Dynamics of Α-Helical Subdomain Rotation in the Intact Maltose ATP-Binding Cassette Transporter

Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

Cédric Orellea, Frances Joan D. Alvareza, Michael L. Oldhamb, Arnaud Orellea, Theodore E. Wileya, Jue Chenb, and Amy L. Davidsona,1

aChemistry Department, and bHoward Hughes Medical Institute, Purdue University, 560 Oval Dr, West Lafayette, IN 47906

Edited by Christopher Miller, Brandeis University, Waltham, MA, and approved October 4, 2010 (received for review May 12, 2010)

ATP-binding cassette (ABC) transporters are powered by a nucleo- events, a ∼10° rotation of the α-helical subdomains relative to tide-binding domain dimer that opens and closes during cycles of the RecA-like subdomains and a ∼14° rotation of the RecA-like ATP hydrolysis. These domains consist of a RecA-like subdomain subdomains relative to the C-terminal regulatory domains (23). and an α-helical subdomain that is specific to the family. Many Analysis of the helical subdomain rotation is crucial for a detailed studies on isolated domains suggest that the helical subdomain ro- understanding of how ATP hydrolysis is stimulated by MBP and tates toward the RecA-like subdomain in response to ATP binding, coupled to transport. We therefore determined the requirements moving the family signature motif into a favorable position to in- for subdomain rotation and defined the sequence of rotational teract with the nucleotide across the dimer interface. Moreover, events in the catalytic cycle. the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the Results rotation in interdomain communication. Electron paramagnetic Positioning Spin Labels to Detect α-Helical Subdomain Rotation in resonance spectroscopy was used to study the dynamics of this MalK. The maltose transporter has been crystallized in an inward- Escherichia coli rotation in the intact maltose transporter MalFGK2. facing conformation in the absence of nucleotide (23), and an This importer requires a periplasmic maltose-binding protein (MBP) outward-facing conformational intermediate, stabilized by bind- that activates ATP hydrolysis by promoting the closure of the ing of both ATP and MBP, that may resemble the transition state

cassette dimer (MalK2). Whereas this rotation occurred during the (22, 24, 25) (Fig. 1A). Alignment of the RecA-like subdomains of BIOCHEMISTRY transport cycle, it required not only trinucleotide, but also MBP, these two structures (Fig. 1B) reveals an inward rotation of the suggesting it is part of a global conformational change in the trans- helical subdomain toward the RecA-like subdomain, as seen in 2þ porter. Interaction of AMP-PNP-Mg and a MBP that is locked in many isolated NBD structures upon ATP binding (5–8). These a closed conformation induced a transition from open MalK2 to two subdomains are connected by the Q-loop (27) that lines part semiopen MalK2 without significant subdomain rotation. Inward of the cleft surrounding the coupling helix of the transmembrane rotation of the helical subdomain and complete closure of MalK2 domains MalF and MalG. The Q-loop is expected to undergo therefore appear to be coupled to the reorientation of transmem- conformational changes upon subdomain rotation that might brane helices and the opening of MBP, events that promote trans- be detected as a change in mobility of an attached spin label fer of maltose into the transporter. After ATP hydrolysis, the helical (SL). Residues 83–87 in the Q-loop were individually mutated subdomain rotates out as MalK2 opens, resetting the transporter in to cysteine. Residues in the helical subdomain (residues V120, an inward-facing conformation. Q122, and A124) and in the RecA-line subdomain (residues A167, L168, and Q171) (Fig. 1B) were also mutated to cysteines, EPR spectroscopy ∣ transport mechanism ∣ membrane protein individually and in pairs, to measure changes in the distance between these two subdomains upon rotation. Residues were TP-binding cassette (ABC) transporters belong to one of the selected for mutation based on distance constraints (28, 29) Alargest protein superfamilies in organisms, and mediate the and side-chain solvent-accessibility [http://mobyle.rpbs.univ- translocation of a wide range of substrates across the membrane paris-diderot.fr/cgi-bin/portal.py?form=ASA (30)], to maximize (1). These transporters typically contain two transmembrane do- spin-labeling efficiency. mains (TMDs) and are energized by a nucleotide-binding domain Plasmids carrying mutation(s) encoding the cysteine substitu- (NBD) dimer that closes and opens during cycles of ATP binding tions in the malK gene were used to transform an E. coli strain and hydrolysis (2, 3). Each NBD consists of a RecA-like subdo- containing a chromosomal deletion of malK, and were tested main, found in numerous ATPases (4), and an α-helical subdo- for function by complementation, as judged by the red color main that is specific to the ABC family (5). Crystallographic of the colonies on maltose MacConkey agar plates (22). By this studies (5–8) and molecular dynamic simulations (9, 10), criterion, all of the substitutions in the helical and RecA-like sub- performed on isolated NBDs, suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP domains were functional, but only one of the Q-loop substitutions binding. This rotation positions the ABC family signature motif (S83C) was functional. The mutants were purified, spin-labeled, to interact with nucleotide across the dimer interface so that the and tested for MBP-stimulated ATPase activity (Table S1). All NBDs can close to hydrolyze ATP. After ATP hydrolysis, it is of the double mutants showed a strong stimulation by MBP,which suggested that the helical subdomain rotates away (5, 11). In is characteristic of the maltose transporter (21). However, only addition, the TMDs contact the NBDs at the interface between four of the double mutants, those that retained reasonably high these subdomains (12–15), suggesting a putative role of the rotation in interdomain communication (16). Here, we used site- Author contributions: C.O. and A.L.D. designed research; C.O., F.J.D.A., M.L.O., A.O., and directed spin labeling electron paramagnetic resonance (EPR) T.E.W. performed research; F.J.D.A., M.L.O., and J.C. contributed new reagents/analytic spectroscopy (17, 18) to study the dynamics of this rotation in tools; C.O., J.C., and A.L.D. analyzed data; and C.O. and A.L.D. wrote the paper. the intact Escherichia coli maltose transporter MalFGK2 (19, 20). The authors declare no conflict of interest. A periplasmic maltose-binding protein (MBP) delivers the sub- This article is a PNAS Direct Submission. strate to the transporter and stimulates its ATPase activity (21). 