ABCMdb

PMID: 24196954

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., [PubMed]

Sentences

No. Mutations Sentence Comment

41 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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. Login to comment 77 ABCB3 p.Glu632Gln 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). Login to comment 78 ABCB3 p.Glu632Gln 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). Login to comment 82 ABCB3 p.Glu632Gln The ATPase-deficient mutant complex T1C/T2Y(E632Q) did not transport peptides, as expected from previous measurements with untagged mutant complexes (11) (Fig. 2B). Login to comment 114 ABCB3 p.Glu632Gln 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). Login to comment 168 ABCB3 p.Glu632Gln ABCB3 p.Glu632Gln 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). Login to comment 169 ABCB3 p.Glu632Gln 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. Login to comment 172 ABCB3 p.Glu632Gln 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. Login to comment 190 ABCB3 p.Glu632Gln 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. Login to comment 197 ABCB3 p.Glu632Gln 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). Login to comment 198 ABCB3 p.Glu632Gln 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). Login to comment 200 ABCB3 p.Glu632Gln 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. Login to comment 229 ABCB3 p.Glu632Gln Interestingly, with T1C-T2Y(E632Q), ADP binding triggers NBD dimerization to the same extent as ATP in the presence of peptide. Login to comment 230 ABCB3 p.Glu632Gln 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. Login to comment