Article pubs.acs.org/biochemistry
Structural Dynamics of the Heterodimeric ABC Transporter TM287/ 288 Induced by ATP and Substrate Binding Tadaomi Furuta,* Yukiko Sato, and Minoru Sakurai Center for Biological Resources and Informatics, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan S Supporting Information *
ABSTRACT: TM287/288 is a heterodimeric ATP-binding cassette (ABC) transporter, which harnesses the energy of ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) to transport a wide variety of molecules through the transmembrane domains (TMDs) by alternating inward- and outwardfacing conformations. Here, we conducted multiple 100 ns molecular dynamics simulations of TM287/288 in different ATP- and substrate-bound states to elucidate the effects of ATP and substrate binding. As a result, the binding of two ATP molecules to the NBDs induced the formation of the consensus ATP-binding pocket (ABP2) or the NBD dimerization, whereas these processes did not occur in the presence of a single ATP molecule or when the protein was in its apo state. Moreover, binding of the substrate to the TMDs enhanced the formation of ABP2 through allosteric TMD−NBD communication. Furthermore, in the apo state, αhelical subdomains of the NBDs approached each other, acquiring a conformation with core half-pockets exposed to the solvent, appropriate for ATP binding. We propose a “core-exposed” model for this novel conformation found in the apo state of ABC transporters. These findings provide important insights into the structural dynamics of ABC transporters.
ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of integral membrane proteins found in all three domains of life (bacteria, archaea, and eukarya)1−5 and are responsible for the ATP-driven translocation of several substrates across membranes. The basic architecture of ABC transporters is based on common structural subunits: two highly conserved nucleotide-binding domains (NBDs) that work as a nucleotide-dependent engine for driving substrate transport and two diverse transmembrane domains (TMDs) that form the translocation pathway (Figure 1A). There are two major types of ABC transporters: ABC importers and ABC exporters. ABC importers such as MalFGK2-E and BtuCD-F, present mostly in prokaryotes, transport essential nutrients and other molecules into cells, whereas ABC exporters such as P-gp, CFTR, Sav1866, and MsbA, which are found in all three domains of life, transport toxins, drugs, ions, peptides, and other diverse molecules toward extracellular regions.3−5 In this study, we mainly focused our attention on the latter. With regard to drug transport, multidrug resistance (MDR) has recently been one of the most fascinating and critical topics in medicine and molecular biology.6 During transport of substrates, the ABC transporter undergoes a conformational transition from the inward-facing (IF) conformation to the outward-facing (OF) one.4 This process is described by the “alternating-access” model proposed by Jardetzky and generally accepted for several transport proteins.8,9 The binding of ATP molecules to the NBDs triggers the dimerization of the NBDs (“ATP sandwich” dimer of the NBDs in a head-to-tail conformation10,11), accompanied © 2016 American Chemical Society
or stimulated by the binding of substrate to the TMDs. This NBD dimerization subsequently induces the transition of the entire structure from the IF to the OF conformation. In this OF conformation, the substrate is expelled toward the extracellular side through the translocation pore located in the TMDs. The subsequent ATP hydrolysis produces ADP and Pi molecules that are released from the NBDs. Consequently, the two NBDs are separated, restoring the initial IF conformation of the whole protein. With regard to ATP hydrolysis, homodimeric ABC transporters have two consensus (hydrolyzable) ATP-binding pockets (ABPs), while heterodimeric ABC transporters have one degenerate (nonhydrolyzable) ABP (hereafter called ABP1) and another consensus ABP (hereafter called ABP2).12 In ABP2, there are seven conserved (canonical) motifs for ATP binding and hydrolysis: the Walker A (P-loop) and Walker B motifs,13 the ABC signature motif (or Cloop),10,14,15 the A-loop,16 the D-loop,10,12 the H-loop (switch motif),17−19 and the Q-loop10,20,21 (Figure 1B). On the other hand, in ABP1, there are also seven motifs; however, several motifs responsible for ATP hydrolysis are mutated (i.e., noncanonical), in particular, the glutamic acid of Walker B, the histidine of the H-loop, and some residues of the ABC signature motif or D-loop. In bacteria, the majority of ABC Received: September 17, 2016 Revised: November 7, 2016 Published: November 10, 2016 6730
DOI: 10.1021/acs.biochem.6b00947 Biochemistry 2016, 55, 6730−6738
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Biochemistry
Figure 1. Structures of TM287/288 and ATP-binding pockets (ABPs) in the NBDs. (A) TM287/288 structure with ATP molecules and substrate (Hoechst 33342). TM287 and TM288 are represented by cyan and pink ribbons, respectively,7 and ATP molecules in ABPs and the substrate are represented by the CPK model (indicated by arrows). (B) ABP1 and ABP2 in the NBDs (view from top, C-terminal regions omitted). Conserved motifs are represented by the stick model, indicated by arrows: the Walker A, Walker B, signature, A-loop, D-loop, H-loop, and Q-loop motifs are colored red, green, blue, yellow, orange, purple, and magenta, respectively. In this figure, compared with the crystal structure,7 the substrate (Hoechst33342) is docked into the TMDs, the ATP molecule is bound to ABP2, and the AMP-PNP molecule is replaced with an ATP molecule (see Materials and Methods).