1To whom correspondence should be addressed. E-mail: [email protected]. In the maltose transporter, closure of the NBD dimer requires This article contains supporting information online at www.pnas.org/lookup/suppl/ both ATP and MBP (22) and involves two separate rotational doi:10.1073/pnas.1006544107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1006544107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 (ADP-Mg2þ-bound) state (22), increased the mobility of S83C- SL relative to the catalytic intermediate, though the spectrum was not superimposable with that of the apo state (Fig. 2). Changes in mobility of SL positioned at V120C and Q122C in the helical subdomain were also seen only following the addition of both AMP-PNP-Mg2þ and maltose-MBP (Fig. 2). In contrast, changes in mobility of SL positioned at L168C and, to a lesser extent, Q171C in the RecA-like subdomain (Fig. S1) are seen upon addition of AMP-PNP-Mg2þ only, in addition to the more substantial changes apparent when both ligands are present. Crystal structures of isolated NBDs indicate that when the NBDs are open, nucleotide binds to the RecA-like subdomain rather than the helical subdomain (31) consistent with the observation that Fig. 1. Structural views of the maltose ABC transporter. (A) Structures of the SLs positioned in the RecA-like subdomain, but not the helical maltose transporter in apo (Left, PDB 3FH6) and catalytic intermediate (Right, subdomain, sensed nucleotide binding. The altered mobilities of PDB 2R6G) states. The transmembrane domains MalF and MalG are both SLs in the helical subdomain in the catalytic intermediate may shown in orange. MBP is in blue. The RecA-like subdomain (gray), α-helical reflect helical subdomain rotation, which brings the SL side subdomain (red), and regulatory domain (purple) of MalK are distinguished. chains closer to residues in the RecA-like subdomain. ATP (green) and maltose (cyan) molecules are present only in the catalytic intermediate. (B) Closeup of the MalK/MalG interface. Structures of the apo and catalytic intermediate state are superimposed based on the RecA- EPR Analysis of Doubly Labeled Mutants in the Apo State. To demon- like subdomain to visualize the helical subdomain rotation and movement strate the rotation of the helical subdomain relative to the RecA- of the coupling helix. MalG is rendered in yellow (apo state) and orange (cat- like subdomain, we tested several different pairs of spin-labels to alytic intermediate state). The coupling helix (or EAA loop) of MalG is shown determine the distance between these subdomains (Table S2). as a cylinder. The RecA-like and helical subdomains of MalK (colored as in A) When the interspin distance is >25 Å, the spectrum of a doubly from the apo and catalytic intermediate states are shown in lighter and dar- labeled protein in continuous wave (cw)-EPR strictly corresponds ker hues, respectively (figure modified from ref. 23). Residue Q82 defining to the sum of the spectra of the noninteracting single mutants. the Q-loop (blue) is shown as stick for the catalytic intermediate state. Interspin distances can be determined at room temperature in Spheres indicate the positions of residues spin-labeled in this study (Q122 – and A167 are indicated). The figure was prepared with Pymol (26). the range of 7 25 Å although the spectral broadening due to dipolar interaction is small above 18–20 Å generating uncertainty activity (≥670 nmol∕ min ∕mg), were selected for further charac- in determining the actual distance and distribution (28, 29). terization. The magnetic dipolar interactions between two spin labels were analyzed in terms of interspin distance populations using the pro- EPR Analysis of Single Site Mutants. EPR spectral lineshapes gram “shortdistances100” (see Materials and Methods). Distances primarily reflect the mobility of the spin label, and are therefore between the spin-labeled subdomains, calculated in the absence sensitive to conformational changes in the local environment. of ligand, were largely consistent with the apo state crystal struc- With spin label at position 83 in the Q-loop, a notable decrease ture (Tables S2 and S3). In all cases a significant percent of the ∼30% in mobility was detected upon addition of both MBP and the non- spins ( ) did not interact, most likely reflecting incomplete hydrolyzable ATP analogue AMP-PNP-Mg2þ (Fig. 2), conditions labeling at both sites, although it is also possible that some frac- promoting the formation of the outward-facing conformation tion of the pairs are outside the measureable range. seen in the crystal structure (Fig. 1A). However, no changes were Inward Rotation of the α-Helical Subdomain in the Intact Maltose detected following addition of either ligand alone (Fig. 2). This Transporter. The double mutant Q122C/A167C showed a substan- result was consistent with our previous work demonstrating that tial increase in spin–spin interaction when both AMP-PNP-Mg2þ closure of the NBD dimer interface requires both nucleotide tri- and maltose-MBP were added (Fig. 3). Distance calculations phosphate and MBP (22) and suggested that the subdomain may showed a change from ∼15 Å in the apo state to ∼7.5 Åin not rotate in response to the addition of either ligand alone be- the catalytic intermediate state for 62% of the population (Fig. 3 cause no mobility changes were seen in SL attached to the hinge and Table S3). No change was seen when maltose-MBP alone 2þ between the two subdomains. Preincubation with ATP-Mg