ABCMdb

PMID: 11027628

Szakacs G, Ozvegy C, Bakos E, Sarkadi B, Varadi A
Transition-state formation in ATPase-negative mutants of human MDR1 protein.
Biochem Biophys Res Commun. 2000 Oct 5;276(3):1314-9., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met Transition-State Formation in ATPase-Negative Mutants of Human MDR1 Protein Gergely Szaka´cs,* Csilla O¨ zvegy,† E´ va Bakos,*, † Bala´zs Sarkadi,* and Andra´s Va´radi†,1 *National Institute of Haematology and Immunology, Membrane Research Group, Hungarian Academy of Sciences, Daro´czi ut 24, H-1113 Budapest, Hungary; and †Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary Received August 26, 2000 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. Login to comment 26 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met We have shown earlier that the replacement of a lysine residue with methionine in the catalytic Walker A motif in either ABC domains of the human MDR1 (K433M and K1076M) eliminated drug-stimulated ATPase activity, while these mutants could still bind ATP [13]. Login to comment 31 ABCB1 p.Lys433Met Recombinant baculovirus transfer vectors carrying wild-type and mutant MDR1 variants (K433M andK1076M) were constructed earlier [13]. Login to comment 59 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met The arrow indicates the position of human MDR1 on the blot. Nucleotide Trapping in the Walker A Mutants in the Presence of AlF4 and BeFx: Effects of Drugs Our earlier results showed that the replacement of lysine with methionine in either of the Nor C-terminal Walker A sequences (mutations K433M and K1076M) resulted in the loss of ATPase activity [13], although these mutations did not interfere with MgATP-binding. Login to comment 64 ABCB1 p.Lys433Met As shown in Fig. 2, both K433M- MDR1 and K10756M-MDR1 was labeled by [␣-32 P]-8-azido-Mg-ATP, when the transition state was stabilized by AlF4 or BeFx. Login to comment 78 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met Labeling was performed by the incubation of 100 ␮g of Sf9 cell membranes expressing K433M MDR1 (lanes 1, 2 and 5, 6) or K1076M MDR1 (lanes 3, 4 and 7, 8), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2, as described under Materials and Methods. Login to comment 82 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met Labeling was performed by the incubation of 200 ␮g of Sf9 cell membranes expressing wild-type MDR1 (lanes 1 and 2), K433M MDR1 (lanes 3 and 4), K1076M MDR1 (lanes 5 and 6), at 37°C for 10 min in the presence of 20 ␮M [␣-32 P]-8-azido-ATP and 2 mM MgCl2. Login to comment 85 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met The arrows indicate the position of human MDR1 and the tryptic fragments containing the N-terminal and C-terminal halves, as revealed by immunostaining of the same the blot. by AlF4 are formed in both ABC domains of the wild-type, as well as of the K433M and the K1076M mutant variants (reactions with BeFx gave similar results, not shown). Login to comment 89 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met We have replaced a key lysine of the Walker A motif (GCGKST in both ABC domains) to methionine (K433M and K1076M). Login to comment 108 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met Drug stimulation of the rate of formation of the occluded nucleotide transitory complexes was preserved in the Walker A mutants, K433M and K1076M (Fig. 2). Login to comment 109 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met In the double mutant, K433M/K1076M, no nucleotide trapping was detected, regardless of the choice of the stabilizing anion (not shown). Login to comment 122 ABCB1 p.Lys433Met As shown earlier, the K to M mutation in the C-terminal ABC domain prevented both halves from vanadate induced trapping (a weak labeling of the N-terminal Walker A mutant (K433M) was seen under nucleotide occlusion conditions in the presence of vanadate; we could not detect this relatively weak label in our previous work [9]). Login to comment 132 ABCB1 p.Lys1076Met However, the results of this study show that a unilateral mutation (K1076M, that prevents the vanadate-dependent labeling, but allows labeling in the presence of AlF4 and BeFx), has the same effect on both sites: the C-terminal mutation results in an altered conformation, and this minor alteration of the mutated active center is "propagated" to the other (native) catalytic site. Login to comment