Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter Koichi Tamura, Hiroshi Sugimoto, Yoshitsugu Shiro, and Yuji Sugita J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b04356 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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The Journal of Physical Chemistry

Title Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter

Authors Koichi Tamura1*, Hiroshi Sugimoto2,3, Yoshitsugu Shiro2,3, Yuji Sugita1,4,5* 1

Computational Biophysics Research Team, RIKEN Center for Computational Science, 6-7-1

Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 2

Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-

1297, Japan 3

Synchrotron Radiation Life Science Instrumentation Team, RIKEN SPring-8 Center, 1-1-1 Kouto,

Sayo, Hyogo 679-5148, Japan 4

Theoretical Molecular Science Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa,

Wako, Saitama 351-0198, Japan 5

Laboratory for Biomolecular Function Simulation, RIKEN Center for Biosystems Dynamics

Research, 6-7-1 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

* Corresponding author: KT: E-mail: [email protected] Tel: +81-78-304-5364 Fax: +81-78-569-8820 YS: E-mail: [email protected] Tel: +81-48-462-1407 Fax: +81-48-467-4532

ORCID IDs: Koichi Tamura (0000-0002-9472-7555) Hiroshi Sugimoto (0000-0002-3140-8362) Yoshitsugu Shiro (0000-0003-0695-8327) Yuji Sugita (0000-0001-9738-9216)

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Abstract The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which use the energy of ATP-binding and hydrolysis for extensive conformational changes. Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs), and then in turn induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied template-based iterative allatom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the B. cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGG[Q/E]) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism.


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Introduction ATP-binding cassette (ABC) transporters are integral membrane proteins which harness free energy gained by ATP binding and hydrolysis for extensive conformational changes to translocate different substrates across the lipid bilayer.1–4 ABC transporters have a common architecture: two transmembrane domains (TMDs) which form a substrate translocation channel, and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (Figure 1a). The TMDs contain coupling helices (CHs) at the interface which transmit the conformational change at the NBDs to the TMDs. Each NBD has a well-conserved2,3 phosphate-binding motif (P-loop)5 and an ABC signature motif (LSGG[Q/E]).6 It is well established that the binding of ATP induces dimerization of the NBDs in a head-to-tail fashion, in which the P-loop of one monomer sandwiches the ATP with the signature motif of the opposite monomer (Figure 1b).7,8 Residues on the ABC signature motif are engaged in extensive interaction with ATP by forming hydrogen bonds and van der Waals contacts. ABC transporters are classified into exporters and importers according to the substrate transport direction.1 Importers, which are found only in prokaryotes, are further classified into type I, type II and energy coupling factor (ECF) transporters.2–4 Each class of importer has a different substrate transport strategy and a specific structural fold. Type I and II importers are associated with a periplasmic binding protein (PBP) that binds substrate in the periplasm and delivers it to the TMDs of the ABC transporter.2–4 Type I importers transport relatively small compounds such as sugars, while type II importers 3 ACS Paragon Plus Environment


are specific for the transport of trace elements.2–4 The ECF transporters share a unique substrate acquisition mechanism and are devoted to the transport of micronutrients.9 Conformational changes of importers are thought to occur in an alternating access model involving large-scale conformational change of the TMD.10 In this mechanism, the TMDs of the transporter alternate between two conformational states: the OF state, in which the extracellular side of substrate-translocation channel is open, and the inwardfacing (IF) state, in which the cytoplasmic side is open (Figure 1c). The OF state is for acceptance of substrate from the extracellular side, while the IF state is for excretion of the substrate to the cytoplasmic side. It has been proposed that an occluded (Occ) intermediate is formed during the conformational transition between the IF and the OF states. Evidently, an occluded intermediate of the vitamin importer BtuCD-F, which belongs to the type II importer family has been captured by X-ray crystallography upon introducing an artificial disulfide bond.11 In the Occ state, the substrate-trapping site is inaccessible to bulk solvent and reverse transport is prevented. For type II importers, substrates translocation by gate open/closure is thought to occur via “peristaltic” or “squeezing” motions rather than rigid body movement of the TMD.11–13 The IF-to-OF conformational transition is induced by ATP binding to NBD, and is thought to be a key feature of the transporters function, and contrasts with channel proteins. Recent progress in structural studies by X-ray crystallography2–4 and cryo-electron microscopy14 filled significant gaps in our understanding of the structure and function of ABC transporters. Those studies provide valuable information on substrate and ATP-binding sites, and on the existence of different conformational states such as the IF and OF states. However, 4 ACS Paragon Plus Environment

