ABCB3 p.Glu602Cys
| Predicted by SNAP2: | A: D (71%), C: D (75%), D: N (61%), F: D (85%), G: D (85%), H: D (75%), I: D (80%), K: D (91%), L: D (85%), M: D (80%), N: D (85%), P: D (91%), Q: D (71%), R: D (91%), S: D (85%), T: D (85%), V: D (75%), W: D (85%), Y: D (85%), |
| 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] Structural arrangement of the transmission interfa... Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5551-6. Epub 2009 Mar 18. Oancea G, O'Mara ML, Bennett WF, Tieleman DP, Abele R, Tampe R
Structural arrangement of the transmission interface in the antigen ABC transport complex TAP.
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5551-6. Epub 2009 Mar 18., [PMID:19297616]
Abstract [show]
The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum-lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and cross-linking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC half-transporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport.
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No. Sentence Comment
57 Remarkably, the X-loop mutations showed a reduced transport activity, with 50% transport activity for E602C and 20% for E602D or E602A (Fig. 2B).
X
ABCB3 p.Glu602Cys 19297616:57:102
status: NEW58 Complete disruption of peptide transport was observed for the E602R mutant.
X
ABCB3 p.Glu602Cys 19297616:58:102
status: NEW63 Altogether, 24 single-cysteine TAP1 mutants were coexpressed with TAP2(E602C) and yielded similar expression levels (Fig. 3A).
X
ABCB3 p.Glu602Cys 19297616:63:71
status: NEW89 TAP cross-linking conditions using copper phenanthroline (CuPhe) were established for the A381/E602C complex (Fig. 4A and Fig. S2).
X
ABCB3 p.Glu602Cys 19297616:89:95
status: NEW100 These findings demonstrate the specificity of the cross-linking between the X-loop (E602C) of TAP2 and CL1/2 of TAP1.
X
ABCB3 p.Glu602Cys 19297616:100:84
status: NEW121 Specific peptide binding and transport of TAP1(Cys-less)/TAP2(E602C) was normalized to 100%.
X
ABCB3 p.Glu602Cys 19297616:121:62
status: NEW129 We chose 2 pairs of mutations, Q277C/E602C and A381C/E602C, containing a single cysteine in each coupling helix of TAP1.
X
ABCB3 p.Glu602Cys 19297616:129:37
status: NEWX
ABCB3 p.Glu602Cys 19297616:129:53
status: NEW131 Strikingly, cross-linking of the A381C/E602C complex impeded both peptide binding and transport, in contrast to the combination with Cys-less TAP1 (Fig. 5B).
X
ABCB3 p.Glu602Cys 19297616:131:39
status: NEW132 Surprisingly, cross-linking of Q277C/E602C arrested TAP in a translocation-incompetent state, in which peptide binding was unaffected (Fig. 5C).
X
ABCB3 p.Glu602Cys 19297616:132:37
status: NEWX
ABCB3 p.Glu602Cys 19297616:132:39
status: NEW133 The transport activity of Cys-less/E602C is only slightly reduced due to irreversible oxidation.
X
ABCB3 p.Glu602Cys 19297616:133:35
status: NEW161 (B and C) The cross-linking of the single-cysteine mutants of CL1 (B) and CL2 (C) in TAP1 with E602C of TAP2.
X
ABCB3 p.Glu602Cys 19297616:161:95
status: NEW201 Membranes (500 g of protein) containing variants of TAP1 in combination with TAP2(E602C) were incubated in the presence or absence of CuPhe for 1 min at 4 °C.
X
ABCB3 p.Glu602Cys 19297616:201:90
status: NEW64 Altogether, 24 single-cysteine TAP1 mutants were coexpressed with TAP2(E602C) and yielded similar expression levels (Fig. 3A).
X
ABCB3 p.Glu602Cys 19297616:64:71
status: NEW90 TAP cross-linking conditions using copper phenanthroline (CuPhe) were established for the A381/E602C complex (Fig. 4A and Fig. S2).
X
ABCB3 p.Glu602Cys 19297616:90:95
status: NEW101 These findings demonstrate the specificity of the cross-linking between the X-loop (E602C) of TAP2 and CL1/2 of TAP1.
X
ABCB3 p.Glu602Cys 19297616:101:84
status: NEW122 Specific peptide binding and transport of TAP1(Cys-less)/TAP2(E602C) was normalized to 100%.
X
ABCB3 p.Glu602Cys 19297616:122:62
status: NEW130 We chose 2 pairs of mutations, Q277C/E602C and A381C/E602C, containing a single cysteine in each coupling helix of TAP1.
X
ABCB3 p.Glu602Cys 19297616:130:37
status: NEWX
ABCB3 p.Glu602Cys 19297616:130:53
status: NEW134 The transport activity of Cys-less/E602C is only slightly reduced due to irreversible oxidation.
X
ABCB3 p.Glu602Cys 19297616:134:35
status: NEW163 (B and C) The cross-linking of the single-cysteine mutants of CL1 (B) and CL2 (C) in TAP1 with E602C of TAP2.
X
ABCB3 p.Glu602Cys 19297616:163:95
status: NEW203 Membranes (500 òe;g of protein) containing variants of TAP1 in combination with TAP2(E602C) were incubated in the presence or absence of CuPhe for 1 min at 4 &#b0;C.
X
ABCB3 p.Glu602Cys 19297616:203:89
status: NEW91 TAP cross-linking conditions using copper phenanthroline (CuPhe) were established for the A381/E602C complex (Fig. 4A and Fig. S2).
X
ABCB3 p.Glu602Cys 19297616:91:95
status: NEW102 These findings demonstrate the specificity of the cross-linking between the X-loop (E602C) of TAP2 and CL1/2 of TAP1.
X
ABCB3 p.Glu602Cys 19297616:102:84
status: NEW124 Specific peptide binding and transport of TAP1(Cys-less)/TAP2(E602C) was normalized to 100%.
X
ABCB3 p.Glu602Cys 19297616:124:62
status: NEW135 Surprisingly, cross-linking of Q277C/E602C arrested TAP in a translocation-incompetent state, in which peptide binding was unaffected (Fig. 5C).
X
ABCB3 p.Glu602Cys 19297616:135:37
status: NEW136 The transport activity of Cys-less/E602C is only slightly reduced due to irreversible oxidation.
X
ABCB3 p.Glu602Cys 19297616:136:35
status: NEW165 (B and C) The cross-linking of the single-cysteine mutants of CL1 (B) and CL2 (C) in TAP1 with E602C of TAP2.
X
ABCB3 p.Glu602Cys 19297616:165:95
status: NEW205 Membranes (500 òe;g of protein) containing variants of TAP1 in combination with TAP2(E602C) were incubated in the presence or absence of CuPhe for 1 min at 4 &#b0;C.
X
ABCB3 p.Glu602Cys 19297616:205:89
status: NEW