Complex Stability During the Transport Cycle of a Subclass I ECF

Jul 28, 2017 - The mechanism of energy-coupling factor (ECF) transporters, a special type of ATP-binding-cassette importers for micronutrients in prok...
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Complex Stability During the Transport Cycle of a Subclass I ECF Transporter Friedrich Finkenwirth, Franziska Kirsch, and Thomas Eitinger Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00390 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Complex Stability During the Transport Cycle of a Subclass I ECF Transporter Friedrich Finkenwirth†, Franziska Kirsch†, and Thomas Eitinger †

*,†

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany

Corresponding Author

* Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany. Telephone: +49 30 2093 49680. Fax: +49 30 2093 49681. E-mail: [email protected]

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ABBREVIATIONS ABC, ATP-binding cassette; DDM, n-dodecyl β-D-maltopyranoside; ECF, energy-coupling factor; EDTA, ethylenediaminetetraacetate; NTA, nitrilotriacetate; SDS PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis.

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ABSTRACT: The mechanism of energy-coupling factor (ECF) transporters, a special type of ATP-binding-cassette importers for micronutrients in prokaryotes, is a matter of controversial discussion. Among subclass II ECF transporters, a single ECF interacts with several substrate-binding integral membrane proteins (S units) for individual solutes. Release-and-catch of the S unit, previously observed experimentally for a subclass II system, was proposed as the mechanism of all ECF transporters. The BioM2NY biotin transporter is a prototype of subclass I systems among which the S unit is dedicated to a specific ECF. Here, we simulated the transport cycle using purified BioM2NY in detergent solution. BioM2NY complexes were stable during all steps. ATP binding was a prerequisite for biotin capture and ATP hydrolysis for subsequent biotin release. The data demonstrate that S units of subclass I ECF transporters do not have to dissociate from holotransporter complexes for high-affinity substrate binding indicating mechanistic differences between the two subclasses.

KEYWORDS Biotin Transporter; Energy-Coupling-Factor Transporter; ABC transporter; Toppling Mechanism.

