ABCMdb

ABCC1 p.Asp792Leu

Predicted by SNAP2:	A: D (95%), C: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: 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] Allosteric interactions between the two non-equiva... J Biol Chem. 2000 Jul 7;275(27):20280-7. Hou Y, Cui L, Riordan JR, Chang X
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
J Biol Chem. 2000 Jul 7;275(27):20280-7., 2000-07-07 [PMID:10781583]

Abstract [show]
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

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No. Sentence Comment
33 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L, were generated by using procedures described previously (11). Login to comment

[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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No. Sentence Comment
161 The variant D792L was unable to mature conformationally and D792A led to an accumulation of equal amounts of mature and immature proteins but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 was essential for MRP1 function [101]. Login to comment

[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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Sentences [show]
No. Sentence Comment
21 However, substitution of the acidic amino acid D792 in Walker B motif with a hydrophobic residue, such as D792L- or D792A-mutated MRP1 [20,23], caused misfolding of the protein and prevented further analysis of the mutated protein. Login to comment
24 However, this mechanism of protein folding may not be applied to the misfolding caused by substitution of the acidic amino acid with a hydrophobic residue, such as D792L- or D792A-mutated MRP1 [20,23]. Login to comment
27 We suspected that D792A or D792L mutation abolished the hydrogen-bond formation between D792 and S685 and resulted in misfolding of the mutated MRP1 protein. Login to comment
94 We suspected that D792A or D792L mutation abolished the hydrogen-bond formation between D792 and S685 and resulted in misfolding of the mutated MRP1 protein. Login to comment

[hide] Mutations of the Walker B motif in the first nucle... Arch Biochem Biophys. 2001 Aug 1;392(1):153-61. Cui L, Hou YX, Riordan JR, Chang XB
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
Arch Biochem Biophys. 2001 Aug 1;392(1):153-61., [PMID:11469806]

Abstract [show]
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.

