Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1 Emily Crowley1, Megan L O’Mara2, Ian D Kerr3 and Richard Callaghan1 Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK Molecular Dynamics Group, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, UK Keywords ABC transporter; bioenergetic coupling; drug resistance; efflux pumps; P-glycoprotein Correspondence R Callaghan, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, UK Fax: +44 1865 221 834 Tel: +44 1865 221 110 E-mail: richard.callaghan@ndcls.ox.ac.uk (Received May 2010, revised July 2010, accepted 27 July 2010) doi:10.1111/j.1742-4658.2010.07789.x Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1 The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half) Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication This data can be reconciled with an atomicscale model of human ABCB1 Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains Introduction ABCB1 (P-glycoprotein) is a member of the ATP-binding cassette (ABC) family of membrane transporters, and is located in the plasma membrane of cells The transporter is localized to a number of tissues associated with absorptive, secretory or barrier roles [1–3], and its primary function is therefore to provide a defensive mechanism against xenobiotics ABCB1 provides this defence by acting as a multidrug efflux pump The expression pattern in physiological tissues enables ABCB1 to play a prominent role in shaping Abbreviations ABC, ATP-binding cassette; AMP-PNP, 5¢-adenylylimidodiphosphate; BM, BODIPY maleimide; CM, coumarin maleimide; FM, fluorescein maleimide; Lext, maximum extent of labelling; NBD, nucleotide-binding domain; TMD, transmembrane domain; TM6, transmembrane helix 6; TM12, transmembrane helix 12 3974 FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS E Crowley et al the pharmacokinetic profile (adsorption, distribution, metabolism and excretion) of many commonly used medications [4] Unfortunately, cancer cells overexpress ABCB1 in order to evade the toxic effects of anticancer drugs, a phenomenon known as multidrug resistance The extraordinary range of compounds recognized by ABCB1 (over 200 known drugs) makes it a powerful mediator of resistance against chemotherapeutic intervention in a number of cancer types The ability to recognize such an array of compounds remains a biological enigma, thereby making the development of inhibitors that may restore the efficacy of chemotherapy in cancer treatment a difficult task The functional unit of ABCB1 consists of two transmembrane domains (TMDs), each comprising six membrane-spanning helices, and two nucleotide-binding domains (NBDs) [5] Much is understood regarding NBD function, owing to the high sequence homology between members of the ABC transporter family Furthermore, several crystal structures of ABC transporters have been solved in the presence and absence of nucleotides, improving our understanding of the mechanism of transport [6–9] However, much remains unclear about structure–function relationships of the TMDs of multidrug resistance pumps, including the location of the drug-binding sites and the molecular mechanism underlying drug translocation The ˚ recent 4–4.3 A resolution crystal structure of fulllength ABCB1 has provided a location for the binding of a purpose-built peptide inhibitor [6] However, more pharmacological information is required to evaluate this inhibitor and how its binding relates to more established substrates or modulators [10–12] Another unresolved issue pertaining to ABCB1 function is the molecular detail of the process of coupling between the NBDs and TMDs The most striking evidence for the presence of coupling between the two domains is the ability of transported drugs to stimulate the basal rate of ATP hydrolysis by ABCB1 [13,14] It is well established that drug binding occurs in the TMDs, and stimulation of hydrolysis therefore requires long-distance communication with the cytosolic NBDs This is supported by evidence that mutations of numerous residues within the TMDs are capable of disrupting the stimulation of ATP hydrolysis [15–19] Moreover, drug translocation and ATP hydrolysis must be coordinated for active efflux This requires interdomain communication in both the TMD to NBD and NBD to TMD directions [20] The latter route has also been demonstrated; for example, binding of the nucleotide analogues ATPcS or 5¢-adenylylimidodiphosphate (AMP-PNP) to the NBDs of ABCB1 was shown to significantly decrease the binding of the Role of TM12 in bioenergetic coupling in ABCB1 UIC2 antibody, which recognizes a conformation-sensitive epitope in the TMD [21,22] Furthermore, the cryo-electron microscopy structure of ABCB1 showed that in the presence of the AMP-PNP the architecture of the TMDs is significantly rearranged [23,24] Together, these experiments demonstrated that global conformational changes occur in the protein and are relayed from the NBDs to the TMDs as a consequence of nucleotide binding and drug binding, respectively, thereby enabling active drug efflux by ABCB1 However, we are yet to understand exactly how these conformational changes are relayed between the TMD and NBD, and how they enable drug translocation Transmembrane helix (TM6) and transmembrane helix 12 (TM12) are likely