ABCMdb

PMID: 17636884

Loo TW, Bartlett MC, Clarke DM
Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments.
Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18., 2007-08-14 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. Login to comment 36 ABCB1 p.Glu556Gln Recently, it was shown that introduction of the E556Q- (NBD1) and E1201Q(NBD2) mutations into P-gp yielded a mutant that did not hydrolyze ATP but exhibited normal affinity for ATP (32). Login to comment 39 ABCB1 p.Leu332Cys ABCB1 p.Val982Cys ABCB1 p.Val982Cys Construction of mutants containing pairs of cysteines that exhibited ATP-sensitive cross-linking in the presence of copper phenanthroline [L332C- (TM6)/L975C(TM12)] (33), 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate (M14M) [L339C(TM6)/ F728C(TM7)] (34), or tris(2-maleimidoethyl)amine (TMEA) [L339C(TM6)/V982C(TM12) and F343C(TM6)/V982C- (TM12)] (31) were described previously. Login to comment 40 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Modifications to the P-gp cDNAs to introduce the catalytic carboxylate mutations, E556Q or E1201Q, were performed by oligonucleotide-directed mutagenesis (35). Login to comment 54 ABCB1 p.Val982Cys In the absence of ATP, mutant L339C(TM6)/V982C- (TM12), but not mutant F343C(TM6)/V982C(TM12), was cross-linked with TMEA. Login to comment 55 ABCB1 p.Leu339Cys In the presence of ATP, however, mutant F343C(TM6)/V982C(TM12), but not mutant L339C- (TM6)/V982C(TM12), was cross-linked with TMEA (31). Login to comment 74 ABCB1 p.Leu332Cys ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB4 p.Leu975Cys ABCB4 p.Phe728Cys The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown. Login to comment 81 ABCB1 p.Leu339Cys Cross-linking with TMEA was inhibited by the presence of ATP in both mutants L339C(TM6)/V982C(TM12) and L339C- (TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Login to comment 89 ABCB1 p.Leu332Cys ABCB4 p.Leu975Cys Mutant L332C(TM6)/L975C(TM12) contains one cysteine residue at the extracellular end of TM6 (L332C) and another at the extracellular end of TM12 (L975C) (Figure 1). Login to comment 94 ABCB4 p.Leu975Cys The mutant L332C(TM6)/L975C- (TM12) exhibited extensive cross-linking only in the presence of ATP (Figure 4). Login to comment 102 ABCB1 p.Glu1201Gln HEK 293 cells expressing histidine-tagged Cys-less P-gp (Cys-less) or mutants containing the E556Q(NBD1), E1201Q- (NBD2), or E556Q(NBD1)/E1201Q(NBD2) mutations were isolated by nickel chelate chromatography. Login to comment 106 ABCB1 p.Leu339Cys ABCB1 p.Glu556Gln Membranes were prepared from HEK 293 cells expressing P-gp mutants L339C- (TM6)/V982C(TM12) or L339C(TM6)/V982C(TM12)/E556Q- (NBD1)/E1201Q(NBD2) (A) and F343C(TM6)/V982C(TM12) or F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) (B). Login to comment 111 ABCB1 p.Glu556Gln Since the E556Q mutation inhibited the verapamil-stimulated ATPase activity of Cys-less P-gp (>90%), the results are consistent with finding that cross-linking between Cys332- (TM6) and Cys975(TM12) requires ATP hydrolysis. Login to comment 114 ABCB1 p.Glu1201Gln It appeared that the E1201Q- (NBD2) mutation caused a long-range conformational change in the TMDs to allow cross-linking between Cys332(TM6) and Cys975(TM12) in the absence of ATP. Login to comment 115 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The results suggest that the conformation of the TM segments is quite sensitive to changes in the ATP-binding site and that the E556Q and E1201Q mutations induce asymmetric changes in the TMDs. Login to comment 116 ABCB1 p.Glu1201Gln The TMDs appear to undergo different conformational changes during ATP binding, during ATP hydrolysis, or in the presence of the E1201Q mutation. Login