ATP Binding to the First Nucleotide Binding Domain of Multidrug Resistance-associated Protein Plays a Regulatory Role at Low Nucleotide Concentration, whereas ATP Hydrolysis at the Second Plays a Dominant Role in ATP-dependent Leukotriene C4 Transport

Abstract

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. The two NBDs possess different properties (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098–13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280–20287) and may play different roles during solute transport. We now report that NBD1 has moderately higher affinity for ATP than NBD2. The consequence of this difference is that the overall K d value for wild-type MRP1 is mainly determined by ATP binding at NBD1. This conclusion is supported by the following: 1) mutation of the cysteine residue at 682 to alanine (C682A) in Walker A motif in NBD1 decreases the K d value, indicating increased affinity for ATP; 2) mutation of the alanine residue at 1331 to cysteine (A1331C) in the Walker A motif of NBD2 does not have an effect on the K d value; and 3) photolabeling of the protein with a cysteine residue in the Walker A motif of NBD1 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD2. In contrast, the K m for ATP in support of LTC4 transport is mainly determined by ATP hydrolysis at NBD2. This conclusion is supported by the following: 1) although mutation of A1331C does not have an effect on the K d value, the K m values measured from LTC4 transport by proteins with this mutation in NBD2 are much higher than the proteins with wild-type NBD2, implying that the A1331C mutation affects ATP binding/hydrolysis at NBD2; and 2) ATP-dependent LTC4 transport by the protein with a cysteine residue in the Walker A motif of NBD2 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD1. Our previous results indicated that ATP binding at NBD1 at low concentration enhanced ATP binding/hydrolysis at NBD2. All of these results support the notion that ATP binding at NBD1 at low concentration plays a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 plays a dominant role in the ATP-dependent LTC4 transport. Multidrug resistance-associated protein (MRP1) transports solutes in an ATP dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. The two NBDs possess different properties (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098–13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280–20287) and may play different roles during solute transport. We now report that NBD1 has moderately higher affinity for ATP than NBD2. The consequence of this difference is that the overall K d value for wild-type MRP1 is mainly determined by ATP binding at NBD1. This conclusion is supported by the following: 1) mutation of the cysteine residue at 682 to alanine (C682A) in Walker A motif in NBD1 decreases the K d value, indicating increased affinity for ATP; 2) mutation of the alanine residue at 1331 to cysteine (A1331C) in the Walker A motif of NBD2 does not have an effect on the K d value; and 3) photolabeling of the protein with a cysteine residue in the Walker A motif of NBD1 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD2. In contrast, the K m for ATP in support of LTC4 transport is mainly determined by ATP hydrolysis at NBD2. This conclusion is supported by the following: 1) although mutation of A1331C does not have an effect on the K d value, the K m values measured from LTC4 transport by proteins with this mutation in NBD2 are much higher than the proteins with wild-type NBD2, implying that the A1331C mutation affects ATP binding/hydrolysis at NBD2; and 2) ATP-dependent LTC4 transport by the protein with a cysteine residue in the Walker A motif of NBD2 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD1. Our previous results indicated that ATP binding at NBD1 at low concentration enhanced ATP binding/hydrolysis at NBD2. All of these results support the notion that ATP binding at NBD1 at low concentration plays a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 plays a dominant role in the ATP-dependent LTC4 transport. It is widely accepted that overexpression of P-glycoprotein (P-gp) 1The abbreviations used are: P-gp, P-glycoprotein; MRP1, multidrug resistance protein; NBD, nucleotide binding domain; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; 8-N3ATP, 8-azidoadenosine 5′-triphosphate; LTC4, leukotriene C4; BHK, baby hamster kidney; NEM, N-ethylmaleimide; ABC, ATP-binding cassette.1The abbreviations used are: P-gp, P-glycoprotein; MRP1, multidrug resistance protein; NBD, nucleotide binding domain; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; 8-N3ATP, 8-azidoadenosine 5′-triphosphate; LTC4, leukotriene C4; BHK, baby hamster kidney; NEM, N-ethylmaleimide; ABC, ATP-binding cassette. and/or multidrug resistance-associated protein (MRP1) confers cancer cells resistance to a broad range of anticancer drugs. Although they pump solutes out of cells in different fashions (i.e. P-gp transports hydrophobic compounds directly (1Sharom F.J. Liu R. Romsicki Y. Biochem. 