candidates to effect coupling, given their direct links to the two NBDs of ABCB1 We have previously constructed, and analysed, a series of TM6s with single cysteine mutations, and demonstrated that this helix plays a prominent role in the coupling process in ABCB1 [25–28] A number of mutations in TM6 caused alterations in drug-stimulated ATP hydrolysis, irrespective of whether they contributed to drug binding Moreover, several residues in TM6 were demonstrated to undergo topographical alterations during conformational changes of ABCB1 In a recent study, we demonstrated, using a similar approach, that the mutation of several residues within TM12 also influences the communication between the TMDs and NBDs [15] The present article describes the conformational changes adopted by TM12 in response to events occurring in the NBDs The data indicate that nucleotide binding and hydrolysis at the NBDs causes conformational changes that are transmitted through TM12 Results Three thiol-reactive fluorescent probes were used to assess the relative accessibility of selected residues in TM12 that had been mutated to cysteine The probes possess distinct physicochemical properties and have been shown to partition to hydrophilic or hydrophobic environments [25] By assessment of the ability of each probe to label residues in TM12, a topographical map of the helix can be generated Furthermore, trapping the protein at distinct stages of the catalytic cycle will reflect how the environment of individual residues in TM12 changes as ABCB1 switches conformational states The maximum extent of labelling of TM12 residues in ABCB1 The maximum extent of labelling (Lext) of selected TM12 mutant isoforms was initially investigated with FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS 3975 Role of TM12 in bioenergetic coupling in ABCB1 E Crowley et al reconstituted protein in the basal (nucleotide-free) state Following incubation with the fluorescent probe, the proteins were resolved by SDS ⁄ PAGE, and the covalent binding of the probe was detected under UV light Figure (lower panel) shows a representative labelling reaction, in this case a time course for the V988C isoform with coumarin maleimide (CM) The gel in the upper panel of Fig shows the same gel but stained with PageBlue to demonstrate purity of the samples and to enable normalization of labelling for protein loading Labelling was time dependent during the 300 incubation, and the extents of labelling were quantified in comparison with that found with cysteine-less ABCB1 and the G324C isoform 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] Furthermore, the complete 150 kDa 100 kDa (i) (ii) (iii) (iv) (v) (vi) (vii) 150 kDa 100 kDa Fig Detection of CM labelling of the V988C isoform SDS ⁄ PAGE analysis of the V988C isoform incubated in the presence of CM for 0–300 The reaction was stopped at various time points by the addition of dithiothreitol Upper panel: the gel protein was visualized with PageBlue staining to indicate sample purity and to enable loading correction Lower panel: the samples were resolved by SDS ⁄ PAGE and the protein was visualized with the BioDocIt system, using a UV light source Molecular mass markers are shown on the left Lane assignments are: (i) 300 min; (ii) 120 min; (iii) 60 min; (iv) 30 min; (v) 10 min; and (vi) Lane (vii) contains the G324C isoform, which has been assigned a 100% value for labelling with BM 3976 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 Consequently, labelling of TM12 isoforms was determined as a percentage of G324C labelling, as outlined in Experimental procedures Additionally, labelling of the cysteine-less ABCB1 isoform was also examined as a negative control Any nonspecific association of the three probes with cysteine-less ABCB1 was subtracted from the specific labelling intensity observed with the single-cysteine-containing isoforms Obtaining full labelling and its accurate quantitation are difficult to achieve in practice, resulting in occasional instances where values for the Lext of single-cysteine isoforms are apparently > 100% The approach does, however, provide strong predictions of relative labelling propensity, reflecting accessibility of the specified residue 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 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 ⁄ = min), 59% for BM (t1 ⁄ = min) and 23% for FM (t1 ⁄ = 45 min) Clearly, this mutant isoform was avidly labelled with the hydrophobic (CM) and zwitterionic (BM) probes, on the basis of the extent and rapid half-life of the interactions In contrast, the hydrophilic FM displayed only partial labelling, with a considerably longer halftime for the reaction Similar analysis was undertaken for each of the TM12 single-cysteine mutants (using multiple protein preparations) in the basal (i.e nucleotide-free) state; the extent and time course of labelling are shown in Table All of the mutant isoforms examined were capable of interacting with CM, which has a high octanol ⁄ water partition coefficient, indicating a preference for hydrophobic regions The central region of TM12, from V982C to M986C, displayed the highest extent of labelling, with Lext values of 75–100% The C-terminal stretch (V988C–F994C) was also capable of interacting with CM, albeit with lower