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experimental determination of structures along the cycle, including intermediates is still challenging, since the transport cycle is composed of multiple and complex steps. In this regards, computational modeling and molecular simulation techniques can contribute to understanding the functional mechanism of the ABC transporter in the atomic level and are therefore integral for studying dynamics. Most recently, we reported the crystal structures of the ABC transporter BhuUV from Gram-negative Burkholderia cenocepacia, which belongs to the type II ABC importer family and transports heme (Fe-porphyrin complex) across the inner membrane.15 The acquisition of heme iron from the host is an essential process for pathogenic bacteria during infection.16–18 It is, thus, essential to reveal the molecular mechanism of their heme transporters on the structural level. BhuUV is a dimer of a dimer (BhuU (TMD) + BhuV (NBD)), in which the coupling helices (CHs) located between TM6 and 7 in NBDs physically link with the TMDs (Figure 2a and S1). Like many other importers in Gram-negative bacteria, BhuUV requires a periplasmic heme-binding protein (PBP), BhuT, for accepting of the heme on the periplasmic side. The crystal structures of BhuUV we previously reported are of the apo IF conformation, complexed with BhuT (BhuUV-T). (Throughout this manuscript, we use the term “apo” for the structure without bound nucleotides and substrates (heme)).15 There, we proposed that the apo IF conformation of BhuUV-T (Figure 1c, left, and Figure 2a) represents a posttranslocation state which is reached after the release of the heme towards the cytoplasm. We postulated that the transport cycle would proceed via the binding of ATP at the NBDs to eventually form the OF state. The conformational transition would include the 5 ACS Paragon Plus Environment


formation of the intermediate Occ state, full dimerization of the NBDs and dissociation of BhuT in the OF state (Figure 1c). The remaining questions in the currently proposed transport mechanism are the detail of the heme translocating process, and the role of ATPbinding and hydrolysis in inducing the large conformational change. Many MD simulations have been conducted on ABC importers and exporters. For type II importer, most works focus on BtuCD-F. The MD simulations start from the OF state or Occ state, focusing on the response of the transporters to the presence of ATP, PBP, or the transporting substrate. There are few unbiased MD studies of conformational change from IF to OF of the ABC transporter.19,20 In this study, we employ template-based iterative molecular dynamics (MD) simulations to generate a high-confidence homology model of the heme importer BhuUV in the OF state. First, we modeled the OF state based on the crystal structure of the nucleotide-bound form of BtuCD. Then the modeled OF state structure and the crystal structure of BhuUV-T in the IF state were used for targeted MD (tMD) to explore the conformational change induced by ATP-binding to the apo IF state which occurs after heme translocation (Figure 1c). The predicted OF and Occ structures are further analyzed by conventional MD simulations. The results of the MD simulations reveal the mechanism of the chemo-mechanical coupling, in which ATPase activity is regulated by the dynamics of the ABC signature motif LSGG[Q/E] and coupled to the large-scale conformational change of the overall structure. The newly proposed mechanism for conformational regulation serves as the basis for understanding chemo-mechanical coupling in ABC transporters. 6 ACS Paragon Plus Environment

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Computational Methods

Overall strategy. In this study, we focus on the IF-to-OF transition of heme importer BhuUV-T (Figure 1c and Figure 2). Accordingly, our simulation systems do not contain the heme substrate. Recently, large conformational changes from IF to OF in the ABC exporter TM287/288 from Thermotoga maritima have been successfully observed by performing a large number of 500-ns unbiased MDs at a relatively high temperature of 375 K.19,20 However, the applicability of the method to the simulation of BhuUV-T seems inadequate because B. cenocepacia is not thermophilic and the high temperature could disturb the structure of the protein. Instead, we and other groups employed biased MD simulations to explore the conformational space of membrane transporters more efficiently.21,22 Here, the unknown OF structure of the heme importer (Figure 2c) is modeled by template-based iterative MD simulations. The technique consists of iterative refinements of a rough model by all-atom MD simulations with explicit solvent and membrane environments. The initial rough model was generated by homology modeling23,24 using the structure of the vitamin B12 transport protein BtuCD in the ATP analog-bound form as template (PDB 4R9U).12 Since 2002 several crystal structures of type II ABC importers have been elucidated, but the BtuCD structure is the only OF structure with bound nucleotides (Table S1). The engineered disulfide bond introduced in the BtuCD structure to suppress basal ATPase activity and conformational fluctuation25 was not included in the homology model. The relatively low sequence identity (~34%)



between BhuUV and BtuCD poses a significant challenge for homology modeling of thermally stable structures in the membrane environment. We therefore extensively refined the predicted structure by a series of template-based MD simulations. Modeling procedures are briefly summarized in the following section, and details are described in SI. The structural stabilities of the predicted structures were examined by relatively long (~1 s) MD simulations without any restraints on protein atoms. Initially, the nucleotide-bound Occ crystal structure of BtuCD-F (PDB 4FI3)11 was considered as a template of the Occ form. However, its lower resolution (3.47 Å) compared to that of the OF form (2.79 Å) could introduce further uncertainties into the modeling process. Instead, the Occ form (Figure 2b) was modeled by a targeted MD (tMD) simulation26 using part of the modeled OF structure as a target (see below). The effect of the binding of ATP to the NBDs was then investigated by comparing the predicted structures with the crystal structure of the apo IF form (Figure 2). Representative MD simulation results are summarized in Table S2.

