ABCB3 p.Glu632Gln
Predicted by SNAP2: | A: D (91%), C: D (75%), D: D (80%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (91%), Y: D (91%), |
Predicted by PROVEAN: | A: D, C: D, D: N, 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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[hide] Catalytic site modifications of TAP1 and TAP2 and ... J Biol Chem. 2006 Dec 29;281(52):39839-51. Epub 2006 Oct 26. Perria CL, Rajamanickam V, Lapinski PE, Raghavan M
Catalytic site modifications of TAP1 and TAP2 and their functional consequences.
J Biol Chem. 2006 Dec 29;281(52):39839-51. Epub 2006 Oct 26., [PMID:17068338]
Abstract [show]
The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.
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No. Sentence Comment
44 Site-directed mutations (D668N in TAP1; E632Q in TAP2) wereintro- duced into the TAP1-his and TAP2 constructs in the pPCR2.1 vector (17) using the QuikChange site-directed mutagenesis kit.
X
ABCB3 p.Glu632Gln 17068338:44:40
status: NEW73 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template.
X
ABCB3 p.Glu632Gln 17068338:73:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:73:43
status: NEW76 The primers used for TAP2(E632D) were E632D forward, 5Ј-CTCATCCTGGAT- GATGCTACTAGTGCC-308; and E632D reverse, 5Ј-GGCACTA- GTAGCATCATCCAGGATGAG-3Ј.
X
ABCB3 p.Glu632Gln 17068338:76:30
status: NEWX
ABCB3 p.Glu632Gln 17068338:76:92
status: NEW78 TAP2(E632DH661Q) was generatedusingTAP2(E632D)asatemplateandusingtheprimers for TAP2(H661Q).
X
ABCB3 p.Glu632Gln 17068338:78:30
status: NEWX
ABCB3 p.Glu632Gln 17068338:78:92
status: NEW79 Sequences encoding TAP2, TAP2(E632Q), TAP2(E632D), TAP2(H661Q), TAP2(E632D/H661Q), and TAP2(E632Q/ H662A) were excised from pPCR2.1 with BglII and XhoI and inserted into pMSCV 2.1 that was digested with BglII and XhoI.
X
ABCB3 p.Glu632Gln 17068338:79:30
status: NEWX
ABCB3 p.Glu632Gln 17068338:79:92
status: NEW113 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins.
X
ABCB3 p.Glu632Gln 17068338:113:17
status: NEWX
ABCB3 p.Glu632Gln 17068338:113:69
status: NEWX
ABCB3 p.Glu632Gln 17068338:113:103
status: NEWX
ABCB3 p.Glu632Gln 17068338:113:179
status: NEW114 Microsomes were prepared of TAP1(D668N)⅐ TAP2, TAP1⅐TAP2(E632Q), or TAP1(D668N)⅐TAP2(E632Q) combinations, or wild type proteins, under conditions in which comparable levels of wild type or mutant proteins were expressed.
X
ABCB3 p.Glu632Gln 17068338:114:71
status: NEWX
ABCB3 p.Glu632Gln 17068338:114:106
status: NEWX
ABCB3 p.Glu632Gln 17068338:114:155
status: NEW117 Consistent with this expectation, a determination of KD values for binding of a fluorescent peptide substrate to TAP1(D668N)⅐TAP2 or TAP1⅐TAP2(E632Q) compared with ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006•VOLUME 281•NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39841 the corresponding wild type complexes (TAP1-his⅐TAP2 and TAP1⅐TAP2, respectively) revealed no difference in calculated affinities (data not shown).
X
ABCB3 p.Glu632Gln 17068338:117:157
status: NEW129 The TAP1(D668N)⅐TAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1⅐TAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays.
X
ABCB3 p.Glu632Gln 17068338:129:81
status: NEWX
ABCB3 p.Glu632Gln 17068338:129:131
status: NEWX
ABCB3 p.Glu632Gln 17068338:129:185
status: NEW130 The WT microsomes used were TAP1⅐TAP2 for comparisons with TAP1⅐TAP2(E632Q), and TAP1-his⅐TAP2 for comparisons with TAP1(D668N)⅐TAP2) and TAP1(D668N)⅐TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:130:83
status: NEWX
ABCB3 p.Glu632Gln 17068338:130:190
status: NEW136 DN indicates TAP1(D668N)⅐TAP2, EQ indicates TAP1⅐TAP2(E632Q), and DN.EQ indicates TAP1(D668N)⅐TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:136:68
status: NEWX
ABCB3 p.Glu632Gln 17068338:136:120
status: NEWX
ABCB3 p.Glu632Gln 17068338:136:182
status: NEWX
ABCB3 p.Glu632Gln 17068338:136:250
status: NEW137 The graph indicates average ϩATP/-ATP ratios from four independent translocation experiments for TAP1(D668N)⅐TAP2, five independent experiments for TAP1⅐TAP2(E632Q), and four independent experiments for TAP1(D668N)⅐TAP2(E632Q) (containing five independent comparisons of expression matched wild type and mutant).
X
ABCB3 p.Glu632Gln 17068338:137:178
status: NEWX
ABCB3 p.Glu632Gln 17068338:137:247
status: NEW142 For the TAP1⅐TAP2(E632Q), analyses, H0: WT ϭ M versus HA: WT Ͼ M, p value ϭ 0.0004465 (highly significant).
