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.