and was added and only a small change of ∼1 Å was seen when MBP, to allow ATP hydrolysis and generate a posthydrolysis AMP-PNP-Mg2þ alone was added. The double mutants A124C/ L168C and A124C/Q171C also showed an increase in spin–spin interaction under the same conditions (Fig. S2 and Table S3), although the distance measurements between these positions are likely to be less accurate because, in the apo state, they lie at the limit of detection by cwEPR. These data are consistent with an inward rotation of the helical subdomain toward the RecA-like subdomain occurring only when both AMP-PNP-Mg2þ and maltose-MBP are bound to the transporter. The final pair, Q122C- L168C, had a wide distribution of distances making it more dif- ficult to interpret the results, but both the apo and catalytic intermediate states could be fit with a bimodal distribution of spins Fig. 2. SLs positioned in the Q-loop and the helical subdomain undergo centered at 8 and 14 Å. The major change upon addition of 2þ changes in mobility in the presence of both AMP-PNP-Mg and MBP. Single AMP-PNP-Mg2þ and maltose-MBP for this SL pair was a shift cysteine substitutions were made in the Q-loop (S83C) and helical subdomain in the distribution of the fit, with a large fraction of the spin label (V120C, Q122C, A124C) of MalK and these mutants were spin-labeled with methanethiosulfonate spin label (MTSL) and purified. A series of EPR spectra shifting from the distribution centered at 14 to 8 Å (Table S3). for each individual mutant, with the indicated additions present at the con- α centrations specified in Materials and Methods, are shown. The addition of Outward Rotation of the -Helical Subdomain in the Intact Maltose ATP-Mg2þ allowed for ATP hydrolysis to occur. No changes in spectral line- Transporter. When studying the distance between the individual shape were seen with SL positioned at A124C. NBDs during the catalytic cycle, we observe a posthydrolytic

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006544107 Orelle et al. Downloaded by guest on September 27, 2021 Fig. 3. Spin-labeled Q122C/A167C mutant reports on the α-helical subdomain rotation. Two cysteine substitutions were made in MalK, at position Q122 in the α-helical and A167 in the RecA-like subdomain, and modified by MTSL. (A) Distances between Q122 and A167 in the structures of the apo and catalytic intermediate are indicated with dashed lines. The two struc- Fig. 4. Locked-closed MBP in the presence of AMP-PNP-Mg2þ stabilized a tures are superimposed based on the RecA-like subdomain and colored as semiopen MalK conformation but did not promote helical subdomain rota- described in Fig. 1B.(B) EPR spectra are color coded according to the ligands tion. Two cysteine substitutions were made in MBP (G69C/S337C) that form a added, overlaid and normalized to maximum amplitude. Enlargements of disulfide bond locking MBP in a closed, maltose-bound conformation (32). EPR spectra highlight the increase in dipolar broadening due to spin-spin 2þ (Inset, Lower), shows an SDS-PAGE gel of the mutant MBP (CL-MBP) prepara- interaction upon addition of AMP-PNP-Mg and maltose-MBP (red). (C) Dis- tion, which contains a small fraction of uncrosslinked MBP (unCL-MBP). EPR tance distributions (x-axis values are in Å) in the presence of different ligands spectra are color coded according to the ligands added (Lower Left) for both “ ” were determined using the program shortdistances100. panels (A) and (B). The EPR spectra in absence of ligand were quasi identical to the spectra obtained with maltose and cross-linked MBP (cyan) and are not

2þ BIOCHEMISTRY conformation requiring ADP-Mg and MBP in which the MalK drawn for clarity. EPR spectra are overlaid and normalized to maximum am- dimer is semiopen (22). The conformation of the helical subdo- plitude. (A) EPR spectra of spin-labeled V16C/R129C. This double mutant was main in this posthydrolytic conformation was studied by adding used previously to report on the distance between the two nucleotide-bind- ATP-Mg2þ and maltose-MBP to the spin-labeled Q122C/A167C ing domains (22). (B) EPR spectra of spin-labeled Q122C/A167C. The enlarge- mutant and allowing hydrolysis to occur (Fig. 3). A decrease in ment of the EPR spectrum highlights the difference in dipolar broadening triggered in the presence of mutant MBP as compared to WT-MBP. spin–spin interaction was observed in the posthydrolysis state (main distance centered at 12.5 Å; Fig. 3 and Table S3) suggesting that the α-helical subdomain of MalK rotated away from the smaller population (18% of interacting spins) at 8 Å, which RecA-like subdomain following ATP hydrolysis. Analysis of most likely resulted from interaction with the small population A124C/Q171C (Fig. S2 and Table S3) also showed a slight dis- of uncrosslinked species in the MBP preparation. The Q122C/A167C mutant was then used to analyze the he- tance increase after ATP hydrolysis (main distance ∼20 Å). lical subdomain movement. Fig. 4B shows that the main distance between spin labels (14–15 Å) was essentially unchanged upon Use of a Locked-Closed MBP to Probe Rotation. To gain a more detailed understanding of subdomain rotation, a mutant of MBP addition of the locked closed CL-MBP, either in the presence or absence of nucleotide, indicating that the helical subdomain (G69C/S337C) was used that forms a disulfide bond designed to does not rotate unless MBP is free to open. Eighteen percent prevent opening of the closed, maltose-bound MBP (32). We of the population was again seen at a distance suggestive of in- tested whether this disulfide cross-linked (CL-)MBP could pro- ward rotation (8 Å) probably resulting from the uncrosslinked mote conformational changes in the NBDs. Our mutant MBP ∼90% species (Table S3). These data indicated that binding of both preparation is cross-linked under nonreducing conditions AMP-PNP-Mg2þ and closed-MBP promoted a transition from (see SDS-PAGE gel in Fig. 4). We tested the effect of this MBP open MalK to semiopen MalK without substantial helical subdo- mutant first on a spin-labeled double mutant (V16C/R129C in main rotation. MalK) previously