X
ABCB3 p.Glu632Gln 17068338:142:25
status: NEWX
ABCB3 p.Glu632Gln 17068338:142:34
status: NEWX
ABCB3 p.Glu632Gln 17068338:142:41
status: NEW143 Additionally, for TAP1⅐TAP2(E632Q), H0: C ϭ M versus HA: C Ͻ M, p value ϭ 0.01052 (significant).
X
ABCB3 p.Glu632Gln 17068338:143:31
status: NEW144 For the TAP1(D668N)⅐TAP2(E632Q) analyses, H0: WT ϭ M versus HA: WT Ͼ M, p value ϭ 0.002718 (highly significant).
X
ABCB3 p.Glu632Gln 17068338:144:32
status: NEWX
ABCB3 p.Glu632Gln 17068338:144:41
status: NEW145 Additionally, for TAP1(D668N)⅐TAP2(E632Q), H0: C ϭ M versus HA: C Ͻ M: p value ϭ 0.07645 (not significant).
X
ABCB3 p.Glu632Gln 17068338:145:42
status: NEW151 For TAP1⅐ TAP2(E632Q), the e9;atp signals were only slightly higher compared with the -atp signals (Fig. 2B), whereas very similar ϩatp and -atp signals were observed with TAP1(D668N)!50; TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:151:22
status: NEWX
ABCB3 p.Glu632Gln 17068338:151:33
status: NEWX
ABCB3 p.Glu632Gln 17068338:151:200
status: NEWX
ABCB3 p.Glu632Gln 17068338:151:214
status: NEWX
ABCB3 p.Glu632Gln 17068338:151:269
status: NEW152 Fig. 2E shows the compiled average ϩatp/ -atp ratios from four independent peptide translocation experiments with TAP1(D668N)⅐TAP2, five independent experiments with TAP1⅐TAP2(E632Q), and four independent experiments with TAP1(D668N)⅐TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:152:196
status: NEWX
ABCB3 p.Glu632Gln 17068338:152:208
status: NEWX
ABCB3 p.Glu632Gln 17068338:152:242
status: NEWX
ABCB3 p.Glu632Gln 17068338:152:266
status: NEW153 The compiled data in Fig. 2E, as well as the individual experiments shown in Fig. 2, A-C, indicated that the TAP1(D668N)⅐TAP2 combination was significantly translocation active, whereas TAP1⅐TAP2(E632Q) and TAP1(D668N)⅐TAP2(E632Q) complexes were significantly impaired relative to wild type TAP.
X
ABCB3 p.Glu632Gln 17068338:153:25
status: NEWX
ABCB3 p.Glu632Gln 17068338:153:33
status: NEWX
ABCB3 p.Glu632Gln 17068338:153:121
status: NEWX
ABCB3 p.Glu632Gln 17068338:153:210
status: NEWX
ABCB3 p.Glu632Gln 17068338:153:245
status: NEW154 Furthermore, the TAP1⅐TAP2(E632Q) mutant microsomes had slightly higher ϩatp/-atp ratios compared with control microsomes, indicating low but measurable activity.
X
ABCB3 p.Glu632Gln 17068338:154:34
status: NEW156 The TAP1(D668N) and TAP2(E632Q) Mutations Enhance Efficiencies of Labeling of TAP Complexes with 8-Azido-nucleotides-The E632Q and D668N mutations target putative ␥-phosphate contact residues; these mutations are not expected to alter nucleotide binding affinities.
X
ABCB3 p.Glu632Gln 17068338:156:25
status: NEWX
ABCB3 p.Glu632Gln 17068338:156:121
status: NEW172 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP.
X
ABCB3 p.Glu632Gln 17068338:172:46
status: NEW176 TAP2 when expressed alone binds weakly to 8-azido-[␥- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity.
X
ABCB3 p.Glu632Gln 17068338:176:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:176:69
status: NEWX
ABCB3 p.Glu632Gln 17068338:176:109
status: NEW177 Low labeling signals were obtained for binding of both TAP2 and TAP2(E632Q) to 8-azido-[$25;-32 P]ATP, which were difficult to quantify (data not shown).
X
ABCB3 p.Glu632Gln 17068338:177:18
status: NEWX
ABCB3 p.Glu632Gln 17068338:177:69
status: NEWX
ABCB3 p.Glu632Gln 17068338:177:85
status: NEW178 When TAP2 or TAP2(E632Q) were expressed in combination with TAP1-eGFP, labeling of TAP2(E632Q) was more efficient than that of TAP2, in analyses with both 8-azido-ATP and 8-azido-ADP (Fig. 3, G and I).
X
ABCB3 p.Glu632Gln 17068338:178:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:178:18
status: NEWX
ABCB3 p.Glu632Gln 17068338:178:88
status: NEW179 The TAP2(E632Q) mutation also enhanced the labeling efficiency ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006•VOLUME 281•NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39843 FIGURE 3.
X
ABCB3 p.Glu632Gln 17068338:179:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:179:85
status: NEW180 Labeling with 8-azido-nucleotides of TAP complexes with a single TAP1(D668N) or TAP2(E632Q) mutation.
X
ABCB3 p.Glu632Gln 17068338:180:85
status: NEW186 F, immunoblotting analyses of microsomes expressing TAP1-eGFP⅐TAP2 (WT, lanes 1 and 3) or TAP1-eGFP⅐TAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[␥-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[␣-32 P]ADP labeling analyses.