values of Lext, in the range 50–60% The lowest labelling observed in the selection of TM12 mutant isoforms was at L976C, with an Lext of 38 ± 5% Lower labelling presumably reflects the location of the residues at the membrane–water interface or significant local steric hindrance The half-lives for the interaction of CM with ABCB1 ranged from 18 to 30 min, but did not reveal further details concerning the accessibility of the residues Of the three FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS E Crowley et al Fig Analysis of probe labelling of mutant TM12 isoforms of ABCB1 For each of the mutant isoforms, densitometric analysis was used to quantify the UV images and values of labelling at each time point These were then expressed as a percentage of the maximal extent of G324C labelling 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 (A) Representative data for labelling of the M986C isoform with CM ( ), FM (d) and BM (s) (B) Representative data for labelling of the F994C isoform with FM in the basal (d), AMP-PNP (s) and vanadate-trapped ( ) conformational states probes used in this investigation, the lipophilic CM has the lowest molecular volume, and the interaction of all but one residue at > 50% suggests that the helix is in a hydrophobic environment BM also displays a high octanol ⁄ water partition coefficient, and is therefore likely to reveal hydrophobic regions of ABCB1 However, unlike CM, this probe contains a delocalized charge and is zwitterionic in nature Presumably, it assumes a more polarized orientation to accommodate this ampiphilicity Like CM, each of the TM12 residues examined was able to undergo covalent modification with BM (Table 1), which also suggests that the helix is located in a hydrophobic environment 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 Either side of this central region was labelled with BM, but to only a partial extent Unlike the case for CM, there Role of TM12 in bioenergetic coupling in ABCB1 was considerable variation in the half-lives of labelling with BM of the TM12 mutant isoforms The rate of labelling (i.e t1 ⁄ 2) was divided into fast (L986C– G992C, average t1 ⁄ $ min) and slow (L976C– G984C, average t1 ⁄ $ 25 min) kinetics between the carboxy-half and the amino-half, respectively So, although the helix is in a predominantly hydrophobic region, there were some differences in topography detected by the amphiphilic BM This may suggest that the carboxy region (i.e cytosolic) lies at an interface with a more hydrophilic domain of ABCB1, as this region displayed more rapid labelling kinetics 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 An alternative explanation for the two distinct kinetic divisions is that the amino region is closely packed with another helix of ABCB1 that imparts steric restrictions on the kinetics of labelling in TM12 The final probe used to examine the topography of TM12 was the large hydrophilic FM; the extents and time courses of interactions are shown in Table The data on extent of labelling data are in broad agreement with the information provided by BM and CM 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 The proximally located S992C was also able to interact with FM, although to only a partial degree The central and amino regions of TM12 displayed low labelling with the hydrophilic probe However, two residues (G984C and M986C) in the central region of TM12 did display labelling above background, albeit with Lext values of approximately 20% This may reflect that these two residues, although in a hydrophobic local environment, are in the vicinity of a more hydrophilic region of ABCB1 The rapid kinetics of labelling of M986C with both BM and FM would also support this local increase in hydrophilicity It is also worth noting that the extent of labelling is affected by numerous factors, including steric effects and local chemistry These may have differential effects on the kinetic parameters for certain residues Do conformational transitions alter the labelling of residues in TM12 of ABCB1? During the drug translocation process, ABCB1 adopts a number of conformational states As drug translocation is coupled to ATP hydrolysis, the conformational transitions will be driven by events at the NBDs If TM12 is involved in the coupling process between the FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS 3977 Role of TM12 in bioenergetic coupling in ABCB1 E Crowley et al Table Propensity for and rate of labelling of ABCB1 with thiol-reactive probes The propensity for labelling of the TM12 mutant isoforms was determined for the thiol-reactive probes CM, BM and FM The reaction was stopped by the addition of dithiothreitol, and proteins were resolved by SDS ⁄ PAGE Densitometric analysis was used to determine the amount of labelling for each ABCB1 isoform The extent (Lext) and half-life (t1 ⁄ 2) of labelling were determined by nonlinear regression of the exponential reaction curve 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 Values represent the means ± standard errors of the mean from at least four independent protein preparations –, no labelling; ND, values where the extent of labelling was too low to accurately assign a value for t1 ⁄ CM BM FM Mutant Lext (%) t1 ⁄ (min) Lext (%) t1 ⁄ (min) Lext (%) t1 ⁄ (min) L976C A980C V982C G984C M986C V988C G989C S992C F994C 38 53 98 73 89 53 64 55 51 29 34 15 29 25 37 15 22 11 66 54 164 84 51 221 21 51 111 29 20 27 22 18 13 – – – 13 ± 21 ± – – 32 ± 129 ± – – – ND ND – – 25 ± 8±3 ± ± ± ± ± ± ± ± ± 14 14 30 10 ± ± ± ± ± ± ± ± ± 12 6 10 18 6 TMDs and NBDs, then it will presumably undergo multiple topographical transitions during the catalytic cycle The previous section outlined the overall topography of TM12, by examining the accessibility of introduced cysteines to maleimide-containing probes The next phase of investigation involved trapping ABCB1 mutant isoforms at distinct conformational stages (e.g nucleotide-bound and immediately posthydrolysis) and reassessing the accessibility to maleimide probes The data thereby identified the dynamic changes produced during transition between various stages of the catalytic cycle 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 vanadatetrapped conformations The nucleotide-bound (prehydrolytic) conformation was achieved by incubation of the mutant isoforms with the nonhydrolysable ATP analogue AMP-PNP, as previously described [24] The posthydrolytic (but pre-ADP or phosphate release) stage was produced by the vanadate-trapping procedure [24] In the basal state, the protein was fully labelled with FM (Lext of 105%); however, Lext was reduced to 47% upon binding of AMP-PNP, and further reduced to 14% following vanadate trapping Accessibility data, as shown in Fig 2B, were obtained (using multiple protein preparations) for each mutant isoform in the three conformations (nucleotidefree, AMP-PNP-bound and vanadate-trapped) Experiments were carried out as described in the previous section, and the Lext and t1 ⁄ parameters were obtained from the labelling time course profiles To simplify analysis, a qualitative representation has been adopted (Table 2) 3978 ± ± ± ± ± ± ± ± ± 14 50 24 63 35 ± ± ± ± ± ± ± ± ± 18 17 12 10 10 24 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 In particular, accessibility of the two residues to FM was negligible in the basal state, and this did not change for the nucleotide-bound and posthydrolytic states There were, however, some subtle changes in accessibility of the two more hydrophobic probes For example, L976C became less accessible to BM, but more accessible to CM, following a shift from the basal to the nucleotide-bound conformation As ABCB1 shifted to the posthydrolytic conformation, the extent of BM labelling returned to the basal level, whereas CM accessibility was retained A980C shifted to a low level of BM accessibility following nucleotide binding by ABCB1, and again, an opposite shift was seen for CM The subsequent transition to a vanadatetrapped state resulted in the highest possible extent of labelling for BM, but with no alteration for CM Overall, nucleotide binding shifts the amino region to a distinctly hydrophobic environment, such that labelling with the zwitterionic BM is, in fact, reduced Given that BM is ampiphilic, this would suggest a shift from a possible interfacial region to a buried hydrophobic one Furthermore, the progression to the posthydrolytic state restored the topographical features seen under basal conditions In complete contrast, V982C did not undergo any alterations of probe accessibility during transition to the nucleotide-bound and posthydrolytic conformational states This was the only residue examined in TM12 that retained an unaltered topography between the states despite the conformational changes within the TMDs induced by the NBDs FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS E Crowley et al Role of TM12 in bioenergetic coupling in ABCB1 Table Relative accessibilities of TM12 residues Accessibilities of cysteines to FM, BM and CM were determined at distinct stages of the catalytic cycle for each ABCB1 isoform The extent of labelling was compared with that of the cysteine-less ABCB1 isoform Basal refers to the nucleotide-free state, whereas the AMPPNP and Vi-trapped states mimic prehydrolytic and posthydrolytic states of the protein, respectively +++, complete labelling (Lext > 75%); ++, partial labelling (Lext = 50–75%); +, weak labelling (Lext < 50%); ), labelling below the amount observed for cysteineless ABCB1 All values were determined as described in Table and obtained from four independent protein preparations ABCB1 isoform L976C A980C V982C G984C M986C V988C G989C S992C F994C Catalytic intermediate CM BM FM Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped ++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ +++ +++ ++ ++ ++ ++ +++ ++ ++ ++ +++ +++ ++ +++ ++ + +++ +++ +++ +++ +++ +++ ++ ++ +++ ++ +++ +++ +++ + ++ + ++ +++ ++ +++ +++ +++ ) ) ) ) ) ) ) ) ) + + ) + ++ ) ) ) ) ) ) ) + ++ + +++ ++ + 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 This was accompanied by a moderate increase in labelling with the zwitterionic BM, but with a reduction in accessibility with the hydrophobic CM This pattern of change suggests a shift towards a more polar environment for this central residue This appeared to be a transient shift in microenvironment, as the posthydrolytic state adopted a topography similar to that in the basal configuration G984C underwent a broadly similar shift in topography as M986C, although the degree of alteration was