Drosophila CDK8: Insights into Their Kinase Domains, the LXXLL Motifs, and Drug Binding Site

International Journal of Molecular Sciences ◽

10.3390/ijms21207511 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7511

Author(s):

Wu Xu ◽

Xiao-Jun Xie ◽

Ali K. Faust ◽

Mengmeng Liu ◽

Xiao Li ◽

...

Keyword(s):

Hydrogen Bond ◽

Hydrogen Bonds ◽

Binding Site ◽

Molecular Dynamic ◽

Kinase Activity ◽

Structural Difference ◽

Drug Binding ◽

Local Structures ◽

Drug Binding Site ◽

Lxxll Motif

Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

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Mutations in Plasmodium falciparumCytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2100-2108.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2100-2108 ◽

Cited By ~ 232

Author(s):

Michael Korsinczky ◽

Nanhua Chen ◽

Barbara Kotecka ◽

Allan Saul ◽

Karl Rieckmann ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Point Mutations ◽

Drug Binding ◽

Single Amino Acid ◽

Malaria Parasites ◽

Drug Binding Site ◽

Sole Agent ◽

Amino Acid Mutations

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYTbc 1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

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