ABCMdb

ABCC1 p.Cys682Ala

Predicted by SNAP2:	A: N (53%), D: D (91%), E: D (91%), F: D (95%), G: D (85%), H: D (95%), I: D (85%), K: D (91%), L: D (91%), M: D (95%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: N (61%), T: D (71%), V: D (85%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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Sentences [show]
No. Sentence Comment
138 The C682A (NBD1) mutation decreased the Kd value for ATP indicating increased affinity for this nucleotide while A1331C (NBD2) did not have any effect, suggesting that ATP binding at NBD1 at low concentration played a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 played a dominant role in the ATP-dependent LTC4 transport [92]. Login to comment

[hide] Effect of multiple cysteine substitutions on the f... Drug Metab Dispos. 2012 Jul;40(7):1403-13. Epub 2012 Apr 16. Qin L, Tam SP, Deeley RG
Effect of multiple cysteine substitutions on the functionality of human multidrug resistance protein 1 expressed in human embryonic kidney 293 cells: identification of residues essential for function.
Drug Metab Dispos. 2012 Jul;40(7):1403-13. Epub 2012 Apr 16., [PMID:22511347]

Abstract [show]
Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (DeltaMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) DeltaMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of DeltaMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of DeltaMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17beta-estradiol 17-beta-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

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Sentences [show]
No. Sentence Comment
64 Name Inserted (ϩ) or Deleted (-) Restriction Enzymes Primer Sequence (5Ј-3Јa 1 C208A N.A. CCCTAATCCCGCCCCAGAGTCCAG 2 C265A -BsmI GGAAGAAGGAAGCCGCCAAGACTAG 3 C375A -PstI GTCACTGCCGCCTTGCAGACCCTCG 4 C388A N.A. CTTCCACATCGCCTTCGTCAGTGG 5 C555A ϩSpoI/NruI CACCTGGGTCGCGACGCCCTTTCTG 6 C563A ϩBalI/MscI GGTGGCCTTGGCCACATTTGCCGTC 7 C682A ϩNarI CCAGGTGGGCGCCGGAAAGTCGTC 8 C730A ϩEspI, SacI ATCCTTTTTGGAGCTCAGCTGGAGG 9 C744A -StuI GATACAGGCCGCTGCCCTCCTCC 10 C984A ϩBalI/MscI TTCCTTTTCATGGCCAACCATGTGTCC 11 C1047A ϩSacI/SstI TTGGCTTCCCGAGCTCTGCACGTGG 12 C1105A ϩPvuI CATTGGTGCCGCGATCGTTATCCTG 13 C1205A N.A. GCGGCTGGAGGCTGTGGGCAACTG 14 C1209A ϩPvuI GTGTGGGCAACGCGATCGTTCTGTTTG 15 C1299A ϩMluI TTCCGGAACTACGCGTTGCGCTACCGAG 16 C1423A ϩPstI CTAGACCATGAAGCTGCAGAAGGC 17 C1439A ϩPflMI CCAGCTTGTGGCCCTAGCCCGGG 18 C1479A N.A. GTTCGAGGACGCCACCGTCCTCAC 19 Rec L N.A. GAAACCATCCACGACCCTAATCCCGCCCCAGAG 20 Rec R N.A. GGATTAGGGTCGTGGATGGTTTCCGAGAACAG 21 KbnL N.A. CATGGTACCATGGCGCTCCGGGGCTTCTGCAGC 22 KbnR N.A. GGCAGGATCCTTGGAGGAGTACACAACCTTC N.A., not applicable. Login to comment