ABCMdb

ABCB3 p.His661Ala

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (91%), E: D (91%), F: D (91%), G: D (85%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (80%), R: D (91%), S: D (85%), T: D (91%), V: D (91%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: 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
73 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
74 The primers used were H661A forward, 5Ј-CTGGTGATTGCTGCCAGGCTGCAGACA-3Ј and H661A reverse, 5Ј-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3Ј. Login to comment
195 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. Login to comment
196 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). Login to comment
208 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. Login to comment
211 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. Login to comment
212 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). Login to comment
213 In each of six- independent flow cytometric analyses, the TAP2(E632Q/ H661A) double mutant was more significantly impaired than the TAP2(E632Q) single mutant. Login to comment
214 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. Login to comment
227 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). Login to comment
235 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. Login to comment
241 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). Login to comment
242 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. Login to comment
295 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. Login to comment
72 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
210 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. Login to comment
226 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). Login to comment
234 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. Login to comment
294 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. Login to comment
70 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. Login to comment
71 The primers used were H661A forward, 5b18;-CTGGTGATTGCTGCCAGGCTGCAGACA-3b18; and H661A reverse, 5b18;-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3b18;. Login to comment
193 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). Login to comment
209 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). Login to comment
224 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). Login to comment
232 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. Login to comment
239 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. Login to comment
292 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. Login to comment