ABCMdb

PMID: 15260484

Zhang DW, Nunoya K, Vasa M, Gu HM, Theis A, Cole SP, Deeley RG
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1.
Biochemistry. 2004 Jul 27;43(29):9413-25., 2004-07-27 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC1 p.Thr581Ala ABCC1 p.Ser585Ala ABCC1 p.Gln580Ala Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Login to comment 7 ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. Login to comment 8 ABCC1 p.Ser604Ala The third, S604A, selectively increased 17 -estradiol 17-( -D-glucuronide) (E217 G) transport. Login to comment 10 ABCC1 p.Asn590Ala Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC4) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Login to comment 47 ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala ABCC1 p.Asn590Gln Ser604 was converted to Ala and Thr, and Asn590 was mutated to Ala, Gln, and Asp. Login to comment 55 ABCC1 p.Thr581Ala ABCC1 p.Gln580Ala ABCC1 p.Ser605Ala ABCC1 p.Ser604Thr Mutations Q580A, T581A, S604T, and S605A were generated using the Transformer- Site-Directed Mutagenesis kit (CLONTECH, Palo Alto, CA). Login to comment 59 ABCC1 p.Thr581Ala ABCC1 p.Gln580Ala ABCC1 p.Ser605Ala ABCC1 p.Ser604Thr They are as follows: Q580A (5'-C ATC CTG GAT GCC GCG ACG GCC TTC GTG TC-3'), T581A (5'-CTG GAT GCC CAG GCG GCC TTC GTG TCT TTG-3'), S605A (5'-C ATG GTC ATC AGC GCG ATC GTG CAG GCG-3'), and S604T (5'-CCC ATG GTC ATC ACT AGT ATC GTG CAG GCG-3'). Login to comment 60 ABCC1 p.Ser585Ala ABCC1 p.Asn597Ala ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala ABCC1 p.Asn590Gln ABCC1 p.Asn590Asp Mutations S585A, N590A, N590Q, N590D, N597A, and S604A were generated using the QuikchangeSite-Directed Mutagenesis kit (STRATAGENE, La Jolla, CA). Login to comment 64 ABCC1 p.Ser585Ala ABCC1 p.Asn590Ala ABCC1 p.Asn590Gln ABCC1 p.Asn590Gln They are as follows: S585A (5'-C CAG ACA GCC TTC GTG GCT TTG GCC TTG-3), N590A (5'- CT TTG GCC TTG TTC GCC ATC CTC CGG TTT CCC-3'), N590Q (5'-CT TTG GCC TTG TTC CAG ATC CTC FIGURE 1: Topology of human MRP1. Login to comment 68 ABCC1 p.Asn597Ala ABCC1 p.Ser604Ala ABCC1 p.Asn590Asp CGG TTT CCC-3'), N590D (5'-CT TTG GCC TTG TTC GAT ATC CTC CGG TTT CCC-3'), N597A (5'-CTC CGG TTT CCC CTG GCC ATT CTC CCC ATG GT-3'), and S604A (5'-CTC CCC ATG GTC ATC GCC AGC ATC GTG CAG GC-3') After confirming all mutations by DNA Thermo Sequenase Cy5.5 and Cy5.0 dye terminator cycle sequencing (Amersham Biosciences) according to the manufacturer`s instructions, DNA fragments containing the desired mutations were transferred into pCEBV7-MRP1. Login to comment 94 ABCC1 p.Asn590Ala To generate the MRP1N590A -pFASTBAC dual vector, pCEBV7-MRP1 containing mutation N590A was digested with BamHI and SphI, and an approximately 1.8 kb fragment comprised of nucleotides 840-2699 of MRP1N590A was isolated. Login to comment 136 ABCC1 p.Phe594Ala Nonconservative mutation of Phe594 to Ala resulted in decreased transport of all substrates tested and loss of photolabeling with LTC4. Login to comment 138 ABCC1 p.Phe594Ala Like the F594A mutation, nonconservative substitution of the two adjacent residues Arg593 and Pro595 also decreased transport of all organic anion substrates tested (35, 49). Login to comment 151 ABCC1 p.Thr581Ala ABCC1 p.Ser585Ala ABCC1 p.Gln580Ala ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala ABCC1 p.Ser604Ala The levels of LTC4 uptake by vesicles prepared from HEK transfectants expressing either wild-type MRP1 or mutations Q580A, T581A, S585A, N597A, S604A, and S605A were proportional to the relative expression levels of the wild-type and mutant proteins. Login to comment 152 ABCC1 p.Asn590Ala The only mutation that affected LTC4 transport was replacement of Asn590 by Ala, which decreased the ability of MRP1 to transport LTC4 by approximately 50-60% (Figure 3A-D). Login to comment 154 ABCC1 p.Thr581Ala ABCC1 p.Ser585Ala ABCC1 p.Gln580Ala ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala Mutations Q580A, T581A, S585A, N597A, and S605A had no detectable effect on transport. Login to comment 155 ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala However, substitution of Ser604 with Ala (S604A) increased the transport efficiency by approximately 2-fold. Login to comment 156 ABCC1 p.Asn590Ala ABCC1 p.Asn590Ala On the other hand, replacement of Asn590 with Ala (N590A) substantially decreased the levels of E217 G transport. Login to comment 157 ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala Thus, mutation S604A altered only the ability of MRP1 to transport the glucuronidated estrogen, whereas mutation N590A influenced the transport of both LTC4 and E217 G. Login to comment 158 ABCC1 p.Asn590Gln ABCC1 p.Ser604Thr ABCC1 p.Asn590Asp Effect of Mutations S604T, N590Q, and N590D on Transport of [3 H]LTC4 and [3 H]E217 G by Wild-Type MRP1. Login to comment 161 ABCC1 p.Ser604Thr ABCC1 p.Asn590Asp Ser604 was mutated to Thr, and Asn590 was converted to Asp and Gln. Login to comment 165 ABCC1 p.Asn590Gln ABCC1 p.Ser604Thr ABCC1 p.Asn590Asp Mutations N590Q, N590D, and S604T had no effect on transport of the two substrates. Login to comment 168 ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala To examine the influence of the N590A and S604A mutations more quantitatively, we compared their effect on the kinetic parameters of transport of both substrates (Figure 5). Login to comment 169 ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala Mutation S604A had no effect on either the apparent Km or Vmax values for LTC4 uptake (Km 150 and 145 nM, and Vmax 89 and 84 pmol/ mg/min for wild-type MRP1 and mutation S604A, respectively) (Figure 5B and Table 1). Login to comment 170 ABCC1 p.Asn590Ala ABCC1 p.Asn590Ala In contrast, the N590A mutation decreased the Vmax and increased the apparent Km value for LTC4 transport (56 pmol/mg/min and 277 nM for mutation N590A) (Figure 5B and Table 1). Login to comment 171 ABCC1 p.Asn590Ala Thus, mutation N590A decreased the Vmax/Km ratio for LTC4 approximately 3-fold. Login to comment 172 ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala For E217 G transport, the Vmax value for mutation S604A was increased by 20-25% relative to wild-type MRP1 (197 pmol/mg/min for wild-type MRP1 vs 241 pmol/mg/min for the mutation), while the apparent Km was decreased ~2.5-fold (1.7 and 0.7 µM for wild-type MRP1 and mutation S604A, respectively) (Figure 5C and Table 1). Login to comment 173 ABCC1 p.Asn590Ala In contrast, substitution of Asn590 with Ala increased the Km value by approximately 65% (2.8 µM) and decreased the Vmax value by approximately 2-fold (93 pmol/mg/min) (Figure 5C and Table 1). Login to comment 174 ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala Thus, as observed with LTC4 as a substrate, mutation N590A decreased the Vmax/Km ratio for E217 G approximately 3-fold, whereas the S604A mutation increased the Vmax/Km ratio for this substrate approximately 3-fold. Login to comment 175 ABCC1 p.Ser604Ala ABCC1 p.Ser604Ala ABCC1 p.Asn590Ala ABCC1 p.Asn590Ala These changes in transport efficiency were mediated predominantly by a decrease in apparent affinity for both LTC4 and E217 G as a result of the S604A mutation, while the N590A mutation increased the apparent Km and decreased the Vmax for E217 G. Login to comment 177 ABCC1 p.Asn590Ala Since transport studies indicated that mutation N590A increased the apparent Km for LTC4, we directly examined the ability of the mutant protein to bind and be photolabeled by [3 H]LTC4. Login to comment 178 ABCC1 p.Asn590Ala The N590A mutation decreased photolabeling, consistent with the results obtained with kinetic analysis of LTC4 uptake (Figure 6B). Login to comment 180 ABCC1 p.Asn590Ala Although photolabeling of the N590A mutant protein was decreased relative to wild type in the absence of ATP, a further decrease was observed in the presence of nucleotide, indicating that the protein retains the ability to shift from a higher to lower affinity state. Login to comment 181 ABCC1 p.Asn590Ala ABCC1 p.Asn590Ala To investigate the effect of mutation N590A on photolabeling of LTC4 more precisely, we took advantage of a