to comment 125 ABCB1 p.Leu332Cys ABCB1 p.Leu332Cys ABCB4 p.Leu975Cys Membranes prepared from HEK 293 cells expressing mutants L332C(TM6)/L975C(TM12), L332C- (TM6)/L975C(TM12)/E556Q(NBD1)/E1201Q(NBD2), L332C- (TM6)/L975C(TM12)/E556Q(NBD1), or L332C(TM6)/L975C- (TM12)/E1201Q(NBD2) were treated with (+) or without (-) 1 mM copper phenanthroline (CuP) for 10 min at 37 °C in the absence (None) or presence of 5 mM ATP (ATP). Login to comment 146 ABCB4 p.Phe728Cys Samples were then subjected to immunoblot analysis. Figure 5C shows that in the absence of vinblastine, almost all of the mutant L339C(TM6)/F728C- (TM7) P-gp was cross-linked in the presence of ADP or ATP. Login to comment 149 ABCB4 p.Phe728Cys In contrast, the cross-linking efficiency of mutant L339C(TM6)/F728C- (TM7) in the absence of vinblastine was reduced in the presence of AMP‚PNP. Login to comment 156 ABCB4 p.Phe728Cys To further inhibit the ATPase activity of mutant L339C(TM6)/F728C- (TM7), the E556Q(NBD1) and E1201Q(NBD2) catalytic carboxylate mutations were introduced. Login to comment 167 ABCB1 p.Leu339Cys Samples were then subjected to immunoblot analysis. Figure 7C shows that there was little difference in the concentration-dependent cross-linking of mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) in the presence or absence of ATP. Login to comment 171 ABCB1 p.Leu339Cys Therefore, we confirmed that mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) did not exhibit ATP hydrolysis by subjecting it to vanadate trapping of nucleotide. Login to comment 172 ABCB1 p.Glu1201Gln Membranes prepared from cells expressing mutant L339C(TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q- (NBD2) were treated with 5 mM ATP, 10 mM MgCl2, and 0.2 mM sodium vanadate (ATP/VO4) for 10 min at 37 °C and then chilled on ice. Login to comment 192 ABCB4 p.Phe728Cys Cross-linking of mutant L339C(TM6)/F728C- (TM7) (Figure 6) was observed to be ~4-fold faster in the presence of ATP than in the presence of AMP‚PNP. Login to comment 199 ABCB1 p.Glu1201Gln The conformational changes in the TMDs appear to be quite sensitive to subtle changes in the ATP-binding sites as the conservative E1201Q mutation had a pronounced effect on the cross-linking pattern of mutant L332C(TM6)/ L975C(TM12) (Figure 4). Login to comment 200 ABCB4 p.Leu975Cys Mutant L332C(TM6)/L975C- (TM12) normally exhibits cross-linking only in the presence of ATP. Login to comment 224 ABCB1 p.Glu1201Gln The catalytic carboxylate mutations had asymmetric effects on cross-linking of mutant L332C(TM6)/L975C(TM12) as introduction of the E556Q(NBD1) mutation inhibited cross-linking even in the presence of ATP while the E1201Q- (NBD2) mutation caused cross-linking even in the absence of ATP. Login to comment 234 ABCB4 p.Leu975Cys The cross-linking pattern of mutant L332C(TM6)/L975C- (TM12)/E556Q(NBD1)/E1201Q(NBD2) indicates that ATP hydrolysis induces conformational changes in the TMDs that are distinct from those involving ATP binding. Login to comment 245 ABCB4 p.Phe728Cys In contrast, we did not observe any detectable difference in the apparent affinity for vinblastine in the presence of ATP (Figures 6 and 7) during cross-linking of mutant L339C(TM6)/F728C- (TM7). Login to comment 247 ABCB1 p.Glu1201Gln (A) The positions of the cysteine residues [L332C(TM6)/L975C(TM12) and F343C(TM6)/V982C(TM12)] that could be cross-linked and the catalytic carboxylate mutations [E556Q(NBD1) and E1201Q- (NBD2)] are shown. Login to comment 254 ABCB1 p.Glu1201Gln The presence of the E1201Q mutation (II), however, appears to mimic some of the changes induced by ATP hydrolysis since Cys332(TM6) could be cross-linked with Cys975(TM12) with copper phenanthroline. Login to comment