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However, to have not directly the affinity of the two NBDs for We have now this and that NBD1 of MRP1 protein has higher affinity for than NBD2. The the two NBDs the different of these the in ATP such as Walker A and and ATP binding J.E. M. J. PubMed Scopus Google Scholar, Cell Biol. 1992; PubMed Scopus Google Scholar, J. I.B. PubMed Scopus Google Scholar). We that the cysteine residue in Walker A in NBD1 and NBD2 of P-gp, whereas it not in the NBD2 Walker A motif of MRP1 these NBD1 and NBD2 to the properties of these two these that for ATP binding as to N-ethylmaleimide indicating that ATP binding at NBD1 plays a much regulatory role at low ATP concentration than high and that ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. NEM, and from from from from The and from from Cell in and Cell hamster cells in with at in A wild-type MRP1 J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). and by the cells with for in in and at J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of MRP1 to and to the in MRP1 are as and The are The of MRP1 in the J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) used as for the The cysteine residue at of 682 to alanine by the and the from Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A1331C the by the The the mutation and the A1331C mutation and the In to that the during the the in J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) by the to The in J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) by the A1331C to The and the in J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) by the and A1331C to and and as Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar). The used a MRP1 Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar), and the with to the to the Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in an and and a the and at of MRP1 and of MRP1 protein to Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of and are indicated in In to the with Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of is indicated in and The in of and of NBD1 and of NBD2 Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to the and in a protein to the and for in a The with the protein with with for at and d values of NBD1 and NBD2 determined in wild-type MRP1 The photolabeling out in a of of MRP1 and of The on for on for with for at and with NBD1 and NBD2 The to and to a are indicated on the MRP1 the MRP1 protein and higher by and by a NBD1 and NBD2 NBD1 and NBD2 of the NBD1 NBD2 The of NBD1 or NBD2 measured by and the of is from the in NBD1 and NBD2 The are the of with of the of NBD1 The of NBD1 measured in A out and a K d value of of the of NBD2 The of NBD2 measured in A and a K d value of has a effect on ATP at cysteine residue in NBD1 than in NBD2. of in of and of indicated in the at for cysteine to the and at for The proteins in a in a The of and and at for The to with of and on for effect of on ATP of wild-type MRP1 of the results in A. The of as effect of on ATP of of the results in C. effect of on ATP of A1331C of the results in E. effect of on ATP of of the results in G. The are indicated on the and the on the the MRP1 has a effect on ATP-dependent LTC4 transport at cysteine residue in NBD2 than in NBD1. of in a and of indicated in the at for the of the of the to LTC4, and and at for The of the transport to the The in The of LTC4 by with as transport of leukotriene the by a I. Jedlitschky G. Buchholz U. Keppler D. J. Biochem. 1994; PubMed Scopus Google Scholar, D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The out in a of LTC4 of and of ATP as indicated in the at for the to and with of transport and and on that with transport The with of transport and in a of The to the determined by in MRP1 Walker A an of Walker A from P-gp, MRP1, and The cysteine residue the and is in the two Walker A of the NBDs of two NBDs have to 1998; Google Scholar, 1998; PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of P-gp by N-ethylmaleimide the of P-gp J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. PubMed Scopus Google Scholar). However, the of for of different for the two NBDs J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the in Walker A of MRP1 is a in NBD1 and an A in NBD2. The role by the residue in in ATP binding is to a to with the of the ATP J.E. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). that it not this is by or However, the two NBDs of MRP1 protein have different at this this may to their different properties with to ATP binding and this to these two in the two NBDs that and protein wild-type MRP1, indicating that these not protein although the of MRP1 protein in the different from The A1331C in NBD2 the K m for ATP in LTC4 to these Walker A in NBD1 and A1331C in NBD2, the ATP-dependent LTC4 from cells these to the K and and that the K of wild-type MRP1 is is to the K m value D.W. Almquist K.C. Deeley R.G. Cole S.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The K m value for NBD1 to whereas the of LTC4 transport increased from to In contrast, the mutation in NBD2, increased the K to whereas the of LTC4 transport not implying that this NBD2 mutation affects the ATP binding/hydrolysis at NBD2. although the K of the NBD1 the K of the much higher than the NBD1 or the to the NBD2 implying that ATP binding/hydrolysis at NBD2 a dominant role in ATP-dependent LTC4 and of of of wild-type and at values 2) and values 2) from m The values 2) and values 2) from in a the is in of of LTC4 of the of MRP1 protein in the is an important the indicated in the of MRP1 proteins different wild-type and the and the the of protein; the determined in a different of MRP1 The in the of MRP1 protein in a of of protein by of from cells The of the LTC4 is wild-type A1331C is with the of the values of wild-type and to in NBD1 (C682A) the whereas to in NBD2 (A1331C) the for ATP K values in that the mutation in increased the affinity for whereas the mutation in NBD2, the affinity for ATP. However, the K value of the to the NBD2 We the following: 1) ATP binding to NBD1 of to NBD1 of whereas ATP binding to NBD2 of to NBD2 of 2) ATP binding and hydrolysis at NBD2 play a dominant role in ATP-dependent LTC4 to the K values of A1331C and that is the the ATP binding to to In to this these proteins with on for these of the of ATP the mainly ATP binding to these that the mutation in the K d from ATP to implying increased affinity for ATP. The K d value of A1331C to wild-type protein implying that the K d value of A1331C mainly to the ATP binding to NBD1. although the K value of higher than the K d value of to the value of conclusion that the K d value mainly to ATP binding to the NBD1. The high K value of as ATP binding at the NBD2 this the different MRP1 with at or and with to NBD1 and NBD2 The of ATP to NBD1 NBD2 at for and with previous results Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), whereas these of at for A1331C and and the of the NBD2 in A1331C and at implying that the of the alanine residue with cysteine in the Walker A motif in NBD2 the affinity for ATP binding at NBD2. The of at and for wild-type and implying that NBD1 may have higher affinity for ATP binding than NBD2. Although the of at for A1331C and not determined of at the NBD2, it that the of NBD1 much higher than that of NBD2 implying that the of the alanine residue with cysteine in Walker A motif in NBD2 the affinity for ATP binding at the of 682 increased affinity for ATP at photolabeling out in of of MRP1 and of The on for on for and with of The proteins on a and to a The of in the MRP1 protein measured by and out values in this m The photolabeling out in of of MRP1 and of The on for on for and with of The proteins on a and to a The of in the MRP1 protein measured by and out values in this in a NBD1 for ATP than results in and and previous M. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that NBD1 of MRP1 may have higher affinity for ATP binding than NBD2. In to the for ATP binding to NBD1 and NBD2, wild-type MRP1 with on for in the of and with to NBD1 and NBD2 and that the K d values of NBD1 and NBD2 are and indicating that NBD1 has higher affinity for ATP binding than NBD2. that the of ATP at NBD1 and NBD2 and at nucleotide higher The at high nucleotide of MRP1 with a in the NBD1 Walker A to than the with a at the of in the or the of NBD1 plays a role in the overall K d values of the ATP of the protein a cysteine residue in the Walker A motif of NBD1 much more sensitive to modification than the protein a cysteine residue in that this the with of and with The of A and and and A1331C and at low of The of this is not However, the in at higher concentration of NEM, values of and It is that the values of the proteins with a cysteine residue in the Walker A motif of such as wild-type and are much than the proteins a cysteine residue in that such