pFASTBAC dual vector, in which the NH2-proximal MRP1 fragment (amino acids 1-932) was modified to contain mutation N590A and coexpressed with a wild-type COOH-proximal fragment (amino acids 932-1531). Login to comment 196 ABCC1 p.Asn590Ala As observed with results obtained from analyses of membrane vesicles prepared from HEK293 cells stably transfected with full-length mutant MRP1N590A , replacement of Asn590 by Ala decreased the transport activity by approximately 50%. Login to comment 198 ABCC1 p.Asn590Ala As shown in Figure 6E, the N590A mutation resulted in a significant reduction of the labeling of LTC4 at both the NH2-and the COOH-proximal halves of MRP1. Login to comment 203 ABCC1 p.Thr581Ala ABCC1 p.Ser585Ala ABCC1 p.Gln580Ala ABCC1 p.Ser604Ala ABCC1 p.Ser604Thr Mutations Q580A, T581A, S585A, S604A, and S604T had no significant effect on the ability of MRP1 to confer resistance to any drug tested. Login to comment 204 ABCC1 p.Asn597Ala In contrast, conversion of Asn597 to Ala resulted in a mutant protein with enhanced resistance to vincristine (3-4-fold) but an approximately 3-fold decrease in the ability to confer resistance to VP-16 without any significant effect on doxorubicin resistance. Login to comment 205 ABCC1 p.Asn590Gln ABCC1 p.Asn590Asp On the other hand, despite the fact that converting Asn590 to either Gln or Asp had no effect on the drug resistance profile of MRP1, substitution of the residue with Ala significantly decreased resistance to vincristine, doxorubicin, and VP-16. Login to comment 206 ABCC1 p.Ser605Ala Replacement of Ser605 by Ala also resulted in approximately a 2-3-fold reduction of resistance to all three drugs tested. Login to comment 215 ABCC1 p.Asn590Ala The studies described previously showed that the N590A mutation modulated the ability of MRP1 to confer drug resistance and to transport conjugated organic anions. Login to comment 216 ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala ABCC1 p.Ser604Ala In contrast, mutations N597A and S605A influenced only the drug resistance profile of MRP1, and mutation S604A affected the transport of only E217 G. Login to comment 220 ABCC1 p.Asn590Ala As observed with the effects of the mutations on LTC4 transport, only replacement of Asn590 with Ala significantly decreased the ability of MRP1 to transport GSH (Figure 7). Login to comment 221 ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala Other mutations, including N597A and S605A, which influenced MRP1-mediated drug resistance, had no effect, suggesting that they modify interactions between MRP1 and the drug rather than altering the ability to bind and transport GSH. Login to comment 222 ABCC1 p.Asn590Ala Effect of Mutation N590A on Photolabeling of MRP1 with 8-Azido-[R32 P]ATP. Login to comment 223 ABCC1 p.Asn590Ala On the basis of all substrates tested, mutation N590A affected the overall activity of MRP1. Login to comment 225 ABCC1 p.Asn590Ala To investigate whether the N590A mutation influenced the binding and/or hydrolysis of ATP at NBD1 and/or NBD2 FIGURE 4: ATP-dependent [3H]LTC4 and [3H]E217 G uptake by membrane vesicles prepared from HEK293 cells transfected with wild-type or mutant MRP1. Login to comment 240 ABCC1 p.Asn590Ala As shown in Figure 8B, substitution of Asn590 with Ala dramatically decreased the binding of the ATP analogue at NBD1. Login to comment 245 ABCC1 p.Asn590Ala However, as shown in Figure 8C, no significant difference could be detected between wild-type and N590A mutant proteins with respect to the ability of the LTC4 to enhance ADP trapping at NBD2 (compare lanes 5 and 6 and lanes 2 and 3 in Figure 8C) (46). Login to comment 249 ABCC1 p.Asn590Ala In contrast, rather than altering substrate specificity, replacement of Asn590 with Ala uniformly decreased the overall activity of MRP1, both with respect to transport of organic anion conjugates and resistance to three different classes of drugs. Login to comment 250 ABCC1 p.Asn590Ala Thus, the consequences of the N590A mutation are similar to those of nonconservative mutations of other