Iterative refinements of the OF form. We employed MODELLER version 9.16 for building the homology models. Sequence alignments were generated by the align2d() function in MODELLER, using the template 3D structure to insert gaps into the alignment. For example, the function avoids placing gaps within the secondary structure. Altogether, we generated four structural models (MODEL1–4). The first OF model with bound ATP, which was based on an alignment involving several manual adjustments (MODEL1), turned out to be unstable after a short MD simulation of ~20 ns: we observed spontaneous 8 ACS Paragon Plus Environment

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closure of the periplasmic gate and distortion of the helices at Arg121 to Gly134 of both NBD monomers (Figure S2a and S2b). The instability, especially in the helical region in the NBDs, seemed to originate from the rather low sequence identity between BhuUV and the vitamin B12 transporter BtuCD which was used as template (Figure S2c). On the other hand, there is a crystal structure of a heme importer of Y. pestis (HmuUV) in the apo OF form (PDB 4G1U)27 having a higher sequence identity (~40%) from which potentially better models can be generated. However, the apo HmuUV structure has a significantly different structural arrangement compared to that of the nucleotide-bound BtuCD and therefore cannot be used on its own as a template for modeling of the nucleotide-bound OF form of the B. cenocepacia heme importer BhuUV. As an alternative, we generated a reference homology model (REF1) by using the apo HmuUV structure as template and replaced the unstable regions in MODEL1 with the corresponding ones of the REF1 structure to create a chimera. Although the helical regions in the new model (MODEL2) turned out to be stable, some other parts were still unstable: the periplasmic gate spontaneously closed (as in MODEL1) and the dimer interface was disrupted (data not shown). We speculated that the structural instability might have arisen from the low sequence identity of TM6 connected to the CHs (Figure S1), causing enlargement of the dimer interface by way of the structural coupling. Then, we built the next model (MODEL3), which involved replacing TM6 in MODEL2 with that in the REF1 structure. Although MODEL3 was stable during the first ~600 ns of the MD simulation, the periplasmic gate gradually closed after this and eventually closed almost completely (Figure S3a). We also observed fraying in the helical region at Ala51 9 ACS Paragon Plus Environment


to Ala70 in one of the BhuU monomers (Figure S3b). The gradual closure of the periplasmic gate involved TM5a of one monomer leaning towards the other (Figure S3a). Then, the final model, MODEL4, was generated by replacing the fraying region and the leaning helix of MODEL3 with the corresponding parts of the opposing monomer. This model remained stable during 1.5 s MD simulation (MD-2ATP-OF) and, importantly, its periplasmic gate stayed open (Figure S4a and S4c). C root mean squared deviation (RMSD) between the template (PDB 4R9U) and the average structure of the predicted OF form calculated from the last 500-ns part of the MD trajectory was 2.2 Å (Figure S4d). More details of the modeling are found in SI.

Modeling of the Occ intermediate. A targeted MD (tMD) simulation26 starting from the equilibrated ATP-bound IF structure was performed in order to model an Occ conformation of BhuUV-T with bound nucleotides. tMD has been widely used to elucidate the conformational transition pathways of proteins including a type II ABC transporter.28 In tMD, a biasing force is applied to pull the protein towards a predefined target structure. The application of the external force enables one to induce conformational changes in proteins very efficiently. A caveat of this method is that it is subjected to a “large-scales-first” bias and that the predicted pathway may deviate from the actual physical one.29,30 We thus disregarded the pathway itself and checked only for stability in the final model. Note that if one chooses the entire MODEL4 OF structure as a target of tMD, the simulation would end up with generating the target structure itself. Alternatively, if one selects a part of the MODEL4 structure as a target, one would obtain 10 ACS Paragon Plus Environment

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protein with the selected part having conformation of the MODEL4 and the remaining part left almost unaffected. In our case, the NBDs and the residues of the cytoplasmic gate of the equilibrated ATP-bound IF structure were pulled so that the domains and the gate closed as in the MODEL4 OF structure (see SI for details). We then performed an equilibrium MD simulation without the bias potential on the protein atoms to check the structural stability of the intermediate state between IF and OF. However, the first model for Occ state was unstable: there was an immediate opening of the cytoplasmic gate and widening of the distances between coupling helices in a short MD simulation (data not shown). Then, another calculation was performed by pulling also TM6 and 7 of both monomers towards the target (MODEL4). The Occ form obtained by this calculation showed a good structural stability, which was confirmed by an equilibrium MD simulation (1.5 s) (MD-2ATP-Occ, Figure S5c). The C-RMSD between the averaged structure along the last 500-ns part of the MD trajectory of the Occ form and the BtuCD-F Occ crystallographic structure was 2.5 Å (Figure S5d).

MD Simulation details. All MD simulations were performed with the development version of GENESIS.31,32 During the simulations, the protonation states of all titratable residues were fixed to their standard states at pH 7, as is the case with the standard MD. Although it has been shown that some ABC exporters function as protonmotive force-driven transporters33 or pH-dependent transport,34 so far the pH-dependent behavior has not been reported for type I and II ABC importers. It might be possible that the protonation state of the carboxylic acid changes when the Asp-Arg salt bridge is 11 ACS Paragon Plus Environment


broken, and that the lower pH in the periplasm plays a role by partially protonating some acidic residues (e.g., D112 of BhuU on the heme translocation channel). Further studies utilizing the continuous constant pH MD method35,36 may help to validate the effect of breakage or formation of salt bridge and pH dependency on the function of the importer. The force field parameter set for water molecules was TIP3P,37,38 and those for protein and lipids were CHARMM36.39,40 We used revised parameters for ATP41 and magnesium ions.42