shown to report on the distance between the two NBDs of the MalK dimer (22). In the apo state, SLs in this Helical Subdomain Rotation in the Reconstituted Transporter. In con- double mutant were too far apart to detect interactions, indica- trast to a detergent-solubilized sample, the reconstituted trans- tive of an open MalK dimer interface (ref. 22 and Fig. 4A). In the 2þ porter does not display basal ATPase activity in the absence of presence of both AMP-PNP-Mg and wild-type MBP, the bulk MBP (33), hence the effect of ATP-Mg2þ binding on helical sub- ∼8 A of the population (65%) was centered at Å (Fig. 4 and domain rotation (rather than just a nonhydrolyzable analogue) Table S3), indicative of a closed MalK dimer interface (22). can be tested in the absence of MBP. Spin-labeled mutants were The remainder of the population (∼35% of interacting spins), reconstituted into nanodiscs, a lipid bilayer system recently devel- as fit by “shortdistances100,” was broadly distributed (Fig. 4A and oped in the laboratory of S. Sligar (34) and demonstrated to be a Table S3), although simulations done using an earlier program suitable membrane mimic for study of the maltose transporter (28), indicated that 85–90% of interacting spins were centered (33). Nanodiscs are soluble disk-shaped membrane patches at 8 Å under these conditions (22). Addition of CL-MBP alone formed by two molecules of membrane scaffold protein wrapped failed to trigger spin-spin interaction, however, when both around a bilayer of phospholipids. Advantages of nanodiscs over 2þ CL-MBP and AMP-PNP-Mg were present, the bulk of the po- proteoliposomes include the accessibility of ligands to both sides pulation of interacting spins (82%) were centered at ∼17 Å. This of the membrane and their ability to be highly concentrated, im- result suggested that the binding of closed MBP in the presence proving EPR data quality (33). As in detergent, motional changes of trinucleotide stabilizes a semiopen configuration at the NBD in SL attached to the RecA-like subdomain were seen upon interface. The distribution of spins seen in the presence of CL- addition of AMP-PNP-Mg2þ in the absence of MBP when the MBP and AMP-PNP-Mg2þ (Fig. 4A and TableS3) also included a transporter was reconstituted into nanodiscs (Fig. S3) demon-

Orelle et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 27, 2021 strating that trinucleotides bind to the reconstituted transporter, though established, lack of structural stabilization of this subdo- even though ATP is not hydrolyzed in the absence of MBP (33). main in absence of trinucleotide. Addition of maltose-MBP alone, AMP-PNP-Mg2þ alone or, We carried out site-directed spin labeling and EPR spectro- more importantly, ATP-Mg2þ alone to the doubly spin labeled scopy to investigate the movement of the helical subdomain in Q122C/A167C mutant did not trigger substantial changes in in- the context of the intact maltose transporter. Our data provided terspin distances with transporter reconstituted in nanodiscs biochemical evidence that this rotation does occur in an intact (Fig. 5A and Table S3). Distance changes consistent with inward ABC transporter. In the absence of ligand, EPR data on the he- rotation of the helical domain occurred only when both lical subdomain conformation were consistent with the inward- AMP-PNP-Mg2þ and maltose-MBP were added (Fig. 5A), and facing structure of the maltose transporter in which the helical the helical subdomain was rotated away from the RecA-like subdomain is rotated outward (23) (Tables S2 and S3). In stark subdomain in the posthydrolysis state (Fig. 5B and Table S3). contrast with the main conclusion drawn from studies of isolated 2þ We also tested the effect of CL-MBP plus AMP-PNP-Mg NBDs, in the intact transporter the binding of trinucleotide to the on the transporter conformation in nanodiscs. Results were again MalK subunits did not promote an inward rotation of the helical consistent with those obtained in detergent, although there was a subdomain relative to the RecA-like subdomain. The apo state significantly higher proportion of interacting spins (∼36%) corre- structure (23) therefore may be representative of the resting state sponding to full rotation of the helical subdomain than seen in of the transporter in vivo, where ATP is likely to be bound. The detergent (∼18%)(Fig. S4 and Table S3). It is possible that transmembrane domains must therefore impose constraints the non-CL-MBP present in the preparation, which will open that prevent both the rotation of the helical subdomain (this 2þ in the presence of AMP-PNP-Mg competed better for binding study) and the closure of the MalK subunits (22) in response 2þ to the reconstituted MalFGK2, accounting for the higher percen- to ATP-Mg binding alone. In contrast, binding of both tage. In summary, the EPR data obtained using transporter AMP-PNP-Mg2þ and maltose-MBP triggered the inward motion reconstituted into nanodiscs essentially recapitulated those ob- of the helical subdomain relative to the RecA-like subdomain, the tained in detergent. same conditions previously shown to trigger the closure of the NBDs in the intact transporter (22). These results indicated that Discussion the rotation played an important role in the transport mechanism, ABC transporters are powered by NBDs that use the energy of though it appeared to be part of a global conformational change ATP binding and hydrolysis to exert a mechanical task. Determin- involving MBP and the TMDs rather than a local response to ing how the conformational changes in these domains coordinate ATP binding. with those of the transmembrane domains is crucial to under- In the presence of AMP-PNP-Mg2þ and maltose-MBP, two stand the molecular mechanism of ABC transporters. Studies different populations of interacting spins were detected in the of the nucleotide-dependent association/dissociation of the two Q122C-A167C pair. Approximately 65% of the population was NBDs during the catalytic cycle (3, 35) indicate a strong coupling centered in a narrow distribution at a distance of ∼7.5 Å, and between these catalytic events