ABCMdb

PMID: 16844693

Sauna ZE, Nandigama K, Ambudkar SV
Exploiting reaction intermediates of the ATPase reaction to elucidate the mechanism of transport by P-glycoprotein (ABCB1).
J Biol Chem. 2006 Sep 8;281(36):26501-11. Epub 2006 Jul 14., 2006-09-08 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E⅐S reaction intermediate of the ATPase reaction. Login to comment 33 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln If this were so, the double mutant (E556Q/ E1201Q) may, in the presence of nucleotide, represent a prehydrolysis transition state of the Pgp-catalyzed ATP hydrolysis reaction. Such a view is consistent with several recent structures of the NBDs of ABC proteins that suggest that ATP acts as molecular glue bringing together the two NBDs to form the ATP sandwich dimer. Login to comment 37 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We also show strong coupling between the NBDs and the transport substrate site(s) in purified and reconstituted wild-type and mutant (E556Q/E1201Q) Pgps. Login to comment 40 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We estimated the activation energies for the formation of the nucleotide triphosphate-trapped (prehydrolysis) state using the mutant (E556Q/E1201Q) Pgp as well as the nucleotide diphosphate-trapped (posthydrolysis) state. Login to comment 48 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Preparation of Crude Membranes from High Five Insect Cells Infected with Recombinant Baculovirus Carrying the Wild-type and Mutant Human MDR1 Gene-High Five insect cells (Invitrogen) were infected with the recombinant baculovirus carrying the human MDR1 cDNA (either wild type or the mutant, E556Q/E1201Q) with a His6 tag at the C-terminal end as described (9). Login to comment 73 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln [␣-32 P]ATP or [␣-32 P]ADP Trapping-Purified wild type and E556Q/E1201Q mutant Pgps reconstituted into liposomes were incubated with 200 ␮M [␣-32 P]ATP for 5 min in the ATPase buffer (see above). Login to comment 103 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln RESULTS The Mutant Pgp E556Q/E1201Q Exhibits Very Low Levels of ATP Hydrolysis but Occludes [␣-32 P]ATP in the Transition State-Several groups have studied the role of the highly conserved glutamates within the Walker B region of the Nand the C-ATP sites of mouse (22) and human (20) Pgp. Login to comment 104 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln In human Pgp, the double mutant E556Q/E1201Q does not show steady state hydrolysis but can trap the nucleotide in the transition state (20), and the results were identical with equivalent mutations in mouse Pgp (22). Login to comment 108 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Since the latter is the more direct method, we overexpressed human Pgp (both wild-type and the E556Q/E1201Q mutant) in High Five insect cells, prepared crude membranes, purified the Pgp, and reconstituted into proteoliposomes as described under "Experimental Procedures." Login to comment 110 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The mutant Pgp (E556Q/E1201Q), on the other hand, shows negligible ATP hydrolysis, and Vi has no effect on the reaction. Login to comment 121 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The E556Q/E1201Q Mutant and Wild-type Pgps Show Comparable Affinities for Nucleotides-As shown in Fig. 1, the mutant Pgp occludes [␣-32 P]ATP such that it cannot be exchanged by a 50-fold excess of cold ATP. Login to comment 124 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Hydrolysis of [␣-32 P]ATP and occlusion of [␣-32 P]ATP/ADP by wild-type and the E556Q/E1201Q mutant Pgp. Login to comment 138 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Determination of the affinities of nucleotides for wild-type and the E556Q/E1201Q mutant Pgps. A, the apparent Kd (8-azido-ATP) was estimated by determining the photolabeling of reconstituted purified Pgps in the presence of increasing concentrations of 8-azido-[␣-32 P]ATP at 4 °C, as described under "ExperimentalProcedures. Login to comment 148 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The graphs depict incorporation of nucleotide in wild-type Pgp (F) and the E556Q/E1201Q mutant Pgp (E). Login to comment 154 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We compared the apparent Kd (8-azido-ATP) for the binding of 8-azido-[␣-32 P]ATP at 4 °C. Fig. 2A shows that both wild-type and the E556Q/E1201Q mutant Pgps have comparable affinities (11.07 Ϯ 0.4 ␮M and 10.7 Ϯ 0.28 ␮M, respectively). Login to comment 163 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The Energetics of Nucleotide Trapping in the E556Q/E1201Q Mutant and Wild-type Pgps-The data in Fig. 2 suggest that the Glu 3 Gln mutant Pgp attains the ATP-trapped state via a conformational change and is not a consequence of inherent greater affinity for nucleotide(s). Login to comment 166 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln Whereas it is impossible to trap the Pgp⅐MgATP transition state of the wild-type protein in the absence of Vi or other transition state analogs of Pi, the mutant Pgp, E556Q/E1201Q, by preventing cleavage of the ␥-phosphate, permits the experimental characterization of the Pgp (E556Q/E1201Q)⅐MgATP reaction intermediate. Login to comment 172 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Thermal titrations of nucleotide occlusion in wild-type and the E556Q/E1201Q mutant Pgps. A, purified wild-type and mutant Pgps (100 ␮g of protein/ml) reconstituted into proteoliposomes were incubated with 50␮M 8-azido[␣-32 P]ATPintheabsenceorpresenceofVifor5minatdifferent temperatures in the range of 4-37 °C. Login to comment 193 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Moreover, these data support the proposition that the mutant Pgp occludes ATP as a reaction intermediate, Pgp(E556Q/E1201Q)⅐MgATP, which may be equivalent to the Pgp⅐MgATP (or the E⅐S) state in the catalytic cycle of Pgp. Login to comment 210 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Kinetics of Nucleotide Occlusion and Inhibition of [125 I]IAAP Binding in the E556Q/E1201Q Mutant Pgp-We have demonstrated above that the formation of the E⅐S and E⅐P states of the ATPase reaction and the concomitant decrease in binding of TABLE 1 Activation energy (Eact) values for the formation of different reaction intermediates during Pgp-mediated ATP hydrolysis Activation energy (Eact) ϭ -(slope) 2.3R. Login to comment 212 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Formation of the Pgp⅐ATP (in the E556Q/E1201Q mutant Pgp) or Pgp⅐ADP⅐Vi (in the wild-type Pgp) reaction intermediates is a slow process that occurs over several minutes (see supplemental Fig. S2). Login to comment 218 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln Reaction Nucleotide trappinga Inhibition of IAAP bindingb Azido-ATP/ADP ATP/ADP kJ/mol Vi-induced nucleotide trapping in wild-type Pgpc 65.3 Ϯ 9.7 43 Ϯ 7.9 49.3 Ϯ 8.8 BeFx-induced nucleotide trapping in wild-type Pgpd 62.4 Ϯ 10.3 33.4 Ϯ 5.9 44.1 Ϯ 6.1 Vior BeFx-independent nucleotide trapping in mutant Pgp (E556Q/E1201Q)e 63.4 Ϯ 8.3 37 Ϯ 4.3 44.1 Ϯ 5.7 a Nucleotide trapping was initiated by using either 8-azido-͓␣-32 P͔ATP or ͓␣-32 P͔ATP. Login to comment 220 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The incubation with wild-type Pgp was carried out in the presence of Vi and in the absence of Vi for the E556Q/E1201Q mutant Pgp. Login to comment 231 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln For purification and reconstitution of the wild type and E556Q/E1201Q double mutant, we used protocol that allows Ͼ90% recovery of verapamil-stimulated ATPase activity (23, 25, 26). Login to comment 233 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln In addition, when we fit the curves taking into account these values, we found that the maximal stoichiometry FIGURE4.Thermaltitrationsoftransportsubstrate,[125 I]IAAP,bindingto Pgp following occlusion of nucleotides in wild-type and mutant Pgps. A, purified Pgps, wild-type (E) and the E556Q/E1201Q mutant (‚) reconstituted intoproteoliposomes(100␮gofprotein/ml),wereincubatedfor5min(under equilibrium conditions for nucleotide trapping) with 1 mM ATP at different temperaturesintherangeof4-37 °C.Thewild-typePgpwasincubatedinthe presence of 250 ␮M Vi, and the mutant Pgp was incubated in the absence of Vi. Login to comment 246 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln Kinetics of nucleotide occlusion and inhibition of [125 I]IAAP binding in wild-type and E556Q/E1201Q mutant Pgps. A, purified Pgps (wild type and the E556Q/E1201Q mutant) reconstituted into proteoliposomes (100 ␮g/ml) were incubated with increasing concentrations of [␣-32 P]ATP at 34 °C for 5 min. Login to comment 251 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Incorporation of [␣-32 P]ADP/ATP into wild type (f) and mutant Pgps (F) is depicted. B, purified mutant Pgp (E556Q/E1201Q) reconstituted into proteoliposomes (100 ␮g/ml) was incubated with increasing concentrations of ATP for 5 min at 34 °C and then transferred to ice. Login to comment 269 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Additionally, Table 1 shows that the activation energy for BeFx-induced trapping (33.4 kJ/mol) is comparable with that obtained for Vi-independent trapping in the E556Q/E1201Q mutant Pgp (37 kJ/mol). Login to comment 289 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The E556Q/E1201Q mutant Pgp can trap [␣-32 P]ATP in the absence of Vi (Fig. 1B) (20, 22), and we demonstrate in this study that this is not due to altered affinities for nucleotides (Fig. 2). Login to comment 313 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The Pgp⅐ATP intermediate is equivalent to the E⅐S complex and captured by the experimental stratagem of mutations (e.g. E556Q/E1201Q mutant) in the ATP sites of Pgp that drastically reduces the hydrolysis of the ␥-phosphate or by BeFx-mediated trapping of nucleotide in wild-type protein. Login to comment