as and indicating that ATP binding to the proteins with a cysteine residue in NBD1 is much more sensitive to modification than the proteins this cysteine The value for A1331C cysteine in both is than that for wild-type protein a cysteine residue in and the value for a cysteine residue in is than that for a cysteine residue in both indicating that the cysteine residue in NBD2 plays a role in the residue in Walker A motif of NBD1 is much more sensitive to the modification in ATP binding than the cysteine residue in at values 2) from value is the concentration of MRP1 values 2) from value is the concentration of MRP1 in a ATP-dependent LTC4 of the a in the Walker A of NBD1 to than the a in the Walker A of results in and that ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. on this a protein a cysteine residue in the Walker A motif of NBD2 much more sensitive to modification than a protein a cysteine residue in that this the of on LTC4 transport by the with of ATP-dependent LTC4 transports by in However, the of the to proteins with a cysteine residue in Walker A motif of NBD2, such as A1331C and are much more sensitive to modification than the proteins this residue in that such as wild-type and that ATP binding and hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. In the value for wild-type MRP1 a cysteine residue in the Walker A motif of is than that for a cysteine residue in both indicating that ATP binding at NBD1 plays a role in ATP-dependent LTC4 residue in Walker A motif of NBD2 is much more sensitive to the modification in ATP-dependent LTC4 transport than the cysteine residue in at in in values 2) from value is the concentration of ATP-dependent LTC4 transport by the MRP1 The in in values 2) from value is the concentration of ATP-dependent LTC4 transport by the MRP1 in a The Walker A and the Walker from and the from the are in ATP binding by proteins J.E. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of a indicated that may to ATP binding J.E. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) the of of with the of the whereas the of with the of the J.E. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of the at the of in Walker A that of them are are and are alanine This the of the of these play a role in ATP is a cysteine residue at this in NBD1 of MRP1 an alanine residue in NBD2 The cysteine residue in NBD1 to alanine and the alanine residue to cysteine in NBD2 The mutation the K m value and K d value indicating that mutation the affinity for ATP. The K m values of A1331C and are higher than that of wild-type are not with their K d values The K d value of A1331C is to that of wild-type whereas the K d value of is to that of results as indicating that the K d value for overall binding to both NBD1 and NBD2 in a MRP1 protein mainly to ATP binding at NBD1. The results in as indicating that the K m value of a MRP1 protein mainly to ATP binding/hydrolysis at NBD2. This that the mutation of A1331C affinity for ATP at NBD2. the of the NBD2 of A1331C or In the results of modification support the The concentration to of of proteins with a cysteine residue in such as wild-type and is much than that of the proteins a cysteine in that such as and indicating that ATP binding to NBD1 plays a dominant role in the K d value of a MRP1 In contrast, the concentration to of ATP-dependent LTC4 transport by the proteins a cysteine residue in NBD2, such as wild-type and is much higher than that of the proteins with a cysteine in NBD2, such as A1331C and implying that ATP binding/hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. We that the ATP to NBD1 of wild-type MRP1 in the of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), implying that NBD1 have higher affinity for than NBD2. In of two MRP1 of NBD1 and of NBD2 with on M. Loe D.W. Grant C.E. Almquist K.C. Cole S.P. Deeley R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that NBD1 have higher affinity for ATP than NBD2. directly this wild-type MRP1 protein with on and with K d values of and ATP for NBD1 and NBD2, results that NBD1 has a moderately higher affinity for ATP than NBD2. The results in that the of ATP at NBD1 NBD2 ATP and to at at higher ATP implying that both NBDs a with ATP in the ATP is a both However, the ATP concentration in the an MRP1 hydrolysis and of ATP this is not may a of low ATP In that ATP bind to this binding a to ATP binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and hydrolysis at NBD2, is to solute transport. In this ATP binding to NBD1 at low nucleotide not at high play a regulatory This of the MRP1 protein that the a range of ATP that are We for of the and for of the