ABCMdb

ABCC1 p.Asn590Gln

Predicted by SNAP2:	A: D (71%), C: D (71%), D: D (71%), E: D (80%), F: D (85%), G: D (66%), H: D (66%), I: D (80%), K: D (85%), L: D (85%), M: D (71%), P: D (85%), Q: D (63%), R: D (85%), S: D (59%), T: D (63%), V: D (85%), W: D (91%), Y: D (75%),
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, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Transmembrane helix 11 of multidrug resistance pro... Biochemistry. 2004 Jul 27;43(29):9413-25. 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 [PMID:15260484]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. 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. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

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Sentences [show]
No. Sentence Comment
47 Ser604 was converted to Ala and Thr, and Asn590 was mutated to Ala, Gln, and Asp. Login to comment
60 Mutations S585A, N590A, N590Q, N590D, N597A, and S604A were generated using the QuikchangeSite-Directed Mutagenesis kit (STRATAGENE, La Jolla, CA). Login to comment
64 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
158 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
165 Mutations N590Q, N590D, and S604T had no effect on transport of the two substrates. Login to comment
205 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