somewhat less striking 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 Labelling of V988C and G989C with the hydrophilic FM was negligible, regardless of the conformational state The refractoriness of labelling to conformational change is clearly demonstrated by G989C 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 Similarly, no interaction between the hydrophilic FM and G989C was observed The variation in physicochemical properties of the three probes suggests that the inherently low labelling at any stage of the catalytic cycle was unlikely to result from the local solvent environment A more likely explanation is steric hindrance to labelling by neighbouring residues or helices in the TMD The labelling properties of V988C– G989C suggest that this region of TM12 undergoes minimal conformational transition Conformational changes – carboxy region Considerably greater changes in accessibility to probes were observed at the extreme carboxy region of TM12, suggesting a more prominent role in mediating conformational transitions In the basal state, none of the probes could effect complete labelling of the S992C isoform However, progression to the nucleotide-bound state resulted in a universal increase in accessibility of the residue to covalent modification by all three probes Further progression to the posthydrolytic state caused a reversion in accessibility in comparison to that seen in the basal state The uniform changes in accessibility to three probes with distinct chemical properties suggest that the adoption of the nucleotidebound state relieves the steric hindrance to labelling found in the basal conformation, and that this is restored as the catalytic cycle continues F994C displays the highest accessibility of any residue in the basal conformation of ABCB1, which may FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS 3979 Role of TM12 in bioenergetic coupling in ABCB1 E Crowley et al reflect localization at the membrane–solute interface There was no alteration in the extent of labelling by BM in any conformational state examined In contrast, there was a dramatic reduction in labelling by the hydrophilic FM as the protein progressed to the nucleotide-bound and posthydrolytic states This was accompanied by a concomitant increase in accessibility to the hydrophobic CM 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 Discussion TM12 has previously been demonstrated to play an integral role in coupling between the drug binding and translocation process (TMD), with the hydrolysis of nucleotide (NBD) [15,29] Moreover, perturbation of TM12 altered not only drug-stimulated ATP hydrolysis, but also the inherent (basal) hydrolytic activity The latter demonstrates that activity of the NBDs, even in the absence of substrate, is subject to some degree of control or modulation by the TMDs of ABCB1 TM6 in the amino-half of ABCB1 has often been regarded as a mirror image of TM12, but, from a purely functional perspective, cysteine introduction within TM12 generated considerably greater functional consequences for ABCB1 than corresponding mutations in TM6 The present study investigated whether the ‘mirror image’ relationship holds true, particularly with respect to the topographical changes in TM12 throughout the catalytic cycle The topographical changes were examined by introducing cysteines at distinct positions in TM12 and assessing their accessibility to covalent modification with thiol-reactive probes In order to determine how changes in the extent and rate of labelling reflect conformational changes in TM12, we used molecular models of ABCB1 [30] in the basal and ATP-bound states as the basis for in silico characterization Homology modelling has previously been used to characterize the effects of mutations in TM12 ⁄ TM6 on the overall function of ABCB1 and to interpret the changes in labelling accessibility that occur in TM6 [27] This approach provided a mechanistic explanation for the role of TM6 in the translocation mechanism of ABCB1, and was reproduced in the present investigation for TM12 The changes in the accessibility to probes of mutated residues within TM12 showed both increases and decreases in the propensity for labelling throughout the catalytic states, suggesting that TM12 undergoes 3980 conformational alterations, or is subjected to changes in its local environment There were two major observations to be drawn from studying the topography of TM12 in the homology model First, the midregion of the helix, i.e V982–G984, was rigid with respect to the intracellular and extracellular sections of the helix in the basal and ATP-bound states of the model, and that this section of TM12 appeared to act as an anchor around which the rest of the helix moved Second, the model shows that the intracellular part of TM12 also contributes residues (between Met986 and Ser992) to the band of hydrophilic residues that line the central aqueous pore in ABCB1 (Fig 3) Both of these observations can be rationalized with the molecular models for ABCB1 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 TM12 is predicted by homology modelling to rotate by approximately a quarter of a turn following ATP binding, which is also in agreement with the biochemical data This rotation is accompanied by a displacement