ABCMdb

PMID: 21315686

Yang R, Hou YX, Campbell CA, Palaniyandi K, Zhao Q, Bordner AJ, Chang XB
Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor.
Biochim Biophys Acta. 2011 Jul;1808(7):1790-6. Epub 2011 Feb 26., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn However, the Vmax values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/ Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Login to comment 4 ABCC1 p.Gln713Met ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Asn Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. Login to comment 27 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn In order to determine the functional roles of the glutamine residue in the Q-loop of human MRP1, we have substituted the glutamine residue in Q-loop of MRP1 with: 1) an asparagine (Q713N and Q1375N) that remains the amide group but with one methylene shorter in asparagine than in glutamine; 2) a methionine (Q713M and Q1375M) that eliminates the amide group but contains paired electrons in the sulfur atom of the methionine residue that might potentially interact with the Mg++ cofactor; 3) a leucine (Q713L and Q1375L) that eliminates the amide group and abolishes the interactions with Mg++ cofactor and the putative hydrolytic water molecule, and used them to determine the consequence of these mutations in ATP-dependent leukotriene C4 (LTC4) transport. Login to comment 74 ABCC1 p.Gln713Asn ABCC1 p.Gln1375Asn ATP-dependent LTC4 transport activity of each individual mutant, including Q713N and Q1375N, after adjusting the membrane vesicles to have similar amount of MRP1 protein, based on the amount of protein shown in Fig. 1B and the ratio listed in Table S2, is similar to that of wt MRP1 (Fig. 1C), indicating that the amide side group of asparagine with one methylene shorter in asparagine than in glutamine plays a similar role as that of glutamine. Login to comment 75 ABCC1 p.Gln713Asn ABCC1 p.Gln1375Asn However, the ATP-dependent LTC4 transport activity of the double mutant Q713N/Q1375N is significantly lower than that of wt MRP1 (Fig. 1C), implying that the double mutations might significantly affect the binding for Mg·ATP in both NBDs. Login to comment 77 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln1375Met Interestingly, substitution of the glutamine residue with a methionine, including Q713M and Q1375M, increased the transport activities, whereas substitution of the glutamine residue with a leucine, including Q713L Fig. 1. Substitution of Q713 in Q-loop of NBD1 or Q1375 in Q-loop of NBD2 with an amino acid that eliminates the amide group did not have a significant effect on the Mg·ATP-dependent LTC4 transport. Login to comment 89 ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met For example, 1.56 μg of wt MRP1+1.44 μg of pDual and 3.00 μg of Q1375M were used to determine the ATP-dependent LTC4 transport activities of wt MRP1 and Q1375M-mutated MRP1. Login to comment 91 ABCC1 p.Gln1375Leu and Q1375L, did not have a significant effect on the ATP-dependent LTC4 transport (Fig. 1C). Login to comment 92 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu In contrast, the double mutants, including Q713M/Q1375M and Q713L/Q1375L, significantly decreased the ATP-dependent LTC4 transport (Fig. 1C). Login to comment 93 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn 3.2. Substitution of the Q713 in NBD1 or Q1375 in NBD2 with an amino acid that eliminates the interaction between this residue and Mg++ cofactor significantly increased the Km value in ATP-dependent LTC4 transport The results in Fig. 1C imply that the double mutants might significantly affect the binding of Mg·ATP. If this would be the case, it meant that individual mutants, including Q713N, Q1375N, Q713M, Q1375M, Q713L and Q1375L, should also decrease the binding of Mg·ATP. Login to comment 96 ABCC1 p.Gln1375Leu The results shown in Fig. S2 clearly indicate that the Vmax and Km (Mg·ATP) values for Q1375L are significantly higher than that for wt MRP1. Login to comment 98 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn ABCC1 p.Gln1375Asn The results in Table 1 indicate that the Vmax values derived from Q713N, Q1375N, Q713 M, Q1375M and Q1375L are significantly higher than that of wt MRP1, whereas the Vmax values of Q713L and the double mutants, including Q713N/ Q1375N, Q713M/Q1375M and Q713L/Q1375L, are not significantly different from that of wt MRP1. Login to comment 100 ABCC1 p.Gln1375Leu In addition, the Km (Mg·ATP) values derived from the double mutants are significantly higher than, except Q1375L, their corresponding single mutants. Login to comment 106 ABCC1 p.Gln713Met ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn ABCC1 p.Gln1375Asn