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INTRODUCTION Energy-coupling factor (ECF) transporters came into focus only recently as a unique type of ABC transporters mediating uptake of micronutrients like vitamins and transition metal ions into prokaryotic cells (see refs. 1-6 for a review). They consist of homodimeric or heterodimeric ABC ATPases, a high-affinity substrate-binding transmembrane protein (S unit), and a transmembrane docking component (T unit) that interacts with the two ATPase molecules and the S unit. The assembly of the ATPases and the T unit represents the ECF, a term created in the 1970's during analyses of vitamin uptake in lactic acid bacteria for an unknown component that is shared by various vitamin transporters 7. Molecular details of ECF transporters were uncovered about thirty years later by investigation of metal-specific 8 and biotin-specific 9 systems. Shortly after, ECF transporters were found to fall into two subgroups 10: Subgroup I systems are encoded by operons and represent dedicated transporters in which a specific ECF interacts with a single S unit. In contrast, a single ECF interacts with different S components to form subgroup II transporters for individual substrates. Although S units for different solutes share only negligible amino acid sequence similarity, their core 3D topology with a six-transmembrane-helix architecture is surprisingly similar as evidenced by X-ray structure determination of BioY (specific for biotin, 11), FolT (folate, 12-14), NikM2 (Ni2+, 15), PanT (pantothenate, 16), PdxU2 (formerly called HmpT, 17; pyridoxamine, 18 ), RibU (riboflavin, 19, 20), ThiT (thiamine, 21), and YkoE (thiamine, 22). Whereas the majority of S units was crystallized in a substrate-bound form in the solitary state, ECF holotransporter structures were reported for A1A2T-FolT 12, 13, A1A2T-PanT 16 , A1A2T-PdxU2 17, 18, and a subgroup I Co2+ transporter (CbiMQO2) 23 in which the S units were substrate-free. The conformation of S units in the solitary and complex-bound state is very similar. Major differences were observed for the conformation of extracytoplasmic loops. Loop 1 connecting transmembrane helices one and two acts as a lid on the substratebinding pocket in most S components except YkoE. Displacement of this loop, which was observed in the holotransporter structures and in substrate-free solitary ThiT 24, destroys the substrate-binding site and is considered a prerequisite for substrate release into the cytoplasm. This assumption is corroborated by the fact that the S units in the available holotransporter structures are oriented almost perpendicular relative to the transmembrane T units. Based on these findings, a toppling mechanism for ECF transporters was proposed in which the S units rotate to an upright orientation for extracytoplasmic highaffinity substrate binding, and topple over for subsequent intracellular substrate release. The toppling hypothesis was tested with the purified subgroup II riboflavin transporter (A1A2T-RibU) from Listeria monocytogenes in detergent solution 25, and the subgroup I biotin transporter BioM2NY from Rhodobacter capsulatus upon reconstitution in lipid nanodiscs 26. The results of fluorescence analyses of the two transporters containing dye-labelled S units strongly support a common step in which ATP binding to the ATPases in the resting state eventually causes rotation of the S components. The following steps differ between the two systems: In the ATP-bound state, the S unit RibU is released from the subgroup II A1A2T-RibU complex, binds its substrate and is recaptured by the ECF. This release-and-catch mechanism 25 is compatible with the early observations of competition of S units for a shared component 7, and with a recent whole-cell transport study using recombinant E. coli producing subgroup II ECF transporter components 27. Dissociation of the S unit BioY from the ECF BioM2N was not observed for the subgroup I biotin transporter 26. This conclusion was drawn from experiments in lipid nanodiscs, an environment that may have prevented detection of complex dissociation. Whether or not ATP hydrolysis is essential for toppling of the substrate-loaded S unit or coupled to an alternate step in the transport cycle is another topic of controversial discussion. Karpowich et al. 25 observed capture of substrate-bound S units by an ATP-loaded ECF variant that cannot hydrolyze ATP. This finding suggests a role of ATP hydrolysis in a later step during toppling and substrate release. Likewise, conditions allowing ATP hydrolysis were required to observe biotin release from the biotin-loaded BioM2NY complex in the nanodisc system 26. In an alternative model by Swier et al. 13, an ATPbound state of the ECF is required solely for rotation of the S unit to result in an upright conformation, and its release from the complex. ATP hydrolysis regenerates the binding platform of the ECF. The substrate-bound S unit topples over spontaneously and combines with the nucleotide-free ECF resulting in destruction of the substrate binding site and substrate release. This model is based on structural comparisons of ECF components from Lactobacillus delbrueckii, the folate-bound FolT1 S unit in isolation and the A1A2T-FolT2 complex containing the substrate-free FolT2, a close relative of FolT1. A closed substrate-binding pocket of the S unit is predicted to prevent stable interaction with the T unit through sterical problems. The authors therefore suppose that high-affinity substrate binding and holotransporter complex formation are mutually exclusive. In light of the different models for the mechanism of ECF transporters, we reinvestigated the transport cycle of BioM2NY in the present study. To exclude the possibility that entrapment in a membrane environment prevents detection of complex dissociation, purified BioM2NY was analyzed in an immobilized state in detergent solution. The data clearly confirm our previous model. Biotin binds to the ATP-loaded BioM2NY complex which does not dissociate at any step of the cycle. Biotin release from the complex depends on ATP hydrolysis.