towards Tyr953 (TM11), the nearest neighbour of Val982 in the closed-state model Despite this motion, Val982 does not form a close contact with Tyr953, and the local environment is therefore unchanged and does not impact on the accessibility of the residue to the fluorescent probes In support of this, no change in labelling was observed In addition, the position of Gly984 does not change between the closed and open states of the model, and would not result in a change in the polarity of the environment This rigidity is clearly reflected in the labelling experiment, which demonstrated little change in residue accessibility among the catalytic states A hydrophilic band of residues in the TMD lines the central cavity of ABCB1 (Fig 3) and presumably contributes to the solvent accessibility of the residues in this region M986C and S992C (Fig 3) on TM12 straddle the boundaries of this hydrophilic band, and also face directly into the presumed translocation pore These two residues were readily labelled by the fluorescent probes, and displayed differences in accessibility between the conformational states examined It has been suggested that conformational transitions may alter the nature of the residues lining the translocation pore [10,31], e.g from hydrophobic to hydrophilic This type of switch may be responsible for the cycling of affinity of ABCB1 for drug substrates during the translocation process [24] Such observations have been made in both the ABCB1 homology models [30] and the low-resolution crystal structure of ABCB1 [6] FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS E Crowley et al Role of TM12 in bioenergetic coupling in ABCB1 A B C Fig Molecular modelling of the TMDs in ABCB1 Representations of the TMDs of ABCB1 obtained from molecular modelling are shown, with the NBDs removed for clarity (A) The TMD of ABCB1 predicted to represent the basal (nucleotide-free) conformation (B) The TMD of ABCB1 predicted to occur in the nucleotide-bound conformation of the protein The two TMDs of ABCB1 are shown with helices from TMD1 (N-terminal) in grey and those from TMD2 (C-terminal) in black The TMDs display a hydrophilic band of residues (cyan) that lines the central cavity, and these are shown in the ‘space-fill’ representation Relative to TM12, the hydrophilic band is located at a depth that corresponds to the region bounded by residues Met986 and Ser992, which are depicted in purple (C) The TMD helices (cylinders) neighbouring, or in the vicinity of, TM12 (ribbon) The helices are shown in the nucleotide-free (bold) or bound (pastel) conformations: orange, TM9; gold, TM10; red, TM11 All other helices have been removed from the diagram to aid clarity The diagram also demonstrates (comparison of bold and pastel representations) that TM12 undergoes relatively little motion in switching between these conformations 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 The nearest neighbouring residue, Tyr953, is shown in red space-fill representation Surprisingly, although FM labels G984C, the homology model suggests that this residue faces into the lipid bilayer 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 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 S992C and F994C are believed to be located at the boundary of the membrane Indeed, Ser992 faces into the translocation pore near the entrance and is highly solvent exposed Consequently, both residues are accessible to labelling by FM Moreover, Phe994 is located within the prominent kink in TM12, which was first identified by the homology model of ABCB1 and subsequently confirmed in the crystal structure [6,30] It is conceivable that this kink may facilitate (or dampen) transmission of movement initiated by events in the NBDs to conformational changes in TM12 For example, upon ATP binding, the NBDs will form a dimer to enable hydrolysis of nucleotide The resultant hydrolytic cleavage of ATP will result in disengagement of the dimer because of the considerable repulsion between ADP and Pi TM12 is directly linked to NBD2, and is therefore ideally placed to transmit these conformational changes The communication would extend in both directions, and the central region of TM12 would act as a stationary element about which the conformational changes occur Similarly, the binding of substrates is thought to stimulate ATP hydrolysis by facilitating conformational changes associated with NBD dimer assembly This might occur through communication between the drug-binding site(s) and TM12 In fact, mutations in TM12 were demonstrated to affect transport or ATPase activity [32,33], in particular, the stimulation of ATP hydrolysis by vinblastine and nicardipine [15] These two compounds are known to interact at pharmacologically distinct (allosterically linked) sites in ABCB1 [34], and this supports the notion of TM12 acting as a key conduit Moreover, the observation that mutations in TM12 could alter stable ATP binding by the NBDs further supports the tight coupling imparted by TM12 on the process of ATP hydrolysis Further biochemical and structural studies will reveal the exact contribution of individual residues in TM12 to drug binding and the role of the TM12 anchor region identified here in allosteric communication A previous investigation has also demonstrated