X
ABCB3 p.Glu632Gln 17068338:186:119
status: NEW191 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J).
X
ABCB3 p.Glu632Gln 17068338:191:30
status: NEWX
ABCB3 p.Glu632Gln 17068338:191:185
status: NEW192 The observation that the TAP2(E632Q) mutation enhances 8-azido-ATP labeling of TAP1 residues (cross-labeling, Ref. 18) is consistent with the possibility that this mutation stabilizes nucleotide-bound conformations of both TAP1 and TAP2, such as would be observed in a transition-state TAP1⅐TAP2 NBD dimer.
X
ABCB3 p.Glu632Gln 17068338:192:30
status: NEWX
ABCB3 p.Glu632Gln 17068338:192:95
status: NEW195 As expected from labeling analyses with the single mutants, labeling of TAP1(D668N)⅐TAP2(E632Q) was more efficient than that of TAP1⅐TAP2 (data not shown), although because fluorescence protein-tagged versions of mutant TAP1 or TAP2 were not available, it was not possible to resolve labeling of the individual subunits.
X
ABCB3 p.Glu632Gln 17068338:195:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:195:21
status: NEWX
ABCB3 p.Glu632Gln 17068338:195:96
status: NEWX
ABCB3 p.Glu632Gln 17068338:195:510
status: NEW196 The TAP2(E632Q) and (E632Q/H661A) Mutations Significantly Impact the Ability of TAP2 to Induce MHC Class I Surface Expression in TAP2-deficient Cells, whereas Small Effects Are Observed with the Counterpart TAP1 Mutations (D668N and D668N/Q701A)-To extend the analyses of peptide translocation activity to assessments of the abilities of the TAP mutants to restore MHC class I surface expression in TAP-deficient human cells, retroviral constructs were generated that encoded TAP1, TAP1(D668N), TAP2, and TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:196:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:196:21
status: NEWX
ABCB3 p.Glu632Gln 17068338:196:95
status: NEWX
ABCB3 p.Glu632Gln 17068338:196:121
status: NEWX
ABCB3 p.Glu632Gln 17068338:196:510
status: NEW197 TAP2-deficient STF-1 cells (25) were infected with the viruses encoding wild type TAP2 or TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:197:94
status: NEW199 Consistent with the marked reduction in the peptide translocation activity of TAP complexes that was induced by the TAP2(E632Q) mutation, the mutant had a significantly reduced ability to restore MHC class I surface expression in TAP2-deficient cells (Fig. 4A).
X
ABCB3 p.Glu632Gln 17068338:199:94
status: NEWX
ABCB3 p.Glu632Gln 17068338:199:121
status: NEWX
ABCB3 p.Glu632Gln 17068338:199:225
status: NEW200 Over 10 independent flow cytometric analyses, STF-1 cells infected with viruses encoding TAP2(E632Q) displayed an average mean fluorescence ratio of 42% relative to that observed with cells that were infected with wild type TAP2 (Fig. 4G).
X
ABCB3 p.Glu632Gln 17068338:200:94
status: NEW202 The average mean fluorescence ratio of the parent uninfected STF-1 cells was 23% relative to cells infected with the wild type TAP2-encoding virus; Fig. 4G), indicating low transport activity of TAP complexes containing TAP2(E632Q) (Fig. 4G).
X
ABCB3 p.Glu632Gln 17068338:202:225
status: NEW208 The peptide translocation assays in insect cells, however, seemed to have lower sensitivity, as the low activity of the TAP2(E632Q) mutant complexes was more readily detectable using TAP activity assays that measured restoration of MHC class I surface expression.
X
ABCB3 p.Glu632Gln 17068338:208:125
status: NEWX
ABCB3 p.Glu632Gln 17068338:208:142
status: NEW211 To additionally explore the effect of the switch region residues on TAP activity, we generated another retroviral construct encoding the TAP2(E632Q/H661A) double mutant, and assessed the ability of the mutant to restore MHC class I surface expression in TAP2-deficient cells.
X
ABCB3 p.Glu632Gln 17068338:211:54
status: NEWX
ABCB3 p.Glu632Gln 17068338:211:142
status: NEW212 The mean fluorescence values of cells expressing TAP2(E632Q/H661A) ranged from 27 to 34% relative to that observed with cells expressing wild type TAP2 (six measurements), only slightly greater than that of the parent STF-1 cells that were TAP2-deficient (mean fluorescence 19-31% relative to wild type) (Fig. 4, C and G).
X
ABCB3 p.Glu632Gln 17068338:212:54
status: NEWX
ABCB3 p.Glu632Gln 17068338:212:63
status: NEWX
ABCB3 p.Glu632Gln 17068338:212:137
status: NEW213 In each of six- independent flow cytometric analyses, the TAP2(E632Q/ H661A) double mutant was more significantly impaired than the TAP2(E632Q) single mutant.
X
ABCB3 p.Glu632Gln 17068338:213:63
status: NEWX
ABCB3 p.Glu632Gln 17068338:213:86
status: NEWX
ABCB3 p.Glu632Gln 17068338:213:137
status: NEWX
ABCB3 p.Glu632Gln 17068338:213:146
status: NEW214 However, in each of the analyses, residual enhancement of MHC class I surface expression was consistently observable in cells expressing the TAP2(E632Q/H661A) double mutant compared with unin- TABLE 1 Apparent affinities of indicated TAP constructs for 8-azido-͓␥-32 P͔ATP and 8-azido-͓␣-32 P͔ADP when expressed as single subunits or in complex with the indicated partner subunit The first four rows of measurements correspond to the derived apparent affinities of the wild type TAP1 or TAP1(D668N) for 8-azido-[␥-32 P]ATP and 8-azido-[␣- 32 P]ADP when expressed individually or in complex with TAP2-eYFP.