amino acids (Arg593 , Phe594 , and Pro595 ) predicted to be near the middle of TM11 (35, 48, 49). Login to comment 269 ABCC1 p.Asn597Ala Consistently, we found that replacement of Asn597 with Ala increased resistance to larger drugs such as vincristine and decreased resistance to smaller substrates such as VP-16. Login to comment 270 ABCC1 p.Ser604Ala ABCC1 p.Ser604Thr In addition, although mutation S604T had no effect on any of the MRP1 functions tested, converting Ser604 to Ala decreased the apparent Km values and increased Vmax for E217 G uptake. Login to comment 272 ABCC1 p.Ser605Ala On the other hand, mutation of Ser605 to Ala decreased the ability of MRP1 to confer resistance to vincristine, VP-16, and doxorubicin, consistent with our previous proposal that hydrogen bonding may be a common form of interaction between MRP1 and its substrates (30-33, 45, 55, 60). Login to comment 273 ABCC1 p.Asn597Ala ABCC1 p.Ser605Ala In addition, mutations N597A and S605A affected only the drug-resistance profile of MRP1. Login to comment 276 ABCC1 p.Asn590Ala The conversion of Asn590 to Ala substantially decreased the ability to confer drug resistance and to transport E217 G, LTC4, and GSH. Login to comment 287 ABCC1 p.Phe594Trp ABCC1 p.Phe594Tyr However, unlike Asn590 , conservative substitution of Phe594 with Tyr or Trp altered the substrate specificity of the protein, suggesting that the latter residue may interact directly with substrate (35). Login to comment 289 ABCC1 p.Asn590Ala Given the pleiotropic effect of substitution of Asn590 with Ala and the lack of an effect of replacement with Asp or Gln, it appears likely that the hydrogen-bonding capacity of the side chain of the residue at position 590 may play a role in defining the conformation of the protein in the vicinity of the substrate binding pocket, possibly by interacting with residues in another helix. On the basis of our recently developed model of the tertiary structure of MSD2 and MSD3 of MRP1, such an interaction appears most likely to involve residues in TM6 and TM10 (48). Login to comment 293 ABCC1 p.Asn590Ala The uniform effect of the N590A mutation on the relative efficiency of transport of several structurally diverse conjugated anions, as well as resistance to three different classes of drugs, suggested that it may have altered a common function, such as the coupling of transport to ATP hydrolysis or nucleotide binding and hydrolysis itself. Login to comment 312 ABCC1 p.Asp1084Asn The D1084N mutation essentially eliminated the transport of LTC4 and prevented the transition of the protein from a high- to low-affinity substrate binding state in the presence of ATP (33). Login to comment 313 ABCC1 p.Asn590Ala The effect of the N590A mutation is less severe, and the mutant protein retains 30-40% of the transport activity of wild-type MRP1. Login to comment 315 ABCC1 p.Asn590Ala Thus, it appears likely that the reduction in transport in the N590A mutation is attributable to a decrease in the binding of ATP by NBD1 and a consequential reduction in ATP binding and hydrolysis at NBD2. Login to comment 316 ABCC7 p.Arg347Asp A nonconservative mutation in CFTR, R347D, that results in a mild form of disease was recently found to affect the ATPase activity of the protein by decreasing its Vmax for ATP 3-5-fold (44). Login to comment 317 ABCC7 p.Arg347Asp R347D is predicted to be in the aqueous pore of the channel in the inner leaflet region of TM6. Login to comment 319 ABCC1 p.Asn590Ala ABCC7 p.Arg347Asp Since there are many functional similarities between the cooperative interactions of the NBDs of MRP1 and CFTR, it is possible that the CFTR R347D mutation may be affecting the same step in the catalytic cycle as the N590A mutation. Login to comment 324 ABCC1 p.Asn590Ala Thus, changes in the position or orientation of TM11 caused by mutations, such as N590A, may be transmitted through the ICD causing a conformational or positional change in NBD1 that alters cooperativity between the two NBDs and results in diminished binding of ATP. Login to comment