As shown in Fig. 2, the mutations at NBD2, including Q1375N, Q1375M and Q1375L, did not have a significant effect on Mg·ATP binding at NBD1, whereas the mutations at NBD1, including Q713N, Q713N/Q1375N, Q713M, Q713M/Q1375M, Q713L and Q713L/ Q1375L, significantly reduced the Mg·ATP binding at NBD1. Login to comment 113 ABCC1 p.Gln713Met ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met In contrast, the labeling at NBD2 with [α-32 P]-8-N3ATP, including wt MRP1, Q713N, Q713M, Q1375M, Q713M/Q1375M and Q713L, is stronger than the corresponding labeling with [γ-32 P]-8-N3ATP, indicating that the bound Mg·ATP was hydrolyzed and trapped there by vanadate. Login to comment 114 ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn ABCC1 p.Gln1375Asn However, the labeling at the mutated NBD2, including Q1375N, Q713N/Q1375N, Q1375M, Q713M/Q1375M, Q1375L and Q713L/Q1375L, with [α-32 P]-8-N3ATP was significantly lower than the corresponding labeling at wt MRP1, implying that much less [α-32 P]-8-N3ATP bound to the mutated NBD2 than to wt MRP1 or the ATP-hydrolysis-product ADP had not been firmly trapped there by vanadate. Login to comment 116 ABCC1 p.Gln713Met ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln713Leu ABCC1 p.Gln713Asn ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Leu ABCC1 p.Gln1375Asn ABCC1 p.Gln1375Asn Construct Vmax (pmol/mg/min)a P value Km (μM Mg·ATP)* P value Wt MRP1 104.3±20.5 65.7±4.2 Q713N 191.7±20.9 0.0135 318.3±2.4 0.0001 Q1375N 280.0±32.7 0.0030 370.0±8.2 0.0001 Q713N/Q1375N 63.7±6.9 0.0566 916.7±20.5 0.0001 Q713M 213.3±29.5 0.0128 278.3±19.3 0.0001 Q1375M 203.3±24.9 0.0123 295.0±7.1 0.0001 Q713M/Q1375M 77.0±12.5 0.1249 1006.3±12.7 0.0001 Q713L 120.0±4.1 0.3489 293.3±30.9 0.0005 Q1375L 390.0±24.8 0.0002 900.0±21.6 0.0001 Q713L/Q1375L 88.3±6.2 0.3505 970.0±80.0 0.0006 a Km (Mg·ATP) and Vmax (LTC4) values (n=3) for wild-type and Q713- or Q1375- mutated MRP1 were derived from the corresponding Michaelis-Menten curves (with variant concentration of ATP at 37 °C for 1 min). Login to comment 123 ABCC1 p.Gln713Leu ABCC1 p.Gln1375Leu If that would be the case, the double mutant Q713L/Q1375L might require higher concentration of Mg++ to perform the ATP-dependent LTC4 transport. Login to comment 125 ABCC1 p.Gln713Leu ABCC1 p.Gln1375Leu As shown in Fig. S3, wt MRP1 transported detectable amount of LTC4 into the membrane vesicles at 0.5-mM MgCl2, whereas Q713L/Q1375L required 5-mM MgCl2 to transport detectable amount of LTC4 into the membrane vesicles. Login to comment 127 ABCC1 p.Gln713Asn We wondered whether a bigger metal cofactor, for example, Mn++ (with a radius of 0.80 Å) or Ca++ (0.99 Å) is bigger than Mg++ (0.66 Å), would increase the nucleotide binding of Q713N. Login to comment 128 ABCC1 p.Gln713Met ABCC1 p.Gln713Asn ABCC1 p.Gln1375Met ABCC1 p.Gln1375Asn Interestingly, the Mn·ATP-dependent LTC4 transport activities of Q713N/ Q1375N and Q713M/Q1375M were significantly higher than that of wt MRP1 (Fig. S4A), whereas the Ca·ATP-dependent LTC4 transport activity of wt MRP1 was significantly higher than that of the double mutants (Fig. S4B). Login to comment 132 ABCC1 p.Gln713Asn ABCC1 p.Gln1375Asn Interestingly, the Mn·ATP-dependent LTC4 transport activity of Q713N or Q1375N is ~191% or 175% of wt MRP1 (Fig. 4), whereas the Mg·ATP-dependent LTC4 transport activity of the corresponding mutant is ~81% or 83% of the wt MRP1 (Fig. 1C). Login to comment 133 ABCC1 p.Gln713Asn ABCC1 p.Gln1375Asn Strikingly, the Mn·ATP-dependent LTC4 transport activity of the double mutant Q713N/Q1375N is ~162% of wt MRP1 (Fig. 4), whereas the Mg·ATP-dependent LTC4 transport activity of this mutant is ~33% of the wt MRP1 (Fig. 1C). Login to comment 134 ABCC1 p.Gln713Met ABCC1 p.Gln713Met ABCC1 p.Gln713Leu ABCC1 p.Gln1375Met ABCC1 p.Gln1375Met ABCC1 p.Gln1375Leu Similarly, the percentages (comparing to wt MRP1 as shown in Fig. 4) of the Mn·ATP-dependent LTC4 transport activities of other mutants, including Q713M, Q1375M, Q713M/ Q1375M, Q713L and Q1375L, are also significantly higher than their corresponding percentages (comparing to wt MRP1 as shown in Fig. 1C) of the Mg·ATP-dependent LTC4 transport activities, suggesting that these mutants might have higher affinity for Mn·ATP than for Mg·ATP. If that would be the case, at lower concentration of divalent cation, wt MRP1 should have higher Mn·ATP-dependent LTC4 transport activity than that of Mg·ATP. Login to comment 157 ABCC1 p.Gln713Leu ABCC1 p.Gln1375Leu The results presented in this manuscript do not support the above conclusion because the Mn·ATP-dependent LTC4 transport activities of all Q-mutants, except Q713L/Q1375L, are significantly higher than that of wt MRP1 (Fig. 4). Login to comment