ABCMdb

ABCB3 p.His661Gln

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
74 The primers used were H661A forward, 5@8;-CTGGTGATTGCTGCCAGGCTGCAGACA-3Ј and H661A reverse, 5Ј-TGTCTGCAGCCTGGCAGCAATCA- CCAG-3Ј. Login to comment
75 TAP2(E632D) and TAP2(H661Q) were generated using TAP2 in pPCR2.1 vector as the template. Login to comment
76 The primers used for TAP2(E632D) were E632D forward, 5Ј-CTCATCCTGGAT- GATGCTACTAGTGCC-3Ј and E632D reverse, 5Ј-GGCACTA- GTAGCATCATCCAGGATGAG-3Ј. Login to comment
77 The primers used for TAP2(H661Q) were H661Q forward, 5Ј-CTGGTGATTGCTC- AAAGGCTGCAGACA-3ЈandH661Qreverse,5Ј-TGTCTGCA- GCCTTTGAGCAATCACCAG-3Ј. Login to comment
78 TAP2(E632DH661Q) was generatedusingTAP2(E632D)asatemplateandusingtheprimers for TAP2(H661Q). Login to comment
79 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. 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
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
244 To further examine this possibility, we analyzed the effects of TAP2(E632D), TAP2(H661Q), and TAP2(E632D/H661Q) mutants upon TAP activity in STF-1 cells. Login to comment
246 The TAP2(E632D) mutation (41% activity relative to wild type) affected TAP activity more significantly than the TAP2(H661Q) mutation (63% relative to wild type), and the double mutant (39% relative to wild type) exhibited a similar level of impairment as the TAP2(E632D) mutation. Login to comment
264 DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment
279 These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment
290 Because the chimeric TAP1⅐T2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu Ͼ Asp and His Ͼ Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E and G). Login to comment
72 The TAP2(E632Q/H661A)wasgeneratedusingTAP2(E632Q)in pPCR2.1 vector as the template. 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
243 To further examine this possibility, we analyzed the effects of TAP2(E632D), TAP2(H661Q), and TAP2(E632D/H661Q) mutants upon TAP activity in STF-1 cells. Login to comment
245 The TAP2(E632D) mutation (41% activity relative to wild type) affected TAP activity more significantly than the TAP2(H661Q) mutation (63% relative to wild type), and the double mutant (39% relative to wild type) exhibited a similar level of impairment as the TAP2(E632D) mutation. Login to comment
263 DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment
278 These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment
289 Because the chimeric TAP1ዼT2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu b0e; Asp and His b0e; Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E 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
261 DISCUSSION Various modifications of TAP2 at Glu632 and His661 , including E632D and H661Q, significantly reduced the catalytic activity of TAP complexes (Figs. 2 and 4, A, C, E, and G). Login to comment
276 These data indicated a more important role for the switch region histidine in mediating ATP hydrolysis within isolated TAP1 NBD; however, at the TAP2 site of full-length TAP complexes, it appears that the TAP2(E632D) mutation causes a slightly greater loss in TAP activity compared with the TAP2(H661Q) mutation (Fig. 4E). Login to comment
287 Because the chimeric TAP1ዼT2MT1C complexes contained non-consensus residues (Asp668 and Gln701 ) at both nucleotide binding sites, the naturally occurring Glu b0e; Asp and His b0e; Gln alterations at these residues apparently did not completely abrogate the ability of the TAP1 site to hydrolyze ATP (consistent with the low but measurable activity of TAP2(E632D/H661Q); Fig. 4, E and G). Login to comment