that, upon ATP binding, the extracellular faces of the two helices can form a zero-length cross-link, indicating a close approach [35] This close approach of the helices is relaxed following progression of ATP hydrolysis FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS 3981 Role of TM12 in bioenergetic coupling in ABCB1 E Crowley et al Moreover, there is a large amount of evidence demonstrating that TM6 and TM12 are intimately involved in numerous aspects of the molecular mechanism of ABCB1 The present investigation focused on TM12, and it is clear that the helix does undergo conformational changes, with the centre of the helix being rigid and motion being amplified at the extracellular and intracellular ends of the helix Experimental procedures Fluorescent labelling of single-cysteine isoforms of ABCB1 Materials Octyl-b-d-glucoside, C219 antibody and Ni2+–nitrilotriacetic acid His Bind Superflow resin were obtained from Merck Chemicals (Nottingham, UK) Dimethylsulfoxide, Na2ATP, AMP-PNP, sodium orthovanadate and cholesterol were purchased from Sigma Aldrich (Poole, UK) Crude Escherichia coli lipid extract was obtained from Avanti Polar Lipids (Alabaster, USA) Insect-Xpress medium was purchased from Lonza (Wokingham, UK) and Excell 405 from SAFC Biosciences (Andover, UK) CM, FM and BM were purchased from Molecular Probes (Leiden, The Netherlands) Site-directed mutagenesis of TM12 in ABCB1 – introduction of cysteines Mutants were constructed with QuikChange or Altered Sites II mutagenesis systems with a pAlter-MCHS or pFastBac1-MCHS template The MCHS cDNA encodes an ABCB1 isoform devoid of cysteines with a C-terminal His6 tag and numerous strategically inserted restriction enzyme sites Full details of the construction of mutant ABCB1 isoforms have been given in previous publications [25,36] Expression, purification and reconstitution of ABCB1 Recombinant baculovirus was generated using the Bac-toBac baculovirus expression system, as previously described [25,36] and according to the manufacturer’s instructions (Invitrogen) Trichoplusia ni (High-five) cells were infected with recombinant baculovirus at a multiplicity of infection of 5, and harvested 72 h postinfection by centrifugation (2000 g, 10 min) For comparative analysis of protein expression, · 106 cells were resuspended in NaCl ⁄ Pi supplemented with 2% (w ⁄ v) SDS, and proteins were resolved by SDS ⁄ PAGE ABCB1 was detected with the C219 antibody following immunoblotting [37] For large-scale expression of ABCB1 isoforms, 1.5 · 109 T ni (High-five) cells were infected, and cell membranes were isolated by nitrogen cavitation and density gradient ultracentrifugation and stored at )80 °C for up to year 3982 [25,36] ABCB1 isoforms were purified by immobilized metal affinity chromatography (Ni2+–nitrilotriacetic acid resin), and reconstituted by the detergent adsorption technique [25,36] Confirmation of reconstitution was performed by examining the relative migration of lipid and protein through sucrose density (0–30% w ⁄ v) gradients Protein concentration following reconstitution was determined with an adapted Lowry colorimetric assay with BSA as standard (DC-Brad Protein Assay; BioRad) [38] The topography of TM12 was assessed by following the labelling kinetics of each single-cysteine mutant isoform with three fluorescent thiol-reactive probes The probes display distinct physicochemical properties, with variations in charge, size and hydrophobicity [25]; for example, CM is hydrophobic, FM is hydrophilic and BM is zwitterionic Purified, reconstituted ABCB1 isoforms (2 lg) were incubated with 10 lm CM, BM or FM for 0, 10, 30, 60, 120 and 300 in the dark at 20 °C The ligand was added from concentrated stocks in dimethylsulfoxide, and the final solvent concentration was maintained at < 0.05% (v ⁄ v) A 100-fold molar excess of probe to protein was used to facilitate labelling and prevent significant depletion of the probes The reaction was stopped by the addition of 100 lm dithiothreitol, which binds avidly to unreacted maleimide probe, and subsequently placed on ice The protein was diluted : with buffer (50 mm Tris ⁄ HCl, pH 7.4, 150 mm NH4Cl, mm MgSO4, 0.02% NaN3) to reduce glycerol content, and centrifuged for 30 at 125 000 g and °C to remove unbound probe The pellet was washed and then resuspended in 20 lL Laemmli sample buffer, and proteins were resolved by 7.5% (v ⁄ v) SDS ⁄ PAGE Nonspecific association of the fluorescent probe with the protein and lipid membrane was determined using a cysteine-free ABCB1 isoform 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 Both the cysteine-less and G324C isoforms were incubated with 10 lm probe for 300 and treated identically to the other isoforms The extent of labelling for each single-cysteine mutant was therefore determined by comparison with G324C In order to calculate the specificity of labelling for each single-cysteine mutant, the background or nonspecific labelling of the cysteine-less isoform was subtracted The propensity for labelling was calculated with the following equation: À Á Liso À Lcys Á  100 L¼À L324C À Lcys FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS E Crowley et al Role of TM12 in bioenergetic coupling in ABCB1 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 The extent