X
ABCB3 p.Glu632Gln 17068338:214:146
status: NEWX
ABCB3 p.Glu632Gln 17068338:214:207
status: NEW216 Rows 7-10 correspond to the derived apparent affinities of the wild type TAP2 or TAP2(E632Q) for 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP when expressed individually or in complex with TAP1-eGFP.
X
ABCB3 p.Glu632Gln 17068338:216:86
status: NEWX
ABCB3 p.Glu632Gln 17068338:216:207
status: NEW217 The last two rows of measurements correspond to the derived apparent affinities of TAP1-eGFP for 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP when expressed in complex with wild type TAP2 or TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:217:58
status: NEWX
ABCB3 p.Glu632Gln 17068338:217:209
status: NEW220 The labeling efficiencies of single subunit TAP2 and TAP2(E632Q) with 8-azido-[␥-32 P]ATP were very inefficient and not accurately quantifiable (ND), as previously described for single subunit TAP2 (18).
X
ABCB3 p.Glu632Gln 17068338:220:58
status: NEWX
ABCB3 p.Glu632Gln 17068338:220:396
status: NEWX
ABCB3 p.Glu632Gln 17068338:220:480
status: NEWX
ABCB3 p.Glu632Gln 17068338:220:592
status: NEW221 Subunit for which KD value is derived Partner subunit KD(ATP) KD(ADP) M TAP1 None 2.5 1.8 Ϯ 0.6 TAP1(D668N) None 1.1 Ϯ 0.6 2.2 Ϯ 2.3 TAP1 TAP2-eYFP 2.6 Ϯ 1.5 0.7 Ϯ 0.5 TAP1(D668N) TAP2-eYFP 1.7 Ϯ 0.6 2.0 Ϯ 0.03 TAP2-eYFP TAP1 1.6 Ϯ 1.3 0.7 Ϯ 0.6 TAP2-eYFP TAP1(D668N) 2.7 Ϯ 0.6 2.2 Ϯ 1.0 TAP2 None NDa 2.3 Ϯ 1.1 TAP2(E632Q) None ND 4.9 Ϯ 0.1 TAP2 TAP1-eGFP 2.1 Ϯ 0.5 1.1 Ϯ 0.01 TAP2(E632Q) TAP1-eGFP 2.1 Ϯ 1.7 2.4 Ϯ 2.7 TAP1-eGFP TAP2 1.5 Ϯ 0.04 1.4 Ϯ 0.3 TAP1-eGFP TAP2(E632Q) 1.1 Ϯ 1.0 1.3 Ϯ 1.4 a ND, not determined.
X
ABCB3 p.Glu632Gln 17068338:221:397
status: NEWX
ABCB3 p.Glu632Gln 17068338:221:481
status: NEWX
ABCB3 p.Glu632Gln 17068338:221:593
status: NEW227 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q).
X
ABCB3 p.Glu632Gln 17068338:227:58
status: NEWX
ABCB3 p.Glu632Gln 17068338:227:80
status: NEW235 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation.
X
ABCB3 p.Glu632Gln 17068338:235:233
status: NEW241 The TAP2(E632D) and (H632Q) Mutations Result in Attenuated TAP Activity, whereas the TAP1(D668E/Q701H) Mutation Induces a Slight Increase in TAP Activity-In TAP1 sequences, Asp668 and Gln701 replace highly conserved glutamic acidandhistidineresidues,respectively,inotherABCtransporters (Fig. 1).
X
ABCB3 p.Glu632Gln 17068338:241:151
status: NEWX
ABCB3 p.Glu632Gln 17068338:241:167
status: NEW242 Our results (Figs. 2 and 4) indicated that TAP1(D668N) andTAP1(D668N/Q701A)mutationsaffected TAP function less significantly than the counterpart TAP2(E632Q) and TAP2(E632Q/H661A) mutations.
X
ABCB3 p.Glu632Gln 17068338:242:151
status: NEWX
ABCB3 p.Glu632Gln 17068338:242:167
status: NEW286 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J).
X
ABCB3 p.Glu632Gln 17068338:286:75
status: NEW295 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport.
X
ABCB3 p.Glu632Gln 17068338:295:124
status: NEW72 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template.
X
ABCB3 p.Glu632Gln 17068338:72:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:72:43
status: NEW112 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins.
X
ABCB3 p.Glu632Gln 17068338:112:17
status: NEWX
ABCB3 p.Glu632Gln 17068338:112:179
status: NEW116 Consistent with this expectation, a determination of KD values for binding of a fluorescent peptide substrate to TAP1(D668N)ዼTAP2 or TAP1ዼTAP2(E632Q) compared with ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites DECEMBER 29, 2006ߦVOLUME 281ߦNUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 39841 at SEMMELWEIS UNIV OF MEDICINE on December 5, the corresponding wild type complexes (TAP1-hisዼTAP2 and TAP1ዼTAP2, respectively) revealed no difference in calculated affinities (data not shown).
