ABCMdb

ABCC1 p.Gln1375Met

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (91%), E: D (85%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (91%), P: D (91%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Glutamine residues in Q-loops of multidrug resista... Biochim Biophys Acta. 2011 Jul;1808(7):1790-6. Epub 2011 Feb 26. 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., [PMID:21315686]

Abstract [show]
Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg.ATP binding and increased the K(m) (Mg.ATP) and V(max) values in Mg.ATP-dependent leukotriene-C4 transport. However, the V(max) 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. 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. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.

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No. Sentence Comment
3 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 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 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
77 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 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
92 In contrast, the double mutants, including Q713M/Q1375M and Q713L/Q1375L, significantly decreased the ATP-dependent LTC4 transport (Fig. 1C). Login to comment
93 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
98 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
106 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 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 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 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
128 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
134 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