and structural rearrangements – 35% in a broad distribution centered at 14.5 Å. Whereas it is in the TMDs (36 38). In addition, structural comparisons of iso- possible that a fraction of the transporters failed to undergo lated NBDs reveal a putative motion within each NBD, charac- any conformational change in response to binding, we find this terized by a rotation of the α-helical subdomain relative to the explanation unlikely as we often see evidence of participation RecA-like subdomain upon ATP binding and hydrolysis (5–8). of the entire population of transporters, as evidenced by changes This motion is also supported by molecular dynamic studies in mobility of spin label (see, for example, SL-S83C in Fig. 2). (10, 11) and the rotation is suggested to transmit conformational Two populations could also have resulted from the dominance changes to the transmembrane domains (16). Interestingly, the of more than one rotamer of the SL at either position 122 or 167 helical subdomain (14, 39) is the most variable region of the in the catalytic intermediate state. The length of the spin label NBDs (40, 41) and might have evolved to accommodate the struc- side chain is 6–7 Å and the coexistence of two rotamers at a single tural constraints imposed by the TMDs in ABC transporters. To site facing in different directions could account for two popula- the best of our knowledge, the only biochemical studies of the tions even though all of the helical subdomains undergo the same subdomain rotation were carried out on isolated NBDs (42, 43). rotation. Finally, given that MalK is a dimer, it is possible that just However, one might wonder whether the relevance and role of one of the two subunits has rotated inward, although both are helical subdomain rotation is overestimated, based on the sole, inward in the crystal structure of the catalytic intermediate, which is largely symmetric (24). After demonstrating the rotation of the helical subdomain in the transport cycle of the maltose transporter, the dynamics of this motion was investigated in more detail. A model is depicted in Fig. 6 to illustrate and summarize these results. Using a MBP locked-closed by cross-linking, we were able to characterize an additional intermediate of the catalytic transport cycle. When the cross-linked MBP interacted with the AMP-PNP-Mg2þ bound transporter, MalK transitioned from an open to a semiopen conformation. During this movement, the helical subdomain Fig. 5. Spin-labeled Q122C/A167C mutant reports on the α-helical subdo- rotated only slightly. Therefore, the subdomain rotation must oc- main rotation in nanodiscs. Mutant transporters, spin labeled at positions cur during the transition from semiopen to closed MalK dimer, Q122C in the helical subdomain and A167C in the RecA-like subdomain, were an event that coincides with the opening of MBP. Because the reconstituted into nanodiscs. EPR spectra in the presence of the indicated crystal structure of the catalytic intermediate (24) indicates that ligands are overlaid and normalized to maximum amplitude. (A) Effect of the open conformation of MBP stabilizes an outward-facing con- ATP-Mg2þ alone or AMP-PNP-Mg2þ plus maltose-MBP on dipolar broadening 2þ 2þ figuration of TM helices, we predict that the inward rotation of in nanodiscs. ATP-Mg can be used in place of AMP-PNP-Mg alone be- the subdomain coincides with the reorientation of TM helices cause the reconstituted transporter does not hydrolyze ATP in the absence of MBP. The EPR spectrum in the presence of maltose-MBP was quasi identical to generate the outward-facing conformation as MBP opens. Fol- to the spectrum obtained in absence of ligand (black) and is not drawn for lowing ATP hydrolysis within the closed NBDs, results from the clarity. (B) Effect of ATP-Mg2þ plus maltose-MBP on dipolar broadening in Q122C-A167C pair suggested that the helical subdomain rotated nanodiscs. ATP is hydrolyzed to ADP, generating a posthydrolysis state. almost fully away from the RecA-like subdomain, whereas the

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006544107 Orelle et al. Downloaded by guest on September 27, 2021 from semiopen to closed, is associated with a large increase in affinity between MBP and FGK2 (25, 45). Likewise, ATP binding contributes to the stabilization of these intermediate states, especially the outward-facing conformation that has a closed nucleotide-binding interface. Because neither ATP alone nor MBP alone can promote this transition, it is likely stabilized by both interactions. ATP hydrolysis would lead to an increase in the free energy of the outward-facing conformation by destabilizing the closed MalK dimer, allowing for a spontaneous return to a lower energy inward-facing state. The NBD–TMD interface in exporters is quite different from that in importers. In importers, NBDs are in close contact with the TMDs, whereas long intracellular loops place the NBDs further from the membrane in exporters (46, 47). Two intracellular loops per dimer subunit form coupling helices, one interacts with residues around the Q-loop of the opposing subunit whereas the second binds a previously unrecognized motif unique to exporters named the “x-loop” (47). The described differences in the NBD/TMD interface of exporters as compared to the maltose importer appear to translate into distinct motions in the TM he- Fig. 6. Model of MalFGK2 transport cycle. (Upper Left) The ATP-bound trans- lices although the net result, alternating access for substrate in the porter is in an inward-facing resting state with open MalK. Helical subdo- TMD coupled to closure of the NBDs, may be conserved (48, 49). mains (red) are in an outward conformation. When maltose-bound MBP Alignment of the RecA-like subdomains of outward-facing interacts with the transporter (Upper Right), ATP-bound MalK adopts a semiopen conformation, but the helical subdomain is still outward. This confor- Sav1866 (50) and inward-facing P-glycoprotein (51) revealed a mation can only be observed if the MBP is maintained closed via cross-link. similar inward rotation of the helical subdomain associated with (Lower Right) The helical domain rotates inward and MalK closes as MBP NBD closure and reorientation of TM helices (Fig. S5). It is not opens, releasing maltose into the binding site of MalF. The transporter is yet clear how the substrates of exporters affect this global confor-