X
ABCB3 p.Glu632Gln 17068338:116:155
status: NEW128 The TAP1(D668N)ዼTAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1ዼTAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays.
X
ABCB3 p.Glu632Gln 17068338:128:129
status: NEW135 DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:135:66
status: NEWX
ABCB3 p.Glu632Gln 17068338:135:117
status: NEW141 For the TAP1ዼTAP2(E632Q), analyses, H0: WT afd; M versus HA: WT b0e; M, p value afd; 0.0004465 (highly significant).
X
ABCB3 p.Glu632Gln 17068338:141:24
status: NEWX
ABCB3 p.Glu632Gln 17068338:141:31
status: NEW150 For TAP1ዼ TAP2(E632Q), the af9;atp signals were only slightly higher compared with the afa;atp signals (Fig. 2B), whereas very similar af9;atp and afa;atp signals were observed with TAP1(D668N)ዼ TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:150:21
status: NEWX
ABCB3 p.Glu632Gln 17068338:150:208
status: NEWX
ABCB3 p.Glu632Gln 17068338:150:224
status: NEWX
ABCB3 p.Glu632Gln 17068338:150:242
status: NEW155 The TAP1(D668N) and TAP2(E632Q) Mutations Enhance Efficiencies of Labeling of TAP Complexes with 8-Azido-nucleotides-The E632Q and D668N mutations target putative ॹ-phosphate contact residues; these mutations are not expected to alter nucleotide binding affinities.
X
ABCB3 p.Glu632Gln 17068338:155:25
status: NEWX
ABCB3 p.Glu632Gln 17068338:155:121
status: NEW171 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP.
X
ABCB3 p.Glu632Gln 17068338:171:46
status: NEW175 TAP2 when expressed alone binds weakly to 8-azido-[ॹ- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity.
X
ABCB3 p.Glu632Gln 17068338:175:18
status: NEWX
ABCB3 p.Glu632Gln 17068338:175:88
status: NEWX
ABCB3 p.Glu632Gln 17068338:175:108
status: NEW185 F, immunoblotting analyses of microsomes expressing TAP1-eGFPዼTAP2 (WT, lanes 1 and 3) or TAP1-eGFPዼTAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[ॹ-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[ॷ-32 P]ADP labeling analyses.
X
ABCB3 p.Glu632Gln 17068338:185:117
status: NEW190 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J).
X
ABCB3 p.Glu632Gln 17068338:190:268
status: NEW194 As expected from labeling analyses with the single mutants, labeling of TAP1(D668N)ዼTAP2(E632Q) was more efficient than that of TAP1ዼTAP2 (data not shown), although because fluorescence protein-tagged versions of mutant TAP1 or TAP2 were not available, it was not possible to resolve labeling of the individual subunits.
X
ABCB3 p.Glu632Gln 17068338:194:95
status: NEW198 Consistent with the marked reduction in the peptide translocation activity of TAP complexes that was induced by the TAP2(E632Q) mutation, the mutant had a significantly reduced ability to restore MHC class I surface expression in TAP2-deficient cells (Fig. 4A).
X
ABCB3 p.Glu632Gln 17068338:198:121
status: NEW201 The average mean fluorescence ratio of the parent uninfected STF-1 cells was 23% relative to cells infected with the wild type TAP2-encoding virus; Fig. 4G), indicating low transport activity of TAP complexes containing TAP2(E632Q) (Fig. 4G).
X
ABCB3 p.Glu632Gln 17068338:201:225
status: NEW207 The peptide translocation assays in insect cells, however, seemed to have lower sensitivity, as the low activity of the TAP2(E632Q) mutant complexes was more readily detectable using TAP activity assays that measured restoration of MHC class I surface expression.
X
ABCB3 p.Glu632Gln 17068338:207:125
status: NEW210 To additionally explore the effect of the switch region residues on TAP activity, we generated another retroviral construct encoding the TAP2(E632Q/H661A) double mutant, and assessed the ability of the mutant to restore MHC class I surface expression in TAP2-deficient cells.
X
ABCB3 p.Glu632Gln 17068338:210:63
status: NEWX
ABCB3 p.Glu632Gln 17068338:210:137
status: NEW215 Rows 7-10 correspond to the derived apparent affinities of the wild type TAP2 or TAP2(E632Q) for 8-azido-[ॹ-32 P]ATP and 8-azido-[ॷ-32 P]ADP when expressed individually or in complex with TAP1-eGFP.
X
ABCB3 p.Glu632Gln 17068338:215:86
status: NEW219 The labeling efficiencies of single subunit TAP2 and TAP2(E632Q) with 8-azido-[ॹ-32 P]ATP were very inefficient and not accurately quantifiable (ND), as previously described for single subunit TAP2 (18).
X
ABCB3 p.Glu632Gln 17068338:219:58
status: NEW226 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q).
X
ABCB3 p.Glu632Gln 17068338:226:58
status: NEWX
ABCB3 p.Glu632Gln 17068338:226:80
status: NEW234 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation.
X
ABCB3 p.Glu632Gln 17068338:234:316
status: NEW285 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J).
X
ABCB3 p.Glu632Gln 17068338:285:75
status: NEW294 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport.