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Sentences [show]
No. Sentence Comment
4 However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. Login to comment
5 We have determined now that this latter effect is entirely due to the D792L substitution. Login to comment
13 Therefore to determine the contribution of NBD1 Walker B aspartate in MRP1 function, we had previously made substitutions at both positions simultaneously, i.e., D792L/D793L (8). Login to comment
17 The results showed that the D792L mutation was responsible for the defective maturation and function while D793L had minimal effect on either. Login to comment
36 The aspartic acid residues at positions of 792 and 793 were mutated either to alanine (Fig. 1B, D792A) or leucine residues (Fig. 1B, D792L and D793L) using the QuikChange Site Directed Mutagenesis kit. Login to comment
42 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). Login to comment
43 The cell lines expressing D792A, D792L, and D793L were generated using the same procedures (9). Login to comment
89 At this level of exposure the mature band is predominantly detected in the wild-type, while none of this band is present in the double mutant, D792L/D793L. Login to comment
90 D793L is indistinguishable from wild-type, whereas D792L is similar to the double mutant indicating that the substitution at this position is primarily responsible for the misprocessing. Login to comment
93 These were more apparent in a longer exposure of the same blot (Fig. 2B), especially in D792L/D793L, where, in addition to the major 170-kDa species, bands of approximately 160, 130, 100, and 30 kDa can be seen. Login to comment
94 Of these four only the 130- and 30-kDa bands are seen in the D792A and D792L lanes. Login to comment
105 To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment
107 By 3 h of chase the wild-type was completely converted to the larger mature form, whereas very little mature D792L and no mature D792L/D793L appeared. Login to comment
124 The following amounts of protein were loaded in each lane: 0.5 ␮g of wild-type MRP1; 8 ␮g of D792A; 18 ␮g of D792L; 0.5 ␮g of D793L; 20 ␮g of D792L/D793L. Login to comment
132 The following amounts were loaded in each lane: 0.5 ␮g of wild-type MRP1; 2 ␮g of D792A; 4 ␮g of D792L; 0.35 ␮g of D793L; 8 ␮g of D792L/D793L. Login to comment
133 The ratio of mature to immature protein in membrane vesicles of D792A and D792L are much higher than in whole cell lysates (Fig. 2A). Login to comment
134 (D) The smaller fragment of D792L/D793L is also core-glycosylated. Login to comment
136 Lanes 1 and 2, 1 ␮g of D793L cell lysates in each lane; Lanes 3 and 4, 4 ␮g of D792A cell lysates in each lane; Lanes 5 and 6, 10 ␮g of D792L/D793L cell lysates in each lane. Login to comment
137 Both the 170-kDa core-glycosylated MRP1 protein from either D793L, D792A, or D792L/D793L and 160-kDa degradation product from D792L/D793L were decreased in size by treatment with endoglycosidase H. pressing either the D792L or D792L/D793L mutants with either lactacystin or ALLN resulted in the total disappearance of the immature forms from nonionic detergent soluble fractions and appearance in insoluble pellets. Login to comment
149 In contrast the pattern of disappearance and appearance of bands was entirely different in the case of the D792L mutant (Fig. 5D), which matured poorly and the D792L/D793L mutant that did not mature at all (Fig. 5F). Login to comment
153 This smaller product of digestion of the immature D792L polypeptide appeared at a lower trypsin to membrane protein ratio (Fig. 5D, lane 6, 1:128). Login to comment
159 The D792L mutant is nearly and the D792L/D792L mutant is completely unable to undergo this conformation change. Login to comment
168 It was impossible to tell if the dysfunction of the D792L mutation was secondary to its inability to mature conformationally. Login to comment
174 Since hydrolysis is believed to drive MRP1 transport it would be expected that the mature D792A protein would not be capable of active transport. Login to comment
175 The data in Fig. 7 confirm this expectation, i.e., there is not significantly more ATP-dependent LTC4 uptake by vesicles containing D792A protein that does mature than by the other variants that do not mature (Fig. 7, D792L and D792L/D793L), nor by the NBD2 mutants (Fig. 7, K1333L and D1454L/E1455L) that do mature but have difficulties to hydrolyze ATP and to trap the hydrolysis product, ADP (8). Login to comment
181 The D792L mutant had minimal ability to trap nucleotide and transport an organic anion but it was also unable to mature during biosynthesis. Login to comment
184 However, when a different substitution, D792A, was made considerable maturation occurred although still less than wild-type (Figs. 2B and 2C). Login to comment
185 Nevertheless, this mutant was not capable of greater transport activity than the completely misprocessed D792L (Fig. 7). Login to comment
195 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment
204 (C) D792A, 0.73 ␮g protein in each lane. Login to comment
205 (D) D792L, 1.1 ␮g protein in each lane. Login to comment
206 (E) D793L, 0.3 ␮g protein in each lane. Login to comment
207 (F) D792L/D793L, 1.1 ␮g protein in each lane. Login to comment
216 This appears to occur with the D792L variant of MRP1 (Fig. 4). Login to comment
88 At this level of exposure the mature band is predominantly detected in the wild-type, while none of this band is present in the double mutant, D792L/D793L. Login to comment
92 These were more apparent in a longer exposure of the same blot (Fig. 2B), especially in D792L/D793L, where, in addition to the major 170-kDa species, bands of approximately 160, 130, 100, and 30 kDa can be seen. Login to comment
104 To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). Login to comment
106 By 3 h of chase the wild-type was completely converted to the larger mature form, whereas very little mature D792L and no mature D792L/D793L appeared. Login to comment
123 The following amounts of protein were loaded in each lane: 0.5 òe;g of wild-type MRP1; 8 òe;g of D792A; 18 òe;g of D792L; 0.5 òe;g of D793L; 20 òe;g of D792L/D793L. Login to comment
131 The following amounts were loaded in each lane: 0.5 òe;g of wild-type MRP1; 2 òe;g of D792A; 4 òe;g of D792L; 0.35 òe;g of D793L; 8 òe;g of D792L/D793L. Login to comment
135 Lanes 1 and 2, 1 òe;g of D793L cell lysates in each lane; Lanes 3 and 4, 4 òe;g of D792A cell lysates in each lane; Lanes 5 and 6, 10 òe;g of D792L/D793L cell lysates in each lane. Login to comment
148 In contrast the pattern of disappearance and appearance of bands was entirely different in the case of the D792L mutant (Fig. 5D), which matured poorly and the D792L/D793L mutant that did not mature at all (Fig. 5F). Login to comment
152 This smaller product of digestion of the immature D792L polypeptide appeared at a lower trypsin to membrane protein ratio (Fig. 5D, lane 6, 1:128). Login to comment
158 The D792L mutant is nearly and the D792L/D792L mutant is completely unable to undergo this conformation change. Login to comment
167 It was impossible to tell if the dysfunction of the D792L mutation was secondary to its inability to mature conformationally. Login to comment
180 The D792L mutant had minimal ability to trap nucleotide and transport an organic anion but it was also unable to mature during biosynthesis. Login to comment
194 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor. Login to comment
215 This appears to occur with the D792L variant of MRP1 (Fig. 4). Login to comment