The

protein

was

embedded

in

a

1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphoethanolamine (POPE) bilayer. The typical simulation system size was 134 × 134 × 197 Å3 (~360,000 atoms) for the BhuUV-T system and 134 × 134 × 154 Å3 (~280,000 atoms) for the BhuUV system. The equations of motion were integrated with the velocity Verlet algorithm with a timestep of 2.5 fs. Bonds including a hydrogen atom were constrained by the SHAKE/RATTLE43 (non-water molecules) and the SETTLE44 (water molecules) algorithms. Temperature and pressure were regulated by the stochastic velocity rescaling thermostat45 and barostat,46 respectively. The thermostat and the semiisotropically coupled barostat were applied every 10 steps. Long range electrostatic interactions were calculated with the particle mesh Ewald method47 and updated every 2 steps. Short range Lennard-Jones interactions were cutoff at 12 Å with a force switching function beginning at 10 Å.48 Details are described in SI. MD trajectories associated with the manuscript are available on the following link https://osf.io/hfb2c/.


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Results

Characteristics of the Predicted Occ and OF Structures of BhuUV(-T). Crystal structure studies of type II importers, including the heme importer and vitamin B12 transporter have identified two gates for substrate (heme and B12) transport, i.e., the cytoplasmic gate and the periplasmic gate. The cytoplasmic gate, specifically the so-called cytoplasmic gate II, is formed by Asn108 and Leu110 (numbered according to BhuUV) from opposing monomers, both of which are well-conserved among type II ABC transporters (Figure 3a).11 In crystal structures of the IF state of the BhuUV-T, the cytoplasmic gate II is widely open, while the periplasmic gate is closed by a salt bridge between Asp200 and Arg204 of opposite subunits (Figure 3a and Figure 4, left). Both residues are conserved among heme importers.15 The PBP BhuT associates to the periplasmic surface of TMD of BhuUV through an electrostatic interaction of Glu94 or Glu231 (BhuT) and Arg84 (BhuU) (Figure S6).15 Our targeted MD simulation predicted the Occ form of BhuUV-T, which is identified as an intermediate state in transition from IF to OF. In this predicted Occ form, both periplasmic and cytoplasmic gates are closed, and BhuT is still bound to TMD (Figure 3b and 4, middle). The MD simulations of the Occ state also showed that the saltbridges involving Arg84 (BhuU) observed in the IF crystal structure are disrupted, and instead new salt-bridge pairs between Glu94 or Glu231 and Arg81 (BhuU) are formed (Figure S6 and S7). Arg81 is widely conserved among type II ABC importer.15 The



central cavity of the Occ form is large enough to accommodate the substrate heme, but in the current simulation it is filled with water molecules (Figure S8a). The predicted Occ structural characteristics are comparable to the crystal structure of the Occ state of the BtuCD-F complex with bound ATP analogs (PDB 4FI3), in which a large central cavity for the substrate is also observed (Figure S8b).11 Details of the conformational change from IF to the unique Occ forms found in our simulation are described in the following section (vide infra). Using the iterative MD techniques performed in a membrane environment, we also predicted the OF structure of BhuUV, in which the periplasmic gate is seen open, while the cytoplasmic gate II is closed (Figure 3c and 4, right). A major difference between the predicted OF form of BhuUV(-T) and the crystal structures of OF of BtuCD (PDB 4R9U)12 (in which the disulfide bond was artificially introduced at the NBD dimer interface), is observed in the distance between the coupling helices (CHs): that in the predicted OF structure is shorter (~2–4 Å) than in the corresponding crystal structure (Figure 5). The shorter distance between the CHs causes a greater tilt of TM6 and 7 of opposing monomers (Figure S9), and a strong tilt of the TM helices. Due to the greater tilt of the TM helices, the periplasmic gate in the predicted OF form of BhuUV remains open during the simulation (Figure S4a). In contrast, in the MD simulation of the ATPbound OF BtuCD performed in this study (BtuCD-2ATP-OF, based on PDB 4R9U), spontaneous closure of the periplasmic gate was observed in early stages of the simulation (Figure S10). Such spontaneous closure was also observed in the previous MD simulations of the apo OF BtuCD (PDB 1L7V) whose periplasmic gate is structurally 14 ACS Paragon Plus Environment

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similar to that of the ATP-bound form (PDB 4R9U).49–51 These observations imply that our predicted structures of BhuUV-T, which was modeled in an explicit membrane environment, are more relevant to physiological conditions, whereas the BtuCD(-F) crystal structures are trapped in slightly unnatural states, possibly due to the usage of detergent solution to solubilize the protein. Indeed, recent single-molecule FRET analysis by Yang et al. revealed that the detergent perturbs the protein structure.13