outward-facing. Next, hydrolysis of one or two ATP promotes a presumably mational change, which encompasses the helical subdomain BIOCHEMISTRY inward-facing conformation of the transporter. MalK is semiopen, and the rotation. Multidrug transporters typically exhibit both basal and helical subdomain outward. Dissociation of either ADP or MBP returns the drug-stimulated ATPase activities (52–54), whereas TAP1/TAP2 transporter to its resting state (Upper Left). Maltose is released inside the cell is an exporter that displays an ATPase activity tightly regulated by either when MalK is in the semiopen or open conformations. peptide substrates (55). ATP binding alone may permit helical subdomain rotation in a drug efflux pump, whereas TAP may MalK subunits adopted a semiopen conformation (22). This re- more closely resemble the maltose transporter with respect to sult suggests that the outward subdomain rotation, like the inward the regulation of ATPase activity. rotation, occurs during the transition between closed and semiopen conformations of MalK. Material and Methods These observations strongly support a role of the helical do- Site-Directed Mutagenesis. Mutagenesis was performed using the main in coupling of conformational changes between NBDs GeneTailor Kit (Invitrogen), according to the manufacturer’s and TMDs and the predicted movements are largely consistent instructions. Single mutations in malK were introduced on the with the available crystal structures (23). The transition between vector pCO-SSM, which encodes a cysteine-free version of MalK the inward- and outward-facing conformations of the transporter previously described (22). Double mutants were constructed by involves concerted motions of transmembrane helices and NBDs. subcloning fragments from each single mutant plasmid, using The main connection between the TMD and the NBD consists of restriction enzymes NheI and BsiWI. The sequences of mutant the coupling helix containing the EAA motif (12, 40) docked into malK genes were verified by DNA sequencing. a cleft on the surface of MalK (Fig. 1B). Although the reorientation of TM helices appears to involve rigid body rotations (23), Purification and Labeling of MalFGK2. Purification was performed as the movement at this interface is likened to a ball and socket joint previously described (22), except for two modifications. Five mM (23) implying a flexibility that would allow for subtle but poten- imidazole was present in the buffer when the spin-labeled tially crucial changes at the interface without disengaging the detergent supernatant was incubated with Talon affinity resin, subunits. ATP binding induces changes in cross-linking patterns which improved the purity of the samples, as judged by Coomas- between NBDs and TMDs in the absence of MBP (15). We sie-staining of SDS-PAGE gels. The gel filtration chromatogra- detected changes in SL mobility at position 168 in the RecA-like phy step, previously used to increase the purity of mutants subdomain in response to nucleotide-binding even though it is used for spin–spin interaction (22), was then omitted. Mutants 20 Å from the site of nucleotide-binding. Although the EAA were labeled with 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl loops move along with MalK during the catalytic cycle, differ- methanethiosulfonate spin label (MTSL), as described (22). ences in cross-linking between EAA loops and Q-loops are seen during the catalytic cycle of MalFGK2 (44). Expression and Purification of G69C/S337C Mutant of MBP. Details are The model depicted in Fig. 6, which bears similarity to the provided in SI Text. mechanism proposed by Shilton (20), has several mechanistic implications. Closed MBP helps to stabilize an inward-facing ATPase Activity. Assays were carried out on proteins reconstituted intermediate state in which MalK2 is semiopen. This conforma- in liposomes, using a coupled assay, as described previously (22). tion is visible by EPR when both locked-closed MBP and 2þ AMP-PNP-Mg are bound to the transporter. Similarly, open Cw-EPR Spectroscopy. X-band EPR was performed at room tem- MBP plus trinucleotide helps to stabilize an outward-facing inter- perature, as described previously (22). Single and double mutants mediate state that is competent for ATP hydrolysis. The transi- spectra shown are 100 and 200 G wide, respectively. Protein sam- tion of bound MBP from closed to open, which coincides with ples were studied either in detergent (22) or following reconstitu- rotation of the helical subdomain and the transition of MalK2 tion in nanodiscs (see SI Text). Samples (transporter ∼90 μM)

Orelle et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 27, 2021 were incubated with one or more of the following ligands for spectra consist of an unbroadened EPR lineshape convolved with 15 min at room temperature before recording spectra: 15 mM a sum of Pake functions for randomly oriented spin pairs at a ATP, 15 mM ADP, 15 mM AMP-PNP, 10 mM MgCl2, 600 μM distance r. The distance distributions from dipolar broadening μ – maltose, 250 M MBP. Spin spin distances were determined are determined by fitting simulated spectra with experimental by fitting the experimental EPR data with simulations using a cus- data using Tikhonov regularization and nonlinear optimization. tom program developed by C. Altenbach (“shortdistances100,” written in Labview 2009 and available at http://sites.google. ACKNOWLEDGMENTS. C.O. thanks Dr. Olivier Dalmas for critical reading of the com/site/altenbach/labview-programs/short-distances). Briefly, manuscript. This work was supported by National Institutes of Health Grants this program utilizes a convolution method in which the interspin GM49261 (A.L.D.) and GM070515-06 (A.L.D. and J.C.), and J.C. is a HHMI distances are simulated as Gaussian distributions. The simulated investigator.