X
ABCB3 p.Glu632Gln 17068338:294:124
status: NEW42 Site-directed mutations (D668N in TAP1; E632Q in TAP2) wereintro- duced into the TAP1-his and TAP2 constructs in the pPCR2.1 vector (17) using the QuikChange site-directed mutagenesis kit. For TAP1, the primers were: 5b18;-GTGTACTTATTCTAGATAA- TGCCACCAGTGCCCTG-3b18; and 5b18;-CAGGGCACTGGTGGC- ATTATCTAGAATAAGTACAC-3b18; (reverse primer).
X
ABCB3 p.Glu632Gln 17068338:42:40
status: NEW70 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template.
X
ABCB3 p.Glu632Gln 17068338:70:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:70:43
status: NEW110 RESULTS The TAP2(E632Q) Mutation Impacts Peptide Translocation More Significantly Than the TAP1(D668N) Mutation-A histidine-tagged version of mutant TAP1(D668N) and untagged TAP2(E632Q) were expressed in insect cells along with the partner wild type subunits, using baculoviruses encoding the desired proteins.
X
ABCB3 p.Glu632Gln 17068338:110:17
status: NEWX
ABCB3 p.Glu632Gln 17068338:110:179
status: NEW111 Microsomes were prepared of TAP1(D668N)ዼ TAP2, TAP1ዼTAP2(E632Q), or TAP1(D668N)ዼTAP2(E632Q) combinations, or wild type proteins, under conditions in which comparable levels of wild type or mutant proteins were expressed.
X
ABCB3 p.Glu632Gln 17068338:111:69
status: NEWX
ABCB3 p.Glu632Gln 17068338:111:103
status: NEW126 The TAP1(D668N)ዼTAP2 complex is able to transport peptide (A), low to residual activity is observable with TAP1ዼTAP2(E632Q) (B), whereas the double mutant (C) did not display measurable activity by these assays.
X
ABCB3 p.Glu632Gln 17068338:126:129
status: NEW127 The WT microsomes used were TAP1ዼTAP2 for comparisons with TAP1ዼTAP2(E632Q), and TAP1-hisዼTAP2 for comparisons with TAP1(D668N)ዼTAP2) and TAP1(D668N)ዼTAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:127:81
status: NEWX
ABCB3 p.Glu632Gln 17068338:127:185
status: NEW133 DN indicates TAP1(D668N)ዼTAP2, EQ indicates TAP1ዼTAP2(E632Q), and DN.EQ indicates TAP1(D668N)ዼTAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:133:66
status: NEWX
ABCB3 p.Glu632Gln 17068338:133:117
status: NEW134 The graph indicates average af9;ATP/afa;ATP ratios from four independent translocation experiments for TAP1(D668N)ዼTAP2, five independent experiments for TAP1ዼTAP2(E632Q), and four independent experiments for TAP1(D668N)ዼTAP2(E632Q) (containing five independent comparisons of expression matched wild type and mutant).
X
ABCB3 p.Glu632Gln 17068338:134:182
status: NEWX
ABCB3 p.Glu632Gln 17068338:134:250
status: NEW139 For the TAP1ዼTAP2(E632Q), analyses, H0: WT afd; M versus HA: WT b0e; M, p value afd; 0.0004465 (highly significant).
X
ABCB3 p.Glu632Gln 17068338:139:24
status: NEW140 Additionally, for TAP1ዼTAP2(E632Q), H0: C afd; M versus HA: C b0d; M, p value afd; 0.01052 (significant).
X
ABCB3 p.Glu632Gln 17068338:140:34
status: NEW148 For TAP1ዼ TAP2(E632Q), the af9;atp signals were only slightly higher compared with the afa;atp signals (Fig. 2B), whereas very similar af9;atp and afa;atp signals were observed with TAP1(D668N)ዼ TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:148:21
status: NEWX
ABCB3 p.Glu632Gln 17068338:148:224
status: NEW149 Fig. 2E shows the compiled average af9;atp/ afa;atp ratios from four independent peptide translocation experiments with TAP1(D668N)ዼTAP2, five independent experiments with TAP1ዼTAP2(E632Q), and four independent experiments with TAP1(D668N)ዼTAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:149:200
status: NEWX
ABCB3 p.Glu632Gln 17068338:149:269
status: NEW169 Based on several reports that the counterpart E632Q mutation of ABC transporter NBDs stabilizes nucleotide-bound NBD dimers (for example, Ref. 28), it is possible that enhanced labeling of TAP1(D668N) reflects the stabilization of NBD interactions both in the presence of 8-azido-ATP and 8-azido-ADP.
X
ABCB3 p.Glu632Gln 17068338:169:46
status: NEW173 TAP2 when expressed alone binds weakly to 8-azido-[ॹ- 32 P]ATP as previously described (18); the TAP2(E632Q) mutation did not enhance the 8-azido-ATP binding affinity.
X
ABCB3 p.Glu632Gln 17068338:173:108
status: NEW174 Low labeling signals were obtained for binding of both TAP2 and TAP2(E632Q) to 8-azido-[ॹ-32 P]ATP, which were difficult to quantify (data not shown).
X
ABCB3 p.Glu632Gln 17068338:174:69
status: NEW183 F, immunoblotting analyses of microsomes expressing TAP1-eGFPዼTAP2 (WT, lanes 1 and 3) or TAP1-eGFPዼTAP2(E632Q)(M,lanes2and4).GandI,microsomesshowninlanes1and2ofFwereusedin8-azido-[ॹ-32 P]ATPlabelinganalysesandmicrosomesshown in lanes 3 and 4 of F were used in 8-azido-[ॷ-32 P]ADP labeling analyses.