NBD Dimerization Induces Closure of Cytoplasmic Gate and Opening of Periplasmic Gate. The cytoplasmic gate II has non-helical regions (extended stretch, exTM3) located prior to the short TM3 units of opposing monomers. The initial step in the transition from the IF state to the Occ state is binding of ATP to the NBD, promoting dimerization of the latter (Figure 3a, ①), and eventually forcing the coupling helices (CHs) and the TM6/7 bundles to lean towards the central cavity (Figure 3a, ② ). The current targeted MD simulations showed that these conformational changes push TM2, causing its C-terminal region to tilt towards the central cavity, bringing to an eventual closure of the cytoplasmic gate II, which is bordered by the exTM3s (Figure 3b). The gate remained tightly closed throughout the unbiased MD simulations, not only for the Occ state (Figure S5a) but also for the OF state (Figure S4b). In spite of such large conformational changes in the NBDs and several TM helices during the transition from the IF to the predicted Occ states, the periplasmic gate remained



closed in the predicted Occ state, as in the case of the IF crystal structure (Figure 4, middle, and Figure S5b). Such state is probably maintained due to the stable interaction of the H5a pair including the salt-bridge (Asp200-Arg204) and a hydrophobic interaction (Leu203-Leu203) at the periplasmic gate of BhuUV-T. In contrast, the periplasmic gate stays open during the transition to the OF state, most probably due to the tilting of TM5 and H5a along with other helices (Figure 4, right, and Figure S4a). The large tilt of these helices on the periplasmic side of the TMD is coupled to the dissociation of BhuT from TMD BhuU, as discussed later. Here, we would like to note that the opening and closing motions of the two gates are highly correlated with the dimerization of the NBD. The average distance during the unbiased MD simulation between the two CHs from each TMD monomer was 37.1, 30.0 and 26.4 Å, for the IF, Occ, and OF states, respectively, as shown in Figure 5. It appears that the dimerized forms of Occ and OF states are somewhat different, with the interNBD distance in the Occ form being larger than that in the OF form (Figure S11). Therefore, we defined the NBD structure of the Occ and OF forms as “partial” and “full” dimerization forms, respectively.

Stoichiometry of ATP in the IF-to-OF Transition of BhuUV-T. The role of bound ATPs in the closure of the cytoplasmic gate II was examined in a series of MD simulations. In the predicted Occ and OF forms of BhuUV(-T), two ATPs are tightly bound to the active sites and contribute to the tight closure of cytoplasmic gate


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II (Figure 3). This mechanism is similar to the one observed in the corresponding crystal structures of BtuCD(-F).11,12 Consistently, when we added two ATPs to the NBDs of the apo IF BhuUV-T complex, the CHs, which directly contact the NBDs, had a tendency to move closer (Figure 6a), although full dimerization was not observed within the current simulation timescale. On the contrary, when we removed two ATPs from the predicted Occ state, spontaneous widening of the CH distance occurred (Figure 6a). Comparably, destabilization of the dimer interface upon removal of the ATPs was also seen in MD of the BtuCD-F system.52 All these observations could highlight the importance of ATP in the narrowing and widening of the CH distance, which clearly correlates with the tendency for formation and destabilization of the cytoplasmic gate II, respectively (Figure 6b), demonstrating mechanical coupling between the NBD and the TMD. Is a single ATP molecule enough to stably close the cytoplasmic gate II? To answer this question, we removed one ATP from an arbitrarily chosen active site of the predicted Occ form. Although the result was not as clear as in the above case, the distance between the CHs consistently widened (Figure 6a), which caused the cytoplasmic gate to transiently open (Figure 6b). These results suggest that one ATP is insufficient to stably close the cytoplasmic gate, and a longer simulation may clarify this.

Dissociation of the PBP from TMD Facilitates Full Dimerization of NBD. ATP binding to NBD would induce conformational transition, first from the IF to the Occ state by closure of the cytoplasmic gate, and then from the Occ to the OF state.



Indeed, a comparison between the predicted Occ and OF structures showed that the CHs and the helix bundles (TM6/7) in the OF form are more tilted than in the Occ form, resulting in opening of the periplasmic gate (Figure 4 and 5). However, these structural changes could not occur in the simulation of the BhuUV-T Occ form without the dissociation of BhuT from BhuU (see below). In our simulation, it was found that the dissociation of BhuT (PBP) from TMD of BhuUV and the structural change on the periplasmic side are coupled, and that these processes are followed by “full” dimerization of the NBDs (Figure 2c). In other words, PBP (BhuT) dissociation facilitates NBD (BhuV) dimerization from “partial” to “full” form. In our MD simulation of the Occ form, BhuT was stably bound to the TMDs, as shown by the distance between the conserved salt-bridge pairs between the TMDs and BhuT (Figure S7). The tight binding of PBP to TMD and the destabilization effect of ATP has been experimentally demonstrated for type II importers.53,54 Our previous pulldown assays performed on BhuUV-T also showed that the binding of ATP, and not its hydrolysis, induces the dissociation of BhuT.15 Therefore, BhuT-dissociation dwell state might have a longer dwelling time than the simulation time scale (1.5 s), since several salt bridges on the interface must be cleaved for the dissociation of BhuT (e.g., Figure S6).