1. Rees DC, Johnson E, Lewinson O (2009) ABC transporters: The power to change. Nat 29. McHaourab HS, Oh KJ, Fang CJ, Hubbell WL (1997) Conformation of T4 lysozyme in Rev Mol Cell Biol 10:218–227. solution. Hinge-bending motion and the substrate-induced conformational transition 2. Kos V, Ford RC (2009) The ATP-binding cassette family: A structural perspective. Cell studied by site-directed spin labeling. Biochemistry 36:307–316. Mol Life Sci 66:3111–3126. 30. Richmond TJ (1984) Solvent accessible surface area and excluded volume in proteins. 3. Moody JE, Thomas PJ (2005) Nucleotide binding domain interactions during the Analytical equations for overlapping spheres and implications for the hydrophobic mechanochemical reaction cycle of ATP-binding cassette transporters. J Bioenerg effect. J Mol Biol 178:63–89. Biomembr 37:475–479. 31. Hung LW, et al. (1998) Crystal structure of the ATP-binding subunit of an ABC trans- 4. Ye J, Osborne AR, Groll M, Rapoport TA (2004) RecA-like motor ATPases—Lessons from porter. Nature 396:703–707. structures. Biochim Biophys Acta 1659:1–18. 32. Zhang Y, Mannering DE, Davidson AL, Yao N, Manson MD (1996) Maltose-binding 5. Karpowich N, et al. (2001) Crystal structures of the MJ1267 ATP binding cassette reveal protein containing an interdomain disulfide bridge confers a dominant-negative an induced-fit effect at the ATPase active site of an ABC transporter. Structure phenotype for transport and chemotaxis. J Biol Chem 271:17881–17889. 9:571–586. 33. Alvarez FJ, Orelle C, Davidson AL (2010) Functional reconstitution of an ABC transpor- 6. Chen J, Lu G, Lin J, Davidson AL, Quiocho FA (2003) A tweezers-like motion of the ter in nanodiscs for use in electron paramagnetic resonance spectroscopy. J Am Chem ATP-binding cassette dimer in an ABC transport cycle. Mol Cell 12:651–661. Soc 132:9513–9515. 7. Smith PC, et al. (2002) ATP binding to the motor domain from an ABC transporter 34. Ritchie TK, et al. (2009) Chapter 11—Reconstitution of membrane proteins in phos- – drives formation of a nucleotide sandwich dimer. Mol Cell 10:139 149. pholipid bilayer nanodiscs. Methods Enzymol 464:211–231. 8. Zaitseva J, et al. (2006) A structural analysis of asymmetry required for catalytic activity 35. Tombline G, Muharemagic A, White LB, Senior AE (2005) Involvement of the “oc- – of an ABC-ATPase domain dimer. EMBO J 25:3432 3443. cluded nucleotide conformation” of P-glycoprotein in the catalytic pathway. Biochem- 9. Campbell JD, Deol SS, Ashcroft FM, Kerr ID, Sansom MS (2004) Nucleotide-dependent istry 44:12879–12886. conformational changes in HisP: Molecular dynamics simulations of an ABC transpor- 36. Rosenberg MF, et al. (2001) Repacking of the transmembrane domains of P-glycopro- – ter nucleotide-binding domain. Biophys J 87:3703 3715. tein during the transport ATPase cycle. EMBO J 20:5615–5625. 10. Jones PM, George AM (2007) Nucleotide-dependent allostery within the ABC transpor- 37. Vergani P, Lockless SW, Nairn AC, Gadsby DC (2005) CFTR channel opening by ATP- ter ATP-binding cassette: A computational study of the MJ0796 dimer. J Biol Chem driven tight dimerization of its nucleotide-binding domains. Nature 433:876–880. – 282:22793 23803. 38. Orelle C, et al. (2008) Conformational change induced by ATP binding in the multidrug 11. Wen PC, Tajkhorshid E (2008) Dimer opening of the nucleotide binding domains of ATP-binding cassette transporter BmrA. Biochemistry 47:2404–2412. ABC transporters after ATP hydrolysis. Biophys J 95:5100–5110. 39. Locher KP, Lee AT, Rees DC (2002) The E. coli BtuCD structure: A framework for ABC 12. Hollenstein K, Dawson RJ, Locher KP (2007) Structure and mechanism of ABC transporter architecture and mechanism. Science 296:1091–1098. transporter proteins. Curr Opin Struct Biol 17:412–418. 40. Dassa E, Bouige P (2001) The ABC of ABCS: A phylogenetic and functional classification 13. Oldham ML, Davidson AL, Chen J (2008) Structural insights into ABC transporter of ABC systems in living organisms. Res Microbiol 152:211–229. mechanism. Curr Opin Struct Biol 18:726–733. 41. Schmitt L, Benabdelhak H, Blight MA, Holland IB, Stubbs MT (2003) Crystal structure of 14. Mourez M, Hofnung M, Dassa E (1997) Subunit interactions in ABC transporters: A the nucleotide-binding domain of the ABC-transporter haemolysin B: Identification of conserved sequence in hydrophobic membrane proteins of periplasmic permeases de- a variable region within ABC helical domains. J Mol Biol 330:333–342. fines an important site of interaction with the ATPase subunits. EMBO J 16:3066–3077. 