X
ABCB3 p.Glu632Gln 17068338:183:117
status: NEW188 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 of TAP1-eGFP in complex (Fig. 3, G and I), to an extent greater than or equal to the enhancement of labeling of TAP2(E632Q) itself (Fig. 3, H and J).
X
ABCB3 p.Glu632Gln 17068338:188:268
status: NEW189 The observation that the TAP2(E632Q) mutation enhances 8-azido-ATP labeling of TAP1 residues (cross-labeling, Ref. 18) is consistent with the possibility that this mutation stabilizes nucleotide-bound conformations of both TAP1 and TAP2, such as would be observed in a transition-state TAP1ዼTAP2 NBD dimer.
X
ABCB3 p.Glu632Gln 17068338:189:30
status: NEW193 The TAP2(E632Q) and (E632Q/H661A) Mutations Significantly Impact the Ability of TAP2 to Induce MHC Class I Surface Expression in TAP2-deficient Cells, whereas Small Effects Are Observed with the Counterpart TAP1 Mutations (D668N and D668N/Q701A)-To extend the analyses of peptide translocation activity to assessments of the abilities of the TAP mutants to restore MHC class I surface expression in TAP-deficient human cells, retroviral constructs were generated that encoded TAP1, TAP1(D668N), TAP2, and TAP2(E632Q).
X
ABCB3 p.Glu632Gln 17068338:193:9
status: NEWX
ABCB3 p.Glu632Gln 17068338:193:21
status: NEWX
ABCB3 p.Glu632Gln 17068338:193:510
status: NEW205 The peptide translocation assays in insect cells, however, seemed to have lower sensitivity, as the low activity of the TAP2(E632Q) mutant complexes was more readily detectable using TAP activity assays that measured restoration of MHC class I surface expression.
X
ABCB3 p.Glu632Gln 17068338:205:125
status: NEW209 The mean fluorescence values of cells expressing TAP2(E632Q/H661A) ranged from 27 to 34% relative to that observed with cells expressing wild type TAP2 (six measurements), only slightly greater than that of the parent STF-1 cells that were TAP2-deficient (mean fluorescence 19-31% relative to wild type) (Fig. 4, C and G).
X
ABCB3 p.Glu632Gln 17068338:209:54
status: NEW218 Subunit for which KD value is derived Partner subunit KD(ATP) KD(ADP) òe;M TAP1 None 2.5 1.8 afe; 0.6 TAP1(D668N) None 1.1 afe; 0.6 2.2 afe; 2.3 TAP1 TAP2-eYFP 2.6 afe; 1.5 0.7 afe; 0.5 TAP1(D668N) TAP2-eYFP 1.7 afe; 0.6 2.0 afe; 0.03 TAP2-eYFP TAP1 1.6 afe; 1.3 0.7 afe; 0.6 TAP2-eYFP TAP1(D668N) 2.7 afe; 0.6 2.2 afe; 1.0 TAP2 None NDa 2.3 afe; 1.1 TAP2(E632Q) None ND 4.9 afe; 0.1 TAP2 TAP1-eGFP 2.1 afe; 0.5 1.1 afe; 0.01 TAP2(E632Q) TAP1-eGFP 2.1 afe; 1.7 2.4 afe; 2.7 TAP1-eGFP TAP2 1.5 afe; 0.04 1.4 afe; 0.3 TAP1-eGFP TAP2(E632Q) 1.1 afe; 1.0 1.3 afe; 1.4 a ND, not determined.
X
ABCB3 p.Glu632Gln 17068338:218:396
status: NEWX
ABCB3 p.Glu632Gln 17068338:218:480
status: NEWX
ABCB3 p.Glu632Gln 17068338:218:592
status: NEW224 The abbreviations are: WT for wild type TAP2, EQ for TAP2(E632Q), EQHA for TAP2(E632Q/H661A), ED for TAP2(E632D), HQ for TAP2(H661Q), and EDHQ for TAP2(E632D/H661Q).
X
ABCB3 p.Glu632Gln 17068338:224:58
status: NEWX
ABCB3 p.Glu632Gln 17068338:224:80
status: NEW232 ATP Hydrolysis at the TAP1 and TAP2 Nucleotide Binding Sites 39846 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 52ߦDECEMBER 29, 2006 fected cells (representative analysis is shown Fig. 4C), indicating that the peptide translocation activity of TAP complexes was not completely impaired by the TAP2(E632Q/H661A) double mutation.
X
ABCB3 p.Glu632Gln 17068338:232:316
status: NEW239 Our results (Figs. 2 and 4) indicated that TAP1(D668N) andTAP1(D668N/Q701A)mutationsaffected TAP function less significantly than the counterpart TAP2(E632Q) and TAP2(E632Q/H661A) mutations.
X
ABCB3 p.Glu632Gln 17068338:239:151
status: NEWX
ABCB3 p.Glu632Gln 17068338:239:167
status: NEW283 Alternatively, blocking hydrolysis at a single site, such as with the TAP2(E632Q) mutant described here, could result in enhanced labeling of both TAP1 and TAP2, if the complexes are trapped in a dimeric conformation (Fig. 3J).
X
ABCB3 p.Glu632Gln 17068338:283:75
status: NEW292 When the ATPase activity of the TAP2 site is reduced to a level below that of the TAP1 site (as might be the case with TAP2(E632Q/H661A)), it is possible that hydrolysis at the TAP1 site drives the residual transport.
