ABCMdb

ABCC1 p.Ser1334Cys

Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (91%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), T: D (80%), V: D (95%), W: D (95%), Y: D (95%),
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, Q: D, R: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] The hydroxyl group of S685 in Walker A motif and t... Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29. Yang R, Scavetta R, Chang XB
The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.
Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29., [PMID:18088596]

Abstract [show]
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 degrees C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the beta-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.

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254 In order to rule out the possibility that the double mutant D1454L/E1455L might rescue the misfolding caused by D14 54L mutation, we have made single mutants including D1454L, D1454N, S1334A, S1334T, S1334C, S1334H, S1334D and S1334N. Login to comment

[hide] Interaction between the bound Mg.ATP and the Walke... Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18. Yang R, Scavetta R, Chang XB
Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18., 2008-08-12 [PMID:18636743]

Abstract [show]
Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.

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No. Sentence Comment
3 In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. Login to comment
4 However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Login to comment
15 In order to test this hypothesis, we have substituted the Walker A S1334 with an A (S1334A), a C (S1334C), a D (S1334D), an H (S1334H), an N (S1334N), or a T (S1334T). Login to comment
36 The forward and reverse primers used to introduce these mutations are as follows: S1334A/forward, 5'-CG GGA GCT GGG AAG GCG TCC CTG ACC CTG GGC-3'; S1334A/ reverse, 5'-GCC CAG GGT CAG GGA CGC CTT CCC AGC TCC CG-3'; S1334C/forward, 5'-CG GGA GCT GGG AAG TGC TCC CTG ACC CTG GGC-3'; S1334C/reverse, 5'-GCC CAG GGT CAG GGA GCA CTT CCC AGC TCC CG-3'; S1334D/forward, 5'-CG GGA GCT GGG AAG GAC TCC CTG ACC CTG GGC-3'; S1334D/reverse, 5'-GCC CAG GGT CAG GGA GTC CTT CCC AGC TCC CG-3'; S1334H/forward, 5'-CG GGA GCT GGG AAG CAC TCC CTG ACC CTG GGC-3'; S1334H/reverse, 5'-GCC CAG GGT CAG GGA GTG CTT CCC AGC TCC CG-3'; S1334N/forward, 5'-CG GGA GCT GGG AAG AAC TCC CTG ACC CTG GGC-3'; S1334N/reverse, 5'-GCC CAG GGT CAG GGA GTT CTT CCC AGC TCC CG-3'; S1334T/forward, 5'-CG GGA GCT GGG AAG ACG TCC CTG ACC CTG GGC-3'; S1334T/reverse, 5'-GCC CAG GGT CAG GGA CGT CTT CCC AGC TCC CG-3'. Login to comment
82 The results in Figure 1A showed that the 190 kDa wild-type MRP1 protein is resistant to endoglycosidase H digestion whereas the minor band is not, indicating that the majority of wild-type MRP1 protein is complex-glycosylated in vivo. All of the mutants, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, have a major band resistant to the endoglycosidase H digestion but sensitive to the PNGase F digestion (Figure 1B), indicating that all of these mutants mainly form complex-glycosylated mature proteins at 37 °C in vivo. Login to comment
88 As shown in Figure 2B, S1334A-, S1334C-, S1334D-, S1334H-, or S1334N-mutated MRP1 almost completely abolished the ATP-dependent LTC4 transport, whereas S1334T mutation, which retained the hydroxyl group at this position, exerted ~175% of wild-type MRP1 transport activity, suggesting that the hydroxyl group at this position plays a crucial role for the ATP binding/hydrolysis and ATP-dependent solute transport by MRP1. Login to comment
112 The intensities of the MRP1 bands were determined by a scanning densitometer. The mean ratios (n ) 2, including the results derived from 400 and 800 ng of protein), considering the amount of wild-type MRP1 as 1, of the mutant proteins are as follows: S1334A, 1.46 ( 0.06; S1334C, 0.84 ( 0.14; S1334D, 1.07 ( 0.69; S1334H, 1.17 ( 0.16; S1334N, 0.97 ( 0.08; S1334T, 1.30 ( 0.11. Login to comment
120 labeling intensity of MRP1 mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, with [R-32 P]-8-N3ATP is weaker than that of wild type or S1334T (Figure 4), indicating that these mutants without the hydroxyl group at this position affect ATP binding, hydrolysis, and vanadate-dependent [R-32 P]-8-N3ADP trapping. Login to comment
121 Comparison of labeling intensities with [R-32 P]-8-N3ATP and [γ-32 P]-8-N3ATP further supports this conclusion: (1) The labeling intensity of MRP1 mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, with [R-32 P]-8-N3ATP is either similar to or weaker than that of [γ-32 P]-8-N3ATP labeling (Figure 4), indicating that the bound ATP in these mutants is not efficiently hydrolyzed. Login to comment
122 (2) The intensity of [γ-32 P]-8-N3ATP labeling on the mutants, including S1334A, S1334C, S1334D, S1334H, and S1334N, is either stronger than wild type or similar to wild type (Figure 4), implying that most of those [γ-32 P]-8-N3ATP labeling might occur at the unmutated NBD1 (8). Login to comment
155 As shown in Figure 6, 2.5 or 10 µM ATP significantly enhanced the vanadate-dependent [R-32 P]-8-N3ADP trapping in wild type MRP1 (Figure 6A) or in S1334T (Figure 6G), whereas there is no enhancement effect in S1334A (Figure 6B), S1334C (Figure 6C), S1334D (Figure 6D), S1334H (Figure 6E), or S1334N (Figure 6F), suggesting that these mutations significantly impaired the nucleotide binding at the mutated NBD2. Login to comment
161 In contrast, the labeling on S1334A, S1334C, S1334D, S1334H, or S1334N is only partially diminished, implying that ATP binding or vanadate-dependent ATP hydrolysis product ADP trapping at the mutated NBD2 is impaired. Login to comment
163 In contrast, mutation of the corresponding residue in NBD2 of MRP1, such as S1334A, S1334C, S1334D, S1334H, or S1334N, has no effect on the protein folding and processing (Figure 1), implying that the stereo structure surrounding the hydroxyl group of S1334 in NBD2 might be different from that of NBD1. Login to comment
175 group (S1334C) exerted slightly higher transport activity than that of S1334A, S1334D, S1334H, or S1334N (Figure 2B), implying that the sulfhydryl group in S1334C might weakly bind to the magnesium cofactor and the -phosphate of the bound ATP. Login to comment
178 The labeling intensities of [R-32 P]-8-N3ATP in wild type or S1334T are stronger than that of [γ-32 P]-8-N3ATP (Figure 4), whereas the labeling intensities of [R-32 P]-8-N3ATP in S1334A, S1334C, S1334D, S1334H, or S1334N are slightly weaker than that of [γ-32 P]-8-N3ATP (Figure 4), suggesting that even though there might be a trace amount of ATP binding at the mutated NBD2, the bound nucleotide might not be efficiently hydrolyzed. Login to comment
180 All of the S1334 mutants, including S1334A (Figure 6B), S1334C (Figure 6C), S1334D (Figure 6D), S1334H (Figure 6E), and S1334N (Figure 6F), lost the ATP-enhanced vanadate-dependent ADP trapping (16, 17) at the mutated NBD2, suggesting that these mutations significantly decreased the affinity for nucleotide at the mutated NBD2. Login to comment
182 The reduced nucleotide binding at the mutated NBD2, such as S1334A, S1334C, S1334D, S1334H, and S1334N, significantly decreased the ability to inhibit the LTC4 binding (Figure 7), similar to the mutations of K684E, G771A, or K1333E (19). Login to comment