42. Gupta S, Chakraborti PK, Sarkar D (2005) Nucleotide-induced conformational change 15. Hunke S, Mourez M, Jehanno M, Dassa E, Schneider E (2000) ATP modulates subunit– in the catalytic subunit of the phosphate-specific transporter from M. tuberculosis: subunit interactions in an ATP-binding cassette transporter (MalFGK2) determined by Implications for the ATPase structure. Biochim Biophys Acta 1750:112–121. site-directed chemical cross-linking. J Biol Chem 275:15526–15534. 43. Horn C, et al. (2008) Monitoring conformational changes during the catalytic cycle 16. Jones PM, George AM (2002) Mechanism of ABC transporters: a molecular dynamics of OpuAA, the ATPase subunit of the ABC transporter OpuA from Bacillus subtilis. simulation of a well characterized nucleotide-binding subunit. Proc Natl Acad Sci USA Biochem J 412:233–244. 99:12639–12644. 44. Daus ML, et al. (2007) ATP-driven MalK dimer closure and reopening and conforma- 17. Klug CS, Feix JB (2008) Methods and applications of site-directed spin labeling EPR tional changes of the “EAA” motifs are crucial for function of the maltose ATP-binding spectroscopy. Methods Cell Biol 84:617–658. cassette transporter (MalFGK2). J Biol Chem 282:22387–22396. 18. Hellmich UA, Glaubitz C (2009) NMR and EPR studies of membrane transporters. Biol 45. Chen J, Sharma S, Quiocho FA, Davidson AL (2001) Trapping the transition state of an Chem 390:815–834. ATP-binding cassette transporter: Evidence for a concerted mechanism of maltose 19. Bordignon E, Grote M, Schneider E (2010) The maltose ABC transporter in the 21(st) – century—towards a structural-dynamic perspective on its mode of action. Mol Micro- transport. Proc Natl Acad Sci USA 98:1525 1530. biol doi: 10.1111/j.1365-2958.2010.07319.x. 46. Chami M, et al. (2002) Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis. J Mol 20. Shilton BH (2008) The dynamics of the MBP-MalFGKð2Þ interaction: A prototype – for binding protein dependent ABC-transporter systems. Biochim Biophys Acta Biol 315:1075 1085. 1778:1772–1780. 47. Dawson RJ, Locher KP (2006) Structure of a bacterial multidrug ABC transporter. – 21. Davidson AL, Shuman HA, Nikaido H (1992) Mechanism of maltose transport in Escher- Nature 443:180 185. ichia coli: Transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci 48. Smriti Zou P, McHaourab HS (2009) Mapping daunorubicin-binding sites in the ATP- USA 89:2360–2364. binding cassette transporter MsbA using site-specific quenching by spin labels. J Biol – 22. Orelle C, Ayvaz T, Everly RM, Klug CS, Davidson AL (2008) Both maltose-binding Chem 284:13904 13913. protein and ATP are required for nucleotide-binding domain closure in the intact 49. Zou P, McHaourab HS (2009) Alternating access of the putative substrate-binding maltose ABC transporter. Proc Natl Acad Sci USA 105:12837–12842. chamber in the ABC transporter MsbA. J Mol Biol 393:574–585. 23. Khare D, Oldham ML, Orelle C, Davidson AL, Chen J (2009) Alternating access in 50. Dawson RJ, Locher KP (2007) Structure of the multidrug ABC transporter Sav1866 from maltose transporter mediated by rigid-body rotations. Mol Cell 33:528–536. Staphylococcus aureus in complex with AMP-PNP. FEBS Lett 581:935–938. 24. Oldham ML, Khare D, Quiocho FA, Davidson AL, Chen J (2007) Crystal structure of a 51. Aller SG, et al. (2009) Structure of P-glycoprotein reveals a molecular basis for catalytic intermediate of the maltose transporter. Nature 450:515–521. poly-specific drug binding. Science 323:1718–1722. 25. Austermuhle MI, Hall JA, Klug CS, Davidson AL (2004) Maltose-binding protein is open 52. Al-Shawi MK, Polar MK, Omote H, Figler RA (2003) Transition state analysis of the in the catalytic transition state for ATP hydrolysis during maltose transport. J Biol coupling of drug transport to ATP hydrolysis by P-glycoprotein. J Biol Chem Chem 279:28243–28250. 278:52629–52640. 26. Schrodinger L (2010) The PyMOL Molecular Graphics System, Version 1 3r1. (DeLano 53. van Veen HW, et al. (1998) A bacterial antibiotic-resistance gene that complements the Scientific, San Carlos, CA). human multidrug-resistance P-glycoprotein gene. Nature 391:291–295. 27. Jones PM, O’Mara ML, George AM (2009) ABC transporters: A riddle wrapped in a 54. Steinfels E, et al. (2004) Characterization of YvcC (BmrA), a multidrug ABC transporter mystery inside an enigma. Trends Biochem Sci 34:520–531. constitutively expressed in Bacillus subtilis. Biochemistry 43:7491–7502. 28. Altenbach C, Oh KJ, Trabanino RJ, Hideg K, Hubbell WL (2001) Estimation of inter- 55. Herget M, et al. (2009) Purification and reconstitution of the antigen transport residue distances in spin labeled proteins at physiological temperatures: Experimental complex TAP: A prerequisite for determination of peptide stoichiometry and ATP strategies and practical limitations. Biochemistry 40:15471–15482. hydrolysis. J Biol Chem 284:33740–33749.

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