of fluorescence labelling for ABCB1 mutant isoforms was also determined in the nucleotide-bound state by trapping with AMP-PNP The ABCB1 nucleotide-bound conformation was generated by the addition of AMP-PNP (2 mm), followed by a 20 incubation at 20 °C Trapping of ABCB1 in the posthydrolytic state was achieved by the addition of 300 lm orthovanadate (Vi) and mm ATP, followed by a 30 incubation at 37 °C in order to generate the ADPỈVi transition state intermediate [22,24,25] Fluorescence labelling was subsequently carried out as detailed in the preceding paragraph The extent of labelling was determined by examining the gel using the BioDocIT Imaging System (UVP), with a UV light source of wavelength 302 nm and a CCD camera The gel was subsequently stained with PageBlue to validate equivalent protein loading Densitometric analysis (scion image) was used to quantify the extent of labelling The maximum extent of labelling (Lmax) and half-time of labelling (t1 ⁄ 2) were determined by nonlinear regression of the exponential reaction curve (graphpad prism 4.0) to plots of labelling as a function of time: À Á L ¼ Lmax À ekt where L is the percentage of labelling, Lmax is the maximum extent of labelling (%), k is the observed rate constant for labelling (min)1), and t is time (min) The labelling rate constant was converted to half-time of labelling according to the following relationship: t1=2 ¼ Ln2=k Statistical analysis All data manipulations and statistical analyses were performed using graphpad prism 4.0 Comparison of datasets for each isoform was performed with Student’s t-test or ANOVA (where n > 3), applying Dunnett’s test, where significance was determined by a P-value < 0.05 Values reported correspond to means ± standard errors of the mean obtained from at least four independent preparations of ABCB1 Homology modelling A homology model of a nucleotide-free, open-state human ABCB1 was developed from the open-state mouse P-glycoprotein crystal structure (3G5U.pdb), using the swissmodel homology modelling server [39], with the aim of producing an open-state homology model of human ABCB1 that would complement the previous closed-state Sav1866-based ABCB1 model [30] The sequence identity between human ABCB1 and mouse P-glycoprotein is 86%, giving a very high degree of confidence to the sequence alignment of the resulting model To verify that the residue threading of this open-state ABCB1 model corresponds to the previously developed closed-state ABCB1 model [30], the sequence alignments were cross-referenced to ensure that there was positional correspondence of the residues in both conformations The series of single-point mutations to cysteine were performed at positions 976, 978, 980, 988, 989 and 990 in the open-state ABCB1 homology model, to give a set of six single-point mutation open-state ABCB1 models These models were developed with the method described in Storm et al [28]; they provide an alternative conformation to the set of closed-state ABCB1 point mutations developed in Crowley et al [15], and allow a comparison of the local environment of each residue in both the open and closed conformation of ABCB1 Acknowledgements E Crowley was generously supported by a Cancer Research UK Studentship (C362 ⁄ A5502) awarded to I D Kerr and R Callaghan M L O’Mara is supported by a University of Queensland Post-doctoral Fellowship References Cordon-Cardo C, O’Brien JP, Boccia J, Casals D, Bertino JR & Melamed MR (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues J Histochem Cytochem 38, 1277–1287 Cordon-Cardo C, O’Brien JP, Casals D, Rittman-Grauer L, Biedler JL, Melamed MR & Bertino JR (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood–brain barrier sites Proc Natl Acad Sci USA 86, 695–698 Leslie EM, Deeley RG & Cole SPC (2005) Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense Toxicol Appl Pharmacol 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tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis Evidence for rotation of a transmembrane helix J Biol Chem 276, 31800–31805 36 Taylor AM, Storm J, Soceneantu L, Linton KJ, Gabriel M, Martin C, Woodhouse J, Blott E, Higgins CF & Callaghan R (2001) Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein Br J Pharmacol 134, 1609–1618 Role of TM12 in bioenergetic coupling in ABCB1 37 Hoedemaeker FJ, Signorelli T, Johns K, Kuntz DA & Rose DR (1997) A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219 Design, expression, and crystal structure at 2.4 A resolution J Biol Chem 272, 29784–29789 38 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 39 Guex N & Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling Electrophoresis 18, 2714–2723 FEBS Journal 277 (2010) 3974–3985 ª 2010 The Authors Journal compilation ª 2010 FEBS 3985 ... single cysteine mutations, and demonstrated that this helix plays a prominent role in the coupling process in ABCB1 [25–28] A number of mutations in TM6 caused alterations in drug- stimulated ATP... Catalytic intermediate CM BM FM Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP Vi trapped Basal AMP-PNP... drug- binding site(s) and TM12 In fact, mutations in TM12 were demonstrated to affect transport or ATPase activity [32,33], in particular, the stimulation of ATP hydrolysis by vinblastine and nicardipine