Catalytic and noncatalytic nucleotide binding sites of theEscherichia coliF1ATPase Amino acid sequences of β-subunit tryptic peptides labeled with 2-azido-ATP

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5289 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5289-5297 ◽

Cited By ~ 219

Author(s):

M Azzaria ◽

E Schurr ◽

P Gros

Keyword(s):

Drug Resistance ◽

Biological Activity ◽

Multidrug Resistance ◽

Amino Acid ◽

Binding Sites ◽

Consensus Sequence ◽

Nucleotide Binding ◽

Atp Binding ◽

Nucleotide Binding Sites ◽

The Core

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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Mammalian multidrug-resistance gene: correlation of exon organization with structural domains and duplication of an ancestral gene

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.17.6488 ◽

1989 ◽

Vol 86 (17) ◽

pp. 6488-6492 ◽

Cited By ~ 35

Author(s):

M Raymond ◽

P Gros

Keyword(s):

Sequence Homology ◽

Binding Sites ◽

Nucleotide Binding ◽

Amino Acid Sequences ◽

Transmembrane Domains ◽

Biologically Active ◽

Mdr1 Gene ◽

Structural Domains ◽

Ancestral Gene ◽

Nucleotide Binding Sites

Analysis of the nucleotide and deduced amino acid sequences of the biologically active mouse mdr1 cDNA clone indicates that the protein is formed by two highly homologous halves, each containing six putative transmembrane domains and a nucleotide-binding site. The duplicated unit shows high sequence homology to the proposed energy-coupling subunit of bacterial periplasmic transport proteins. We have cloned and characterized the mouse mdr1 gene and have analyzed the genomic organization of the two homologous halves forming the mdr1 protein. The gene spans 68 kilobases, is split into 28 exons, and the two homologous halves are encoded by 14 and 13 exons. The transcriptional initiation site of the gene has been mapped and putative TATA and consensus CAAT sequences have been found at positions -27 and -83, respectively. Discrete structural domains of the mdr1 protein are encoded by separate exons: Ten of the 12 putative transmembrane domains are encoded by individual exons and the two nucleotide-binding sites are each encoded by three exons. The exon/intron organization of the gene is conserved in the two highly homologous regions encoding the nucleotide-binding sites. The conservation of certain pairs of introns, together with the high degree of sequence homology, indicate that the mouse mdr1 gene originated from the duplication of an intron-containing ancestral gene.

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