Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state.
Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.
Zhang
Da-Wei
DW
Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Gu
Hong-Mei
HM
Situ
Donna
D
Haimeur
Anass
A
Cole
Susan P C
SP
Deeley
Roger G
RG
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000838:Anions; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004351:Drug Resistance; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000596:Amino Acids; D000838:Anions; D001216:Asparagine; D001224:Aspartic Acid; D004958:Estradiol; D017997:Leukotriene C4; D008239:Lysine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012694:Serine; C025483:estradiol-17 beta-glucuronide; C421109:multidrug resistance-associated protein 1