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MATERIALS AND METHODS Purification of ECF Transporter Components. Escherichia coli UT5600 was used for production of BioM2NY complexes (with an N-terminal deca-His-tag on BioM, a C-terminal cMyc-tag on BioN and a C-terminal FLAG-tag on BioY) and transporter variants were purified as previously described 9, 28. Briefly, recombinant E. coli were grown in 2-liter culture volume in Lysogeny Broth. The harvested cells were disrupted in a French pressure cell, membranes were pelleted by ultracentrifugation and subsequently solubilized in DDM-containing buffer. BioM2NY complexes were purified by nickel-chelate affinity chromatography via the deca-His-tag on BioM. The eluates were concentrated in Amicon filtration units (MerckMillipore), passed through PD-10 columns (GE Healthcare) and stored in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 15 % (vol./vol.) glycerol, 0.05 % (wt./vol.) DDM. For stimulation of biotin binding to Bio(ME161Q)2NY, 2 mM ATP, 10 mM MgCl2 and 6.7 µM biotin were added during solubilization of the membranes and all purification steps. Mass Spectrometry. The amount of biotin bound to BioM2NY complexes upon isolation was quantified via an HPLCcoupled electrospray ionization time-of-flight mass-spectrometric (ESI-ToF MS) assay as previously described 29. Briefly, 250 µl samples containing 10 µM BioM2NY in detergent solution were heat-denatured. After pelleting denatured protein, 50 µl of the supernatant (representing the equivalent of 500 pmol BioM2NY) was subjected to ESI-ToF MS. For biotin quantification, a freshly prepared biotin calibration solution was used as described 29. Size-Exclusion Chromatography. Purified BioM2NY complexes (2.5 mg in storage buffer) were concentrated in Amicon filtration units to a final concentration of 50 µM, and 500 µl were loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with DTT (2 mM)-containing storage buffer in an ÄKTA system equipped with a UV detector (GE Healthcare). Upon elution from the column, peak fractions representing elution volumes between 11.5 ml and 13 ml were pooled and split into four equal samples with a protein concentration of approximately 5 µM. Where indicated, effectors (2 mM ATP, 50 µM biotin or both) were added and the mixtures were kept on ice before being concentrated to 50 µM and reloaded onto the column. Chromatograms were analyzed with the UNICORN software (GE Healthcare). Stability and Activity of Immobilized BioM2NY. His-tagged BioM2NY variants (2.5 nmol in 1 ml of storage buffer) were immobilized using 250 µl Ni-NTA-matrix. For stability assays, ADP (2 mM), ATP (2 mM), biotin (50 µM), MgCl2 (5 mM) and combinations thereof as indicated were added, the samples were incubated for 12-16 h at 4 °C, washed once with 5 ml storage buffer, once with 5 ml storage buffer containing 100 mM imidazole, and were eluted with 1 ml storage buffer containing 300 mM imidazole. Upon concentration to a volume of 125 µl, aliquots (15 µl) were subjected to SDS PAGE. For [3H]biotin-binding assays samples were treated as above with the exception that [3H]biotin (50 µM, 89 Ci/mmol), [3H]biotin plus ATP (2 mM) plus EDTA (0.5 mM), and [3H]biotin plus ATP plus MgCl2 (5 mM), respectively, were the effectors. [3H]biotin in 50 µl of the concentrates was quantified by liquid scintillation counting. Aliquots (15 µl) were subjected to SDS PAGE.

RESULTS AND DISCUSSION Biotin content of BioM2NY in detergent solution. Previous mass spectrometric analyses had failed to detect biotin in purified wild-type BioM2NY 26. The lack of biotin was ascribed to the presence of ATP upon disruption of the recombinant E. coli cells resulting in ATP hydrolysis and concomitant substrate release. Alternatively, the transporter complexes may have lost their vitamin substrate as a consequence of solubilization from membranes and storage in detergent solution. To distinguish between the two possibilities and test the feasibility of investigating individual steps of the transport cycle in detergent solution, we compared the biotin content of wild-type (BioMwt)2NY and the (BioME161Q)2NY variant in the as-isolated state. This variant lacks the so-called catalytic carboxylate, retains the ability to bind Mg2+-ATP but is unable to hydrolyze the nucleotide 26. The results are summarized in Figure 1. The purified BioM2NY forms displayed a comparable subunit stoichiometry. Apparently, slightly lesser amounts of BioY were detected by SDS PAGE compared to BioN suggesting that subfractions of BioM2N had lost their BioY S unit. In agreement with previous findings, biotin was not detectable in Bio(Mwt)2NY but was detected by mass spectrometry in Bio(ME161Q)2NY. Under conditions of standard purification, approx. 5 % of the transporter complexes were loaded with a biotin molecule assuming that the total protein is organized in BioM2NY stoichiometry. Since this assumption ignores the presence of BioY-free complexes, the true percentage of biotinloaded Bio(ME161Q)2NY complexes is higher. Addition of Mg2+-ATP and biotin during all purification steps resulted in approx. 10 % of biotin-bound Bio(ME161Q)2NY complexes. As shown in Figure 1, these findings suggest that a significant fraction of the complexes was capable of binding ATP leading to reorientation of BioY and subsequent biotin binding. Hence, the central steps of the transport cycle can be analyzed in detergent solution. Effects of substrates on complex stability. In the next series of experiments, we analyzed the response of wild-type BioM2NY, purified in the absence of additives, to the addition of ATP and/or biotin using size exclusion chromatography. The results are illustrated in Figure 2. The red fractions obtained during the original chromatography (Figure 2A) were pooled and split, the resulting samples were treated as indicated in Figure 2B and re-chromatographed. It was clearly evident by the elution profiles and SDS PAGE that the addition of ATP did not affect complex stability neither in the absence of 5