Dynamics of the Catalytic Serine Residues and NBD Dimerization. The ATPase site of the ABC transporter consists of a P-loop (Gly34 to Ser41), Walker B, and other motifs (Q-loop and switch motif) of one NBD, and an LSGG[Q/E] 18 ACS Paragon Plus Environment

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motif of the second NBD (Figure 1b). One of the most significant observations from our MD simulation of the Occ form was the orientational changes of the catalytically important serine residues (Ser147)55 in the LSGG[Q/E] motifs of the NBDs (Figure 7a, upper panel). Namely, the side chain of Ser147 in either monomer occasionally flipped its direction between the -phosphate of ATP and the glutamate residue of the LSGG[Q/E] motif (Figures 7a, 7c and S12a). The former can be assigned to a catalytic form, while the latter to non-catalytic form. The change in orientation between these two forms clearly correlates with a association/dissociation of the NBD dimer, which is illustrated by the plot of the distance between the P-loop and the LSGG[Q/E] motif (Figure 7a). The “partial” dimerization in the Occ state, which sandwiches the bound ATP, creates space and allows for the non-catalytic conformation of the serine residue to exists at the interface. In addition, during our MD simulation the main chain conformation of the LSGG[Q/E] region did not show large fluctuation (Figure S13), suggesting that the flipping motion originates from a rigid body motion of the NBD rather than from flexibility of the LSGG[Q/E] region. This rigid character of the LSGG[Q/E] motif is advantageous, since it directly transmits the environmental change of LSGG[Q/E] in the catalytic site to the motion of the NBD domain as a single unit, as required for chemomechanical coupling. Another interesting finding is that in the Occ state the side chain flipping occurs more frequently in one of the nucleotide-binding sites (NBS) than in the other (Figure 7a). Here, we designate the former as NBS1 and the latter as NBS2. The difference in side-chain mobility of Ser147 originates from the asymmetrical NBD dimerization in the 19 ACS Paragon Plus Environment


Occ state. The distance between the P-loop and the LSGG[Q/E] motif in NBS1 is larger than that in NBS2, resulting in more frequent dissociation of the side chain of the serine residue (Figure 7a, lower panel). In contrast, in the OF state, Ser147 in each monomer has a stable orientation toward the -phosphate of ATP (Figure 7b, upper panel and Figure S12b), presumably due to the stable “full” dimerization of the NBDs (Figure 7b, lower panel). The dimeric interface of the NBDs in the “full” dimerization state (Figure S11) has shorter NBD-NBD distance and can hold the serine residue in a catalytic orientation (Figure 7b). The motion and interaction of Ser147 possibly have an important role in modulating the catalytic power of the protein in the functional cycle, as discussed below.


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Discussion The two key questions toward understanding the transport cycle of the heme ABC transporter are: when do ATP-binding and hydrolysis occur, and how does the protein transmit the structural change by those events in NBDs to the conformational transition of the overall structure. Our previous crystallographic and biochemical studies of BhuUV-T suggested that ATPs bind to the post-translocation state of BhuUV-T to induce the conversion of IF to OF form of TMD, as well as release the BhuT (PBP) to initiate the transport cycle. However, mechanistic details of these processes were yet to be established, because the ATP-bound OF and the Occ forms have not been previously observed. Another question to be answered is how NBD suppresses the undesired hydrolysis reaction during the transition from IF form. To address these issues, we performed MD simulations, in which we predicted two experimentally unknown conformations (Occ and OF) of the heme ABC importer BhuUV-T, and proposed the conformational change of dimerization process of NBD and IF-to-OF transition upon the ATP-binding. Based on our MD simulations and previous structural studies on BhuUV-T, we propose a mechanism for the protein conformational changes following heme translocation as detailed below: 1. In the IF state, the protein is in an ATP-binding dwell and the catalytic sites are “empty” (without bound ATP), as represented by the crystal structure of BhuUV-T (PDB 5B58). 21 ACS Paragon Plus Environment


2. Binding of ATP to NBD (BhuV) of the IF form triggers a conformational change to the Occ form. The Occ form is apparently in a BhuT-dissociation dwell, because the bound BhuT hinders further tilting of the TM helices and the narrowing of the interCHs space required to complete the ATP-induced IF-to-OF transition. The structural constraint holds the NBDs somewhat apart, due to structural coupling between the NBDs and the CHs, stabilizing “partial” dimerization. As a result, the catalytic sites are in a “loose” state, in which the Ser residues (Ser147) of LSGG[Q/E] motifs frequently flip away from the -phosphate of ATP, and thereby destabilizing the catalytic conformation. 3. Upon transitioning from the Occ to the OF states, dissociation of BhuT is followed by the full tilting of the TM helices and complete dimerization of the NBDs. These transitions restrict the conformational freedom of the sidechains of the two Ser147 residues, so that the signature motif can recognize the ATP more specifically in its catalytic geometry. Higher ATPase activity is achieved upon “full” dimer engagement of the NBDs. At this stage, the catalytic sites are in a “tight” state (Figure 8). The impairment of ATPase activity by BhuT in the Occ state is seemingly inconsistent with the experimental data which showed ATPase activity of BhuUV-T is slightly higher (× 1.4) than that of BhuUV without BhuT. However, the inconsistency can be explained in case the rate-limiting step of ATPase reaction is different (e.g., the OF-to-IF conformational transition after the hydrolysis reaction or the release of ADP and inorganic phosphate) and that the process is accelerated by re-binding of BhuT. 22 ACS Paragon Plus Environment

