Transition-state formation in ATPase-negative mutants of human MDR1 protein.
In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Both mutations have been demonstrated to cause a loss of drug transport, drug-stimulated ATPase, and vanadate-dependent nucleotide trapping activity. Here we show that these mutants still allow transition state formation (nucleotide trapping) when fluoro-aluminate or beryllium fluoride is used as a complex-stabilizing anion. Drug stimulation of nucleotide trapping was found to be preserved in both mutants. Limited trypsin digestion revealed that whenever MDR1-nucleotide trapping occurred, both ABC domains were involved in the formation of the catalytic intermediates. Our results show that details of the MDR1-ATPase cycle can be studied even in ATPase-negative mutants. These data also demonstrate that the conformational alteration caused by a mutation in one of the ABC domains is propagated to the other, nonmutated domain, indicating a tight coupling between the functioning of the two ABC domains.
Szakács
G
G
National Institute of Haematology and Immunology, Membrane Research Group, Hungarian Academy of Sciences, Daróczi ut 24, Budapest, H-1113, Hungary.
Ozvegy
C
C
Bakos
E
E
Sarkadi
B
B
Váradi
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000535:Aluminum / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001386:Azides / Q000378:metabolism; D001608:Beryllium / Q000378:metabolism; D001692:Biological Transport; D017124:Conserved Sequence / Q000235:genetics; D005459:Fluorides / Q000378:metabolism; D005461:Fluorine / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; C053629:fluoroaluminum; D005461:Fluorine; D014638:Vanadates; C035982:beryllium fluoride; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000535:Aluminum; D014357:Trypsin; D000251:Adenosine Triphosphatases; D001608:Beryllium; D005459:Fluorides