X
ABCB3 p.Glu632Gln 17068338:292:124
status: NEW[hide] Analyses of conformational states of the transport... J Biol Chem. 2013 Dec 27;288(52):37039-47. doi: 10.1074/jbc.M113.504696. Epub 2013 Nov 6. Geng J, Sivaramakrishnan S, Raghavan M
Analyses of conformational states of the transporter associated with antigen processing (TAP) protein in a native cellular membrane environment.
J Biol Chem. 2013 Dec 27;288(52):37039-47. doi: 10.1074/jbc.M113.504696. Epub 2013 Nov 6., [PMID:24196954]
Abstract [show]
The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.
Comments [show]
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No. Sentence Comment
41 The hydrolysis-deficient TAP2 mutant TAP2-EYFP(E632Q) was made by introducing the E632Q mutation into TAP2-EYFP using the QuikChange site-directed mutagenesis kit.
X
ABCB3 p.Glu632Gln 24196954:41:47
status: NEWX
ABCB3 p.Glu632Gln 24196954:41:82
status: NEW77 A fluorophore-labeled TAP substrate (RYWANATKFITCSR) was incubated with control cells, cells expressing wild-type TAP complexes, T1C-T2Y, or an ATPase-deficient mutant complex, T1C-T2Y(E632Q) (11).
X
ABCB3 p.Glu632Gln 24196954:77:185
status: NEW78 The glutamate-to-glutamine mutation at the residue C-terminal to the Walker B motif, as in TAP2(E632Q), impairs ATP hydrolysis and NBD dimer separation in many ABC transporters (8, 16, 17).
X
ABCB3 p.Glu632Gln 24196954:78:96
status: NEW82 The ATPase-deficient mutant complex T1C/T2Y(E632Q) did not transport peptides, as expected from previous measurements with untagged mutant complexes (11) (Fig. 2B).
X
ABCB3 p.Glu632Gln 24196954:82:44
status: NEW114 As can be seen in Fig. 3, C and D, with the ATPase-deficient TAP mutant complex T1C/T2Y(E632Q), the addition of ATP, but not ADP, markedly increased the FRET signal even at 4 &#b0;C (Fig. 3C).
X
ABCB3 p.Glu632Gln 24196954:114:88
status: NEW168 We expected that the ATPase-deficient mutant TAP1-TAP2(E632Q) would also arrest the catalytic cycle of TAP in a prehydrolysis reaction intermediate in the presence of both ATP and peptide because TAP1-TAP2(E632Q) complexes are able to bind nucleotides and peptide with similar affinities as wild-type TAP (11).
X
ABCB3 p.Glu632Gln 24196954:168:55
status: NEWX
ABCB3 p.Glu632Gln 24196954:168:206
status: NEW169 As shown in Fig. 2B, TAP1-TAP2(E632Q) complexes were impaired for peptide transport into sf9 cell microsomes because of a deduced deficiency in ATPase activity.
X
ABCB3 p.Glu632Gln 24196954:169:31
status: NEW172 At 4 &#b0;C (Fig. 5G), similar FRET efficiencies were observed with T1C-T2Y(E632Q) complexes in the presence of both nucleotides and peptide, compared with that observed in the presence of peptide alone.
X
ABCB3 p.Glu632Gln 24196954:172:76
status: NEW190 B-H, FRET efficiency changes were measured in permeabilized cells expressing the T1C-T2Y or T1C-T2Y(E632Q) complexes in the presence of nucleotide alone, peptide RKL alone, nucleotide af9; the peptide RKL, or, additionally, in the presence of vanadate (Vi) at 4 &#b0;C or 27 &#b0;C as indicated.
X
ABCB3 p.Glu632Gln 24196954:190:100
status: NEW197 The T1C-T2Y(E632Q) complex is more sensitive to nucleotides than the wild-type TAP complex, as suggested by the findings of ATP-induced enhancement in FRET efficiency, even at 4 &#b0;C (Fig. 3C), and a more significant enhancement in FRET at 27 &#b0;C compared with wild-type complexes (normalized FRET efficiency changes of b03;3.6 and b03;2%, respectively).
X
ABCB3 p.Glu632Gln 24196954:197:12
status: NEW198 These findings are consistent with our previous study, which showed that, compared with wild-type TAP1-TAP2, the TAP2(E632Q) mutation enhances labeling of both TAP1 and TAP2 with 8-azido-ATP (11).
X
ABCB3 p.Glu632Gln 24196954:198:118
status: NEW200 We found that nucleotide-induced TAP conformational change is temperature-dependent, indicated by higher FRET efficiencies of both the wild-type and T1C-T2Y(E632Q) mutant at 27 &#b0;C compared with 4 &#b0;C in the presence of nucleotide, both in the presence and in the absence of peptide.
X
ABCB3 p.Glu632Gln 24196954:200:157
status: NEW229 Interestingly, with T1C-T2Y(E632Q), ADP binding triggers NBD dimerization to the same extent as ATP in the presence of peptide.
X
ABCB3 p.Glu632Gln 24196954:229:28
status: NEW230 These findings are consistent with the possibility that the E632Q mutation of TAP2 lowers the energy barrier for the conformational change so that either ADP or ATP suffices to induce full closure.
X
ABCB3 p.Glu632Gln 24196954:230:60
status: NEW