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Regarding the IF-to-OF pathway after substrate translocation, the conformational change proposed for BhuUV-T in this study is similar to that in the molybdate importer MolBC, but slightly different from those for BtuCD-F and HmuUV, although all are classified into the type II ABC importer family.4,11, 56–59 For example, crystal structures of both BhuUV and MolBC have been determined in the IF form without ATP15,57 and share a common structural feature. In addition, the closure of the cytoplasmic gate II in the proposed IF-to-OF transition pathway of BhuUV-T is consistent with the electron paramagnetic resonance (EPR) spectroscopy-based observation in MolBC.56 On the other hand, the Occ state in the proposed transport cycle of the vitamin B12 transporter BtuCD-F has distinct features from the Occ model proposed in the current study.4,11 Namely, the mechanism of the former is based on the crystal structures of BtuCD without ATP solved in the OF form and with a closed cytoplasmic gate I in the Nterminal part of TM5 of opposing monomers (Table S1). The ATP-binding dwell of BtuCD-F is an asymmetric apo Occ state based on the PDB 2QI9,58 and the transport cycle does not involve a state corresponding to the PBP-dissociation dwell (Figure S14b, middle). It is not clear whether or not there are some mechanistic differences among the type II ABC importer family. However, it seems reasonable to consider that the binding of ATP to the NBDs of BtuCD-F would force dimerization of the NBDs, resulting in closure of cytoplasmic gate II, as observed in the crystal structure (PDB 4FI3). Therefore, the structure of the ATP-bound Occ state of BtuCD-F, in which both periplasmic and cytoplasmic gate II are closed, would be present between the asymmetric Occ and the OF forms (Figure 8 and S14b). The Occ form of BtuCD-F might be very similar to the Occ 23 ACS Paragon Plus Environment


state that was predicted for BhuUV-T in this study. Our finding on the role of the Ser residue in the chemical reaction is well consistent with the previous result based on the high-resolution structures of type I maltose importer in complex with ADP-VO4 or ADP-AlF4; i.e., the side chain of Ser and backbone amido group of Gly in LSGG[Q/E], together with other residues in the P-loop and the Q-loop, stabilize the geometry of the transition state intermediate for ATP hydrolysis. A pentavalent intermediate of γ-phosphate is formed in the hydrolysis reaction and the lytic water is activated by the acidic residue at the end of the Walker B motif.60 Biochemical analysis of mouse multidrug resistance 3 P-glycoprotein (MDR3/ABCB4) also displayed the mechanism in which the Ser residue in the LSGG[Q/E] motif contributes to the stabilization of the transition state.55 Future work may include the hybrid quantum mechanical/molecular mechanical (QM/MM) calculation61,62 to directly examine the catalytic role of the Ser residue on LSGG[Q/E] and other residues in the nucleotide-binding site. A recent QM/MM study of BtuCD-F showed the mechanism of ATP-hydrolysis initiated by the attack of the activated lytic water, followed by proton transfer to conserved Glu adjacent to the Walker B motif as a catalytic base.63 Since conformational sampling of proteins by direct QM/MM MD simulations is still limited to sub-ns timescale, QM/MM simulations of ABC transporters with large conformational changes upon ATP hydrolysis64 are computationally difficult. Alternatively, a variational QM/MM free energy optimization approach would offer an advantage in implementing

thermal fluctuations of the protein more efficiently.65–68

In contrast to the distinct fold of TMDs of ABC transporters, NBDs are highly 24 ACS Paragon Plus Environment

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conserved in sequence and structure among all ABC transporters. Especially, the LSGG[Q/E] signature motif is the hallmark of the family. Moreover, as suggested by Oldham and Chen,69 the role of LSGG[Q/E] in orienting the γ-phosphate of ATP in proper geometry resembles that of the “Arg finger” of the biomolecular motor F1-ATPase, which controls the catalytic activity and interface of the α and β subunit.70 Therefore, it is reasonable to assume that the basic mechanism of chemo-mechanical coupling is widely shared among the ABC transporters and other ATPase proteins.



Conclusions

The catalytic cycle of ABC transporters consists of a series of steps. For type II heme ABC importer BhuUV-T, it has been proposed that the binding of ATP to NBDs is utilized for the dimerization of NBD, concomitant with release of apo-PBP to reset the transporter to the OF state for the next cycle. This study is the first attempt to simulate the conformational change of the IF form upon the ATP-binding in type II importers. The template-based iterative MD simulation and the targeted MD simulation predicted two structurally unknown conformations of the transporter, and revealed the details and order of the conformational change from IF to OF upon ATP-binding. The newly predicted structures, Occ and OF, provided an insight into how the binding of ATP is coupled to the conformational change of the protein. It was found that the dimerization of the NBDs in the predicted Occ form is incomplete due to structural constraints imposed by bound BhuT. As a consequence, the catalytically important Ser residue of the conserved LSGG[Q/E] signature motif flips between catalytic and non-catalytic orientations. Dissociation of BhuT enables tight dimerization of the NBDs and proper Ser orientation, leading to active recognition of ATP by the transporter. These results demonstrated the novel structural property of the LSGG[Q/E] motif and its role in avoiding ATP hydrolysis during the Occ state, in which both ends of the channel are closed and NBDs remains associated with ATPs. The proposed chemo-mechanical coupling for heme transporter


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may provide basis for the understanding of transport mechanisms in other members of the ABC transporter family.