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biotin nor in its presence. Significant complex dissociation was observed, however, when biotin was added in the absence of ATP. Under those conditions, an additional peak at 14.4 ml in the elution profile occured that contained elevated amounts of BioY. Biotin-containing solitary BioY was chromatographed on the same column and eluted at 14.4 ml as the major peak, and at 9 ml and 11.5 ml as much smaller peaks representing oligomers (data not shown). Since the BioM2NY peak and the BioY peak overlap (Figure 2B, right-hand bottom panel), it is not surprising that the fraction around 14.4 ml does not contain only BioY, which is the dominant protein, but also BioM and BioN. By comparison of the "red" part of Figure 2A and the panels in Figure 2B it became evident that BioM2NY did not elute as a monodisperse complex since the peaks in Figure 2B were broader than those of the collected fractions shown in Figure 2A. Our findings are compatible with the cartoons shown in Figure 2B. Complex dissociation does not take place under any condition unless biotin is present at high concentration in the absence of ATP. This nonphysiological state is irrelevant in a living cell. In order to investigate the influence of biotin and nucleotides on complex stability in more detail, the compounds were added to BioM2NY immobilized to Ni-NTA matrix. Upon washing, the complexes were eluted with imidazole-containing buffer and the eluates were analyzed by SDS PAGE. Figure 3 confirms the previous observation that the presence of biotin results in dissociation of BioY from the wild-type complex in the absence of ATP, but not in its presence. ATP-hydrolysis activity depends on the presence of a divalent cation. It was not surprising that Mg2+-ATP did not prevent biotin-induced complex decomposition. At 4 °C, BioM2NY retains approx. 10 % of its ATPase activity compared to 37 °C. Therefore, the long incubation of >12 h in Ni-NTA solution with effectors at 4 °C probably resulted in almost complete hydrolysis of ATP. In contrast, Mg2+-ATP did antagonize the biotin effect when Bio(ME161Q)2NY was analyzed, since this variant binds, but cannot hydrolyze the nucleotide 26 (Figure 3). ATP hydrolysis by wild-type complexes produces Mg2+-ADP in the nucleotide-binding site of BioM. A role of Mg2+-ADP for complex stabilization or destabilization was not observed. It did neither cause release of BioY in the absence of additional effectors nor did it prevent this dissociation upon addition of biotin (Figure 3).

Figure 1. Influence of effectors on the biotin content of purified BioM2NY. The complexes were isolated via Ni2+-chelate affinity chromatography. Biotin contents were quantified by mass spectrometry. In BioM variants with the E161Q replacement an ATP-bound state is maintained since ATP-hydrolysis activity is abolished. The cartoons in the lower part illustrate the predicted topologies of the complexes.

