ABCMdb

ABCC1 p.Ser1334Thr

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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[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
7 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. 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
16 Indeed, all of these mutants, except S1334T, almost completely abolished the ATP-dependent LTC4 transport, indicating that the interaction of the hydroxyl group at S1334 with Mg·ATP † This work was supported by a grant from the National Cancer Institute, National Institutes of Health (CA89078 to X.C.). 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
89 The Km (Mg·ATP) Value of S1334T Is Significantly Higher than That of Wild-Type MRP1. Login to comment
93 As shown in Figure 3 and Table 1, the Km(Mg ·ATP) of S1334T (104 µM Mg·ATP) is significantly higher (with a P value of 0.0024) than that of wild-type MRP1 (61 µM Mg·ATP). Login to comment
94 In addition, the Vmax (LTC4) value of S1334T (618 pmol mg-1 min-1 ) is also significantly higher (with a P value of 0.0016) than that of wild-type MRP1 (342 pmol mg-1 min-1 ), consistent with the results derived from a solution containing 4 mM ATP and 10 mM MgCl2 (Figure 2B). Login to comment
97 Indeed, all of the mutants, except S1334T, almost completely abolished the ATP-dependent LTC4 transport (Figure 2B), perhaps owing to affecting the ATP binding/hydrolysis. Login to comment
98 In order to prove this hypothesis, a similar amount of MRP1 protein in 10 µg of membrane protein (see Figure 4 legend) was used to label them with either [R-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP (with the same specific activity as [R-32 P]- Table 1: Km (Mg·ATP) and Vmax (LTC4) Values of Wild-Type and Mutant MRP1s Vmax (pmol mg-1 min-1)a Km (µM)a MRP1 342.0 ( 50.3 61.0 ( 3.3 S1334T 618.3 ( 11.9 104.0 ( 8.3 a Km (Mg·ATP) and Vmax (LTC4) values (n ) 3) for wild type and S1334T were derived from the corresponding Michaelis-Menten curves shown in Figure 3. Login to comment
99 The P values for comparison of Vmax (LTC4) and Km (Mg·ATP) of wild type versus S1334T are 0.0016 and 0.0024. Login to comment
101 However, the labeling intensity of wild-type MRP1 or S1334T with [R-32 P]-8-N3ATP, including the intact [R-32 P]-8-N3ATP bound at NBD1 and the vanadate-trapped ATP hydrolysis product [R-32 P]-8-N3ADP at NBD2 (8), is stronger than that of [γ-32 P]-8-N3ATP labeling including the intact [γ-32 P]-8-N3ATP bound at NBD1 and NBD2 and the 32 P-phospharylated products (8), implying that the majority of the bound ATP at NBD2 is hydrolyzed. 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
115 FIGURE 3: S1334T mutation increases the Km (ATP) for ATP-dependent LTC4 transport. Login to comment
118 Since the amounts of MRP1 proteins determined in Figure 2A were different, the amounts of membrane vesicles used in these experiments were adjusted to a similar amount by adding varying amounts of membrane vesicles prepared from parental BHK cells: 3 µg of wild-type MRP1; 2.308 µg of S1334T plus 0.692 µg of BHK. 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
123 In order to prove this hypothesis, S1334A and S1334T mutations were introduced into pDual/N-half/C-half expression plasmid (12, 13) and expressed in Sf21 cells. Membrane vesicles prepared from Sf21 cells (Figure 5A) were used to do ATP-dependent LTC4 transport and the photoaffinity labeling with either [R-32 P]-8-N3ATP or [γ-32 P]-8-N3ATP. Login to comment
124 As shown in Figure 5B, the ATP-dependent LTC4 transport activity of wild type N-half plus S1334T-mutated C-half is more or less similar to that of wild-type, whereas the transport activity of the wild type N-half plus S1334A-mutated C-half is significantly reduced. Login to comment
125 The [γ-32 P]-8-N3ATP labeling of wild type NBD1-containing N-half fragment is much higher than the corresponding labeling in wild type or S1334T-mutated NBD2-containing C-half fragment (Figure 5C), whereas the [R-32 P]-8-N3ATP labeling of wild type or S1334T-mutated NBD2-containing C-half fragment in the presence of vanadate is much higher than the corresponding labeling in NBD1-containing N-half fragment (Figure 5C), implying that ATP bound to wild type or S1334T-mutated NBD2 is efficiently hydrolyzed. Login to comment
134 The intensities of the MRP1 bands were determined by a scanning densitometer. The mean ratios (n ) 2, including the results derived from 200 and 300 ng of protein), considering the amount of wild type MRP1 as 1, of the mutant proteins are as follows: S1334A, 0.68 ( 0.06; S1334T, 0.90 ( 0.06. Login to comment
137 Wild type MRP1 or S1334A- or S1334T-mutated MRP1 was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells (12, 25). 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
160 In order to test this hypothesis, membrane vesicles containing these mutants were used to label them with 3 H-LTC4 in the presence or absence of 1 mM ATP and vanadate. As shown in Figure 7, the 3 H-LTC4 labeling on wild type MRP1 and S1334T as well as E1455Q is almost completely inhibited by the presence of ATP and vanadate. Login to comment
176 In contrast to other mutants, substitution of the S1334 with a threonine (S1334T) increased ATP-dependent LTC4 transport to ~175% of the wild-type MRP1 (Figures 2B and 3 and Table 1), indicating that the interactions of the hydroxyl group at the position of 1334 with the Mg2+ cofactorandthe -phosphateoftheboundMg·ATP(1,2,4-7) play a crucial role for ATP binding/hydrolysis at NBD2 and ATP-dependent LTC4 transport. 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
179 Indeed, a certain amount of [R-32 P]-8-N3ATP hydrolysis product [R-32 P]-8-N3ADP was trapped in wild-type or S1334T-mutated NBD2, whereas there was no detectable amount of [R-32 P]-8-N3ADP trapped in S1334A-mutated NBD2 (Figure 5C). Login to comment
181 In contrast, although substitution of the S1334 with a threonine (S1334T) introduced an extra methyl group, but kept the hydroxyl group at the original position, the S1334T mutation retained the ATP-enhanced vanadate-dependent ADP trapping (Figure 6G), indicating that the interactions of the hydroxyl group at position 1334 with the Mg2+ cofactor and the -phosphate of the bound Mg· ATP (1, 2, 4-7) play a very important role for ATP binding at NBD2. Login to comment