The role of the conserved glycines of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers

Zsófia Szentpétery, András Kern, K. Liliom, B. Sarkadi, A. Váradi, E. Bakos

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Abstract

A key element of the structural model of ABC-ATPases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the γ-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide- as well as the transported substrate-protein interactions were studied. We found that these transport- and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide + oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.

Original languageEnglish
Pages (from-to)41670-41678
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number40
DOIs
Publication statusPublished - Oct 1 2004

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Glycine
Nucleotides
Adenosine Triphosphate
Binding Sites
Communication
Adenosine Triphosphatases
Substrates
Sf9 Cells
ATP-Binding Cassette Transporters
Vanadates
Glutathione Disulfide
Structural Models
Etoposide
Mutant Proteins
Aspartic Acid
Insects
Catalytic Domain
Proteins
Phosphates
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "The role of the conserved glycines of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers",
abstract = "A key element of the structural model of ABC-ATPases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the γ-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide- as well as the transported substrate-protein interactions were studied. We found that these transport- and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide + oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.",
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T1 - The role of the conserved glycines of ATP-binding cassette signature motifs of MRP1 in the communication between the substrate-binding site and the catalytic centers

AU - Szentpétery, Zsófia

AU - Kern, András

AU - Liliom, K.

AU - Sarkadi, B.

AU - Váradi, A.

AU - Bakos, E.

PY - 2004/10/1

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N2 - A key element of the structural model of ABC-ATPases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the γ-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide- as well as the transported substrate-protein interactions were studied. We found that these transport- and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide + oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.

AB - A key element of the structural model of ABC-ATPases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the γ-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide- as well as the transported substrate-protein interactions were studied. We found that these transport- and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide + oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.

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