To analyze the significance of the substrate-binding site in BioY for the destabilizing property of biotin, we investigated the behavior of the BioM2N(YD164N) complex. This variant lacks the strongly conserved carboxylate within transmembrane helix VI of BioY. The carboxylate was shown in crystals of Lactococcus lactis BioY to interact with the ureido ring of biotin in the substrate-binding pocket 11, and to be essential for biotin binding to R. capsulatus BioY 29, the object of the present study, as well as BioY proteins from other organisms 30. Even in the absence of ATP, BioM2N(YD164N) complexes did not dissociate by addition of biotin (Figure 3). The data suggest that the closed vitamin-binding pocket of biotin-containing wildtype BioY prevents its interaction with ATP-free BioM2N; they confirm on the other hand, that stable complexes are formed with the ATP-containing BioM2N. These findings are only partly in agreement with the transport model of Swier et al. 13 which excludes substrate binding to S units in holotransporter complexes under any conditions. 6

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Figure 2. Size-exclusion chromatography of detergent-solubilized BioM2NY. (A) Fractions from 11.5-13 ml (highlighted in red) around the peak (at 12.1 ml) were pooled, split and (B) re-chromatographed upon addition of substrates as indicated. Aliquots of the peak fractions and adjacent fractions were analyzed by SDS PAGE.

Biotin capture and release. [3H]biotin binding assays were performed to determine the significance of ATP binding and ATP hydrolysis for substrate capture and release by detergent-solubilized, immobilized wild-type BioM2NY. As in the case of its non-radioactive counterpart (Figure 3), [3H]biotin caused dissociation of BioY from ATP-free BioM2N (Figure 4). Addition of ATP, which could not be hydrolyzed in the presence of the divalent-metal-ion scavenger EDTA, was a prerequisite for [3H]biotin binding to the holotransporter complex. In the presence of Mg2+-ATP, i.e. under conditions that allow ATP hydrolysis, only background amounts of [3H]biotin were captured and BioY dissociated from BioM2N (Figure 4). Considering the fact that Mg2+-ATP plus biotin did not result in dissociation of BioY from holotransporter complexes, if ATP hydrolysis was blocked (Bio(ME161Q)2NY, Figure 3), and our previous work with nanodisc-embedded BioM2NY, that retains biotin under these conditions 26, we propose the following order of events: ATP binding induces a conformation of the holotransporter ready for substrate binding, and ATP hydrolysis is required for subsequent substrate release. ATP consumption under the artificial experimental conditions led to complex dissociation. The complex stability of BioM2NY during all steps of the transport cycle observed in our previous work in the nanodisc system 26 and in the present study in detergent solution may represent a specific feature of subgroup I transporters in which S units are dedicated to a specific ECF. In contrast, a releaseand-catch mechanism is a lucid explanation for promiscuity of subgroup II ECFs. Future structural and dynamic analyses of 7

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additional members of the two subgroups are needed to uncover the molecular details that distinguish between the two mechanisms.

Figure 3. Role of substrates for complex stability of BioM2NY. Purified complexes in detergent solution were immobilized to Ni2+chelate affinity matrix (2.5 nmol BioM2NY, 250 µl Ni-NTA in storage buffer, 1 ml total volume) and incubated in the presence of substrates as indicated; ADP, 2 mM; ATP, 2 mM; biotin, 50 µM; MgCl2, 5 mM. Upon washing, elution and concentration, aliquots were analyzed by SDS PAGE. The BioME161Q and BioYD164N replacements eliminate ATPase activity and biotin binding, respectively. The vertical line between the first and second lane in the middle panel indicates that additional lanes were contained in the original gel.

Figure 4. Biotin capture by immobilized BioM2NY. The experimental setup was essentially as described in the legend to Figure 3.

[3H]biotin (50 µM), ATP (2 mM), MgCl2 (5 mM) and EDTA (0.5 mM) as indicated were added to immobilized complexes. Aliquots of washed, eluted and concentrated complexes in detergent solution were analyzed by liquid scintillation counting and SDS PAGE.

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Biochemistry

Author Contributions

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FF, FK und TE conceived the study; FF, FK and TE analyzed data; FF and FK performed the experiments; FF and TE wrote the paper. All authors revised and approved the manuscript. Funding Sources

This work was financially supported by grant EI 374/4-2 within Paketantrag PAK459 from the Deutsche Forschungsgemeinschaft to TE. Notes

The authors declare no competing financial interests.

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