Supporting Information Available Supporting Methods, Supporting Tables S1–S3, and Supporting Figures S1–S18. This material is available free of charge via the Internet at https://pubs.arc.org.


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Acknowledgments K.T. thanks J. Jung, C. Kobayashi, Y. Matsunaga, and M. Kamiya for sharing their expertise on running simulations on the K computer. This work was supported by the RIKEN pioneering project “Dynamic Structural Biology” (to Y.Sugita), MEXT Grantin-Aid for Scientific Research on Innovative Areas Grant Number 26119006 (to Y.Sugita), JP26220807 (to Y.Shiro), JP15H01655, JP17H05896 (to H.Sugimoto), and RIKEN Special Postdoctoral Researcher Program (to K.T.). This research used computational resources of the K computer provided by the RIKEN Center for Computational Science through the HPCI System Research project (to K.T., Project ID: hp170027, hp180009, to Y.Sugita, ra000009). The computer resources of RIKEN HOKUSAI BigWaterfall were also used. Molecular figures were prepared with VMD71 and PyMOL.72



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Figure 1. Architecture and alternating access mechanism of ABC transporter. (a) A shared architecture of ABC transporter. Transmembrane domains (TMDs) are embedded in the biological membrane, whereas nucleotide-binding domains (NBDs) are exposed to the cytoplasm. They are physically connected by the coupling helices. (b) Dimerization interface of NBDs as seen from the periplasmic side. They dimerize upon binding of ATP to the P-loop in a head-to-tail fashion, where the P-loop of one monomer binds to the LSGG[E/Q] motif of the opposing monomer. (c) An alternating access mechanism of a heme ABC transporter BhuUV-T. The conformational transition from the inward-facing (IF) to the outward-facing (OF) form involves the binding of ATP to the NBDs and the formation of the occluded (Occ) state.



Figure 2. Structures of heme importer BhuUV-T. (a) Crystal structure of BhuUV-T complex (PDB 5B58) in the inward-facing conformation. Transmembrane domains (TMDs, dimer of BhuU) are shown in blue or red. Nucleotide-binding domains (NBDs, dimer of BhuV) are shown in gray or orange. Periplasmic binding domain (PBP, BhuT) is shown in yellow. (b,c) Alternate conformations of BhuUV-T predicted in this study. (b) The occluded and (c) the outward-facing conformations.


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Figure 3. Closure of the cytoplasmic gate II coupled to NBD dimerization. The cytoplasmic gate is open in the inward-facing (IF) conformation (a), while it is closed in the occluded (Occ) (b) and the outward-facing (OF) (c) conformations. The upper panels show the transporter from the membrane space and the lower ones from the periplasmic space. Only TM2–3 and 6–7 are shown for the TMDs. Carbon, nitrogen, and oxygen atoms are in yellow, blue, and red, respectively.



Figure 4. Opening of the periplasmic gate upon the binding of ATP. The panels show the transporter from the periplasm space. In the inward-facing (IF) state (left, PDB 5B58), the periplasmic gate is sealed by TM5 and 5a of opposing monomers. The interdomain salt bridges between Asp200 and Arg204 stabilize the dimer interface. The situation is not changed in the predicted occluded (Occ) state (middle), while in the predicted outward-facing (OF) state (right), the gate is wide open due to the large tilt of the TM helices.


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Figure 5. Comparison of the distance between the coupling helices of type II ABC importers in different conformational states. (a) The predicted OF (magenta) and Occ (sky blue) average structures along the last 500-ns equilibrium MD simulations are superimposed on the IF crystal structure (green). Only TM2–3 and TM6–7 of the TMDs are shown. Distances between the coupling helices (CHs) for each structure are denoted at the bottom. (b) Distribution of distance between the CHs in the last 500-ns equilibrium MD simulation for each conformation.



Figure 6. Structural changes in the equilibrium MD simulations. (a,b) Temporal changes of distance between (a) the coupling helices and (b) Leu110 of opposing monomers during the MD-IF-2ATP (IF + 2ATP), MD-apo-Occ (Occ – 2ATP), and MDOcc-1ATP (Occ – 1 ATP) trajectories.


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Figure 7. Alternate configurations of Ser147. (a,b) The upper panel shows the distance between OG atom of Ser147 and O2G atom of ATP and the lower one shows the distance between P-loop and LSGGE for the occluded state (a) and the outward-facing states (b). Here, the two nucleotide-binding sites (NBSs) are designated as NBS1 and NBS2, and the results for NBS1 and NBS2 are indicated by magenta and green lines, respectively. (c) Two possible orientations adopted by the sidechain of Ser147 observed in the simulation of the occluded state.



Figure 8. Proposed chemo-mechanical coupling model. The horizontal axis describes progress of conformational changes (e.g. distance between coupling helices), whereas the vertical axis indicates the ATPase activity of the protein. In this model, the progress of conformational changes is coupled to the ATPase activity of the protein (the curved arrow) so that as the conformation transition proceeds from the inward-facing (IF) state to the outward-facing (OF) one, the ATPase activity gradually increases.


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TOC Graphic


Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC

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