ABCMdb

PMID: 22028339

Nagao K, Takahashi K, Azuma Y, Takada M, Kimura Y, Matsuo M, Kioka N, Ueda K
ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding.
J Lipid Res. 2012 Jan;53(1):126-36. Epub 2011 Oct 25., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Chambenoit et al. reported that substituting the Walker A lysine residue in either NBD with methionine (K939M or K1952M) abolishes the ability of apoA-I to bind to ABCA1-expressing cells and inhibits cholesterol secretion to apoA-I (12). Login to comment 26 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment 27 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment 45 ABCA1 p.Lys939Met Because a 3-Å crosslinker can cross-link apoA-I with ABCA1 (19) and the K939M mutant cannot bind or be cross-linked to apoA-I (22), it is likely that apoA-I interacts with specific conformations of the ECDs that are linked by the two disulfide bonds and formed in an ATP-dependent manner. Login to comment 46 ABCA1 p.Lys939Met Because a 3-&#c5; cross-linker can cross-link apoA-I with ABCA1 (19) and the K939M mutant cannot bind or be cross-linked to apoA-I (22), it is likely that apoA-I interacts with specific conformations of the ECDs that are linked by the two disulfide bonds and formed in an ATP-dependent manner. Login to comment 88 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 ␮g/ml apoA-I. Login to comment 89 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 òe;g/ml apoA-I. Login to comment 119 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment 120 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment 135 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment 137 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment 140 ABCA1 p.Lys939Met The K939M mutation reduced the photoaffinity labeling of incubated with 5 ␮M [␣32 P]8N3ATP for 10 min at 37°C and then UV irradiated on ice after the free nucleotides were removed (Fig. 4B). Login to comment 142 ABCA1 p.Lys939Met The K939M mutation decreased the photoaffinity labeling by approximately 40%, and vanadate did not affect the labeling. Login to comment 143 ABCA1 p.Lys1952Met On the other hand, the K1952M mutation drastically decreased the photoaffinity labeling by approximately 95%. Login to comment 144 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment 145 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met There was no photoaffinity-labeled band with ABCA1-K939M,K1952M. Login to comment 146 ABCA1 p.Lys1952Met Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment 147 ABCA1 p.Lys939Met ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 p.Lys1952Met These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment 149 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment 160 ABCA1 p.Lys939Met Then cells were fixed and NBD2 with [␣32 P]8N3ATP, apparently consistent with the decrease in the photoaffinity labeling of the full-length K939M mutant, although immunoprecipitation and subsequent protease digestion make it difficult to quantitatively compare the mutants (Fig. 6B). Login to comment 161 ABCA1 p.Lys939Met Nevertheless, there was no signal for the K939M mutant with [␥32 P]8N3ATP. Login to comment 162 ABCA1 p.Lys1952Met In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment 163 ABCA1 p.Lys939Met Then cells were fixed and NBD2 with [ॷ32 P]8N3ATP, apparently consistent with the decrease in the photoaffinity labeling of the full-length K939M mutant, although immunoprecipitation and subsequent protease digestion make it difficult to quantitatively compare the mutants (Fig. 6B). Login to comment 164 ABCA1 p.Lys939Met Nevertheless, there was no signal for the K939M mutant with [ॹ32 P]8N3ATP. Login to comment 165 ABCA1 p.Lys1952Met In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment