ABCMdb

PMID: 20731718

Crowley E, O'Mara ML, Kerr ID, Callaghan R
Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1.
FEBS J. 2010 Oct;277(19):3974-85. doi: 10.1111/j.1742-4658.2010.07789.x. Epub 2010 Aug 20., [PubMed]

Sentences

No. Mutations Sentence Comment

45 ABCB1 p.Val988Cys Figure 1 (lower panel) shows a representative labelling reaction, in this case a time course for the V988C isoform with coumarin maleimide (CM). Login to comment 47 ABCB1 p.Gly324Cys Labelling was time dependent during the 300 min incubation, and the extents of labelling were quantified in comparison with that found with cysteine-less ABCB1 and the G324C isoform. Login to comment 48 ABCB1 p.Gly324Cys The G324C mutant was assigned as the positive control and given a value of 100%, as this residue is located on an external loop and is freely accessible to each of the probes used [25,28]. Login to comment 49 ABCB1 p.Gly324Cys Furthermore, the complete labelling of the G324C mutant with the zwitterionic and hydrophilic probes BODIPY maleimide (BM) and fluorescein maleimide (FM), respectively, demonstrated that the protein was not preferentially oriented in one direction within the proteoliposomes. Login to comment 50 ABCB1 p.Gly324Cys Consequently, labelling of TM12 isoforms was determined as a percentage of G324C labelling, as outlined in Experimental procedures. Login to comment 55 ABCB1 p.Met986Cys Labelling of each isoform was analysed by densitometry and plotted as a function of time, as shown for the M986C isoform for the three probes in Fig. 2A. Login to comment 56 ABCB1 p.Met986Cys Nonlinear regression of the exponential reaction curve estimated that the maximum extent of labelling for the representative curve of the M986C isoform in the basal state was 78% for CM (t1 / 2 = 8 min), 59% for BM (t1 / 2 = 4 min) and 23% for FM (t1 / 2 = 45 min). Login to comment 61 ABCB1 p.Val982Cys ABCB1 p.Met986Cys The central region of TM12, from V982C to M986C, displayed the highest extent of labelling, with Lext values of 75-100%. Login to comment 62 ABCB1 p.Val988Cys ABCB1 p.Phe994Cys The C-terminal stretch (V988C-F994C) was also capable of interacting with CM, albeit with lower values of Lext, in the range 50-60%. Login to comment 63 ABCB1 p.Leu976Cys The lowest labelling observed in the selection of TM12 mutant isoforms was at L976C, with an Lext of 38 ± 5%. Login to comment 67 ABCB1 p.Val988Cys Detection of CM labelling of the V988C isoform. Login to comment 68 ABCB1 p.Val988Cys SDS / PAGE analysis of the V988C isoform incubated in the presence of CM for 0-300 min. Login to comment 74 ABCB1 p.Gly324Cys Lane (vii) contains the G324C isoform, which has been assigned a 100% value for labelling with BM. Login to comment 79 ABCB1 p.Val982Cys ABCB1 p.Met986Cys ABCB1 p.Val988Cys A similar stretch of TM12 (namely V982C-V988C) displayed the greatest propensity to be labelled with BM, with only isoform M986C being not completely labelled by the probe. Login to comment 82 ABCB1 p.Gly984Cys ABCB1 p.Leu976Cys The rate of labelling (i.e. t1 / 2) was divided into fast (L986C- G992C, average t1 / 2 $ 8 min) and slow (L976C- G984C, average t1 / 2 $ 25 min) kinetics between the carboxy-half and the amino-half, respectively. Login to comment 85 ABCB1 p.Phe994Cys This hypothesis is supported by the fact that F994C, which is proximal to the membrane surface, has a considerably greater Lext (111 ± 35%) for BM than the near neighbours examined. Login to comment 89 ABCB1 p.Phe994Cys Only one residue displayed avid labelling with FM, namely F994C (Lext of 129 ± 24%), and this is at the extreme carboxy-end of TM12, in proximity to the aqueous environment. Login to comment 90 ABCB1 p.Ser992Cys The proximally located S992C was also able to interact with FM, although to only a partial degree. Login to comment 92 ABCB1 p.Gly984Cys ABCB1 p.Met986Cys However, two residues (G984C and M986C) in the central region of TM12 did display labelling above background, albeit with Lext values of approximately 20%. Login to comment 94 ABCB1 p.Met986Cys The rapid kinetics of labelling of M986C with both BM and FM would also support this local increase in hydrophilicity. Login to comment 103 ABCB1 p.Gly324Cys These were then expressed as a percentage of the maximal extent of G324C labelling. Login to comment 104 ABCB1 p.Gly324Cys The degree of labelling (% of G324C level) was plotted as a function of time (min) and fitted with an exponential reaction curve, using nonlinear least squares regression. Login to comment 105 ABCB1 p.Met986Cys (A) Representative data for labelling of the M986C isoform with CM ( ), FM (d) and BM (s). Login to comment 106 ABCB1 p.Phe994Cys (B) Representative data for labelling of the F994C isoform with FM in the basal (d), AMP-PNP (s) and vanadate-trapped ( ) conformational states. Login to comment 111 ABCB1 p.Phe994Cys The data in Fig. 2B show a representative time course for labelling of the F994C mutant isoform with FM in the basal, nucleotide-bound and vanadate-trapped conformations. Login to comment 118 ABCB1 p.Val982Cys ABCB1 p.Leu976Cys Conformational changes - amino region of TM12 As shown in Table 2, the amino region of TM12 (L976C-V982C) was not associated with large alterations in topography. Login to comment 121 ABCB1 p.Leu976Cys For example, L976C became less accessible to BM, but more accessible to CM, following a shift from the basal to the nucleotide-bound conformation. Login to comment 123 ABCB1 p.Ala980Cys A980C shifted to a low level of BM accessibility following nucleotide binding by ABCB1, and again, an opposite shift was seen for CM. Login to comment 128 ABCB1 p.Val982Cys In complete contrast, V982C did not undergo any alterations of probe accessibility during transition to the nucleotide-bound and posthydrolytic conformational states. Login to comment 136 ABCB1 p.Gly324Cys The Lext for labelling is expressed as the fraction of specific labelling of single-cysteine isoforms over the specific labelling of the G324C positive control. Login to comment 139 ABCB1 p.Gly989Cys ABCB1 p.Val982Cys ABCB1 p.Gly984Cys ABCB1 p.Gly984Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Met986Cys ABCB1 p.Met986Cys ABCB1 p.Met986Cys ABCB1 p.Val988Cys ABCB1 p.Phe994Cys ABCB1 p.Ser992Cys Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide. Login to comment 143 ABCB1 p.Gly984Cys ABCB1 p.Met986Cys G984C underwent a broadly similar shift in topography as M986C, although the degree of alteration was somewhat less striking. Login to comment 144 ABCB1 p.Gly989Cys ABCB1 p.Val988Cys Conformational changes - proximal to the central region The region immediately proximal to the centre of TM12 (V988C-G989C) showed avid labelling by both of the lipophilic probes (BM and CM) in the basal configurations, and there were no significant alterations in accessibility upon progression of the catalytic cycle. Login to comment 145 ABCB1 p.Gly989Cys ABCB1 p.Val988Cys Labelling of V988C and G989C with the hydrophilic FM was negligible, regardless of the conformational state. Login to comment 146 ABCB1 p.Gly989Cys The refractoriness of labelling to conformational change is clearly demonstrated by G989C. Login to comment 147 ABCB1 p.Gly989Cys In particular, this residue displayed the lowest overall accessibility to covalent modification, regardless of the conformational state. At no stage of the catalytic cycle was either CM or BM able to fully label G989C, which was the only residue to exhibit this property. Login to comment 148 ABCB1 p.Gly989Cys Similarly, no interaction between the hydrophilic FM and G989C was observed. Login to comment 151 ABCB1 p.Gly989Cys ABCB1 p.Val988Cys The labelling properties of V988C-G989C suggest that this region of TM12 undergoes minimal conformational transition. Login to comment 153 ABCB1 p.Ser992Cys In the basal state, none of the probes could effect complete labelling of the S992C isoform. Login to comment 157 ABCB1 p.Phe994Cys F994C displays the highest accessibility of any residue in the basal conformation of ABCB1, which may Table 2. Login to comment 164 ABCB1 p.Gly989Cys ABCB1 p.Val982Cys ABCB1 p.Gly984Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Met986Cys ABCB1 p.Val988Cys ABCB1 p.Phe994Cys ABCB1 p.Ser992Cys ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface. Login to comment 168 ABCB1 p.Phe994Cys Clearly, F994C undergoes considerable changes in accessibility, suggestive of a move from a relatively hydrophilic region to a more lipophilic one as ABCB1 binds and hydrolyses nucleotide. Login to comment 183 ABCB1 p.Val982Cys ABCB1 p.Gly984Cys The homology models predict that both V982C and G984C, located within the centre of the helix, experience little change in molecular environment upon ATP binding, which is in agreement with the biochemical data. Login to comment 191 ABCB1 p.Met986Cys ABCB1 p.Ser992Cys M986C and S992C (Fig. 3) on TM12 straddle the boundaries of this hydrophilic band, and also face directly into the presumed translocation pore. Login to comment 196 ABCB1 p.Gly984Cys Surprisingly, although FM labels G984C, the homology model suggests that this residue faces into the lipid bilayer. Login to comment 197 ABCB1 p.Gly984Cys However, G984C is not in a very densely packed region, and it may be possible for FM to gain access to the residue via the translocation pore. Login to comment 198 ABCB1 p.Gly984Cys In addition, the loss of labelling of G984C with FM following progression to a vanadate-trapped state suggests that labelling is not optimal and therefore is very sensitive to even minor environmental changes. Login to comment 199 ABCB1 p.Phe994Cys ABCB1 p.Ser992Cys S992C and F994C are believed to be located at the boundary of the membrane. Login to comment 228 ABCB1 p.Val982Cys ABCB1 p.Gly984Cys The structures are shown in the panel as viewed from the translocation pore; the relative environments of V982C (cyan) and G984C (blue) are unaltered by nucleotide binding. Login to comment 257 ABCB1 p.Gly324Cys The G324C mutation, located on a freely accessible extracellular loop, has previously been demonstrated to be freely accessible to each maleimide probe [28]; labelling of the isoform containing this mutation was therefore assigned the value of 100% after 300 min. Login to comment 258 ABCB1 p.Gly324Cys Both the cysteine-less and G324C isoforms were incubated with 10 lm probe for 300 min and treated identically to the other isoforms. Login to comment 259 ABCB1 p.Gly324Cys The extent of labelling for each single-cysteine mutant was therefore determined by comparison with G324C. Login to comment 261 ABCB1 p.Gly324Cys The propensity for labelling was calculated with the following equation: L ¼ Liso À Lcys À Á L324C À Lcys À Á 100 where L is extent of labelling (%), Liso is the extent of isoform labelling, Lcys is labelling of the cysteine-less isoform, and L324C is labelling of the G324C isoform. Login to comment