NBD region acquires a partially closed asymmetric structure [hereafter called the “one-side-open” structure (Figure 1B)], where ABP1 is not completely closed; it is not necessary, however, for this degenerate pocket (ABP1) to undergo complete closure because of its nonhydrolyzable nature. This one-side-open structure is also called the “C2 state” in studies on CFTR, the most investigated heterodimeric ABC protein, and the actual conformation of the “C2 state” is one of the major open questions of the CFTR cycle.51,52 In this study, we focused upon the elucidation of the structure and dynamics of TM287/288. The results obtained will help us understand the properties of cognate heterodimeric ABC transporters that ubiquitously exist in humans. We paid special attention to the effect of ATP binding that would initiate the functional cycle of TM287/288. For this purpose, multiple 100 ns MD simulations were conducted for three different states of ATP binding: apo (ATP-free), 1ATP (one ATP only in ABP1), and 2ATP (ATPs in both ABPs). Moreover, to clarify the effect of substrate binding, we conducted similar MD simulations for a state (2ATP+S) in which a substrate (Hoechst 33342) was docked into the binding site of the TMDs (Figure 1A). We will reveal that the binding of ATP to ABP2 is indispensable for dimerization of the NBDs, and a novel conformation of the NBDs that could effectively take up ATP molecules is induced in the apo state.
transporters are homodimeric. In contrast, in mammals (or humans), half of the ABC transporters are heterodimeric: TAP1/TAP2 (ABCB2/3), CFTR (ABCC7), MRP1 (ABCC1), SUR1 (ABCC8), etc. (Figure S1). There are ∼50 human ABC transporters (classified into seven subfamilies, A−G),22 and more than 20 among them have asymmetry within their conserved motifs.23 Recently, a number of molecular dynamics (MD) simulations have been conducted for ABC transporters (reviews by Ferreira et al.6 and George et al.24). With regard to homodimeric ABC transporters, several studies have been conducted on Sav1866,25−30 MsbA,31−33 and P-gp,34−41 specifically, the NBD dimerization from the IF conformation, asymmetric opening of the NBDs from the OF conformation, the behaviors of the TMDs, etc. In these studies, the crystal structures42−45 were used for the initial structures in simulations. With regard to heterodimeric ABC transporters, several MD studies for CFTR46−49 and BmrCD50 have also been performed. However, the structures used in these studies were based on homology modeling, because available crystal structures were lacking. In our previous study on CFTR,47 its overall structure in the apo (ATP-free) state simulation converged to a structure similar to the “closed-apo” one found for MsbA.45 However, this structure is thought to be inappropriate taking protein function into consideration, because ATP molecules are highly unlikely to enter ABPs (see the following discussion). Such an unfavorable result might derive from the fact that the initial structure of the heterodimeric CFTR was constructed by referring to the homodimeric structure of the mouse P-gp.42 Currently, more sophisticated simulations based on corresponding crystal structures are needed for accurate and comprehensive understanding of the functional mechanisms of heterodimeric ABC transporters involving CFTR. The crystal structure of TM287/288, the first heterodimeric ABC transporter structure, was recently determined by Hohl et al. 7 TM287/288 is an exporter from a thermophilic eubacterium, Thermotoga maritima. In this crystal structure, an ATP analogue (AMP-PNP) is bound to ABP1, thus keeping it slightly closed, while ABP2 remains open. As a result, the
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MATERIALS AND METHODS
Preparation of MD Starting Structures. The atomic coordinates of TM287/288 were obtained from Protein Data Bank (PDB) entry 3QF4.7 This crystal structure was characterized by an inward-facing (IF) conformation, in which one AMP-PNP molecule with an Mg2+ ion was bound to ABP1, and four water molecules were bound to the intracellular side of the TMDs. First, the AMP-PNP molecule was replaced with an ATP molecule; the resultant structure had one ATP molecule and was termed “1ATP”. Second, one ATP and one Mg2+ ion (MgATP) were added to ABP2 on the basis of the structural alignment of NBD2 of TM287/288 and chain A of the MJ0796 structure (PDB entry 1L2T);11 the resultant 6731
DOI: 10.1021/acs.biochem.6b00947 Biochemistry 2016, 55, 6730−6738
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Biochemistry
Figure 2. RMSDs and final structures of the NBDs in the apo and 1ATP states. RMSDs of the apo state are plotted in panels A (310 K) and B (323 K), and those of the 1ATP state are plotted in panels D (310 K) and E (323 K). The final structures in the apo state (first run at 323 K) and the 1ATP state (first run at 310 K) are depicted in panels C and F, respectively, where α-helical subdomains (HSDs) are represented by the black ribbon model; other domains are colored gray, and the ATP molecule in ABP1 is represented by the CPK model. The approached HSDs in the apo state are indicated by arrows in panel C.
the γ-phosphate was protonated (−3e), whereas it should be deprotonated under physiological conditions; the total charge of ATP was −4e. In this study, we modified the original ATP force field through deprotonation of the γ-phosphate and even redistribution of the partial charge over phosphate oxygen atoms. The lipid parameters of Kukol were used for the DPPC lipid.54,55 For each system, energy minimization was first performed until the maximal force was 2 Å) in the 2ATP+S state was shifted to the shorter distance side compared with the one in the 2ATP state. In addition, with regard to substrate binding, the peak height of the 2 Å distance increased by ∼30% at 310 K: peak values in the 2ATP and 2ATP+S states were 20.7 and 48.8%, respectively. On the other hand, the heights at 323 K were similar (∼30%): those values were 29.2 and 31.3%, respectively. At the higher temperature, the protein would be largely fluctuating, which might cancel out the effect of substrate binding on the observed heights. In this section, for comparison between 2ATP and 2ATP+S states with regard to allostery, we adopted the ATP−C distance, which corresponds to the endpoint interaction of the allosteric pathway responsible for TMD−NBD communication. It is known that the ATPase activity of ABC transporters increases depending on substrate concentration, i.e., stimulation of “basal ATPase activity”.42,69 According to the results presented above, binding of the substrate to the TMDs enhances the probability of the formation of a structure in which an ATP molecule is sandwiched between conserved motifs, a structural prerequisite for ATP hydrolysis. This model is consistent with experimental results of basal ATPase activity, although further investigation is required to elucidate the process of TMD−NBD communication and the effect of substrate binding. Rotation of the Catalytic Residue Glu517 on ABP2. Glu517, located in the Walker B motif, is inferred to act as a catalytic base, but in the crystal structure of TM287/288, it points away from the γ-phosphate position of a potentially bound ATP at NBD2.7 Thus, a conformational change of Glu517 is required for the occurrence of ATP hydrolysis. Here, we calculated the minimal distances between the γ-phosphate of ATP and Oε1/2 of Glu517 in TM288 on the ABP2 side in 6734
DOI: 10.1021/acs.biochem.6b00947 Biochemistry 2016, 55, 6730−6738
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Biochemistry the 2ATP and 2ATP+S states (Figure 5 and Figure S5). As a result, large conformational changes (rotations) of Glu517 were
Figure 6. “Core-exposed” model in the transport cycle of heterodimeric ABC transporters. “Apo”, “1ATP”, and “2ATP” model structures are represented by the pink, green, and cyan ribbon models, respectively. The ATP-binding “core” motif of Walker A is colored red [on the (apo) degenerate ABP] and orange [on the (apo or 1ATP) consensus ABP]. ATP and Mg2+ are represented by the CPK model. The binding of ATP and release of ADP and Pi are denoted by curved arrows.
Figure 5. Distances between ATP and Glu517 and conformations of Glu517 in the initial and 98.9 ns MD structures. (A) Time course of the minimal distances between ATP and Glu517 (Oε1/2) in five runs at 310 K in the 2ATP+S state. Data from the fourth run are colored red. (B) Conformations of Glu517 in the initial and 98.9 ns MD structures in the fourth run in the 2ATP+S state at 310 K. The protein, Glu517, and ATP at 98.9 ns are represented by the ribbon (pink), stick (orange for carbon atoms), and CPK models, respectively. For comparison, the protein and Glu517 in the initial step are represented by the ribbon (gray) and stick (green for carbon atoms) models, respectively.
ABC transporters, several “fully separated” NBD structures have been found in P-gp/ABCB1,42,72,73 MsbA,45 and PglK,74 and “partially contacted” NBD structures have been found in MsbA (“closed-apo”)45 and ABCB10.70 We define an ABP half-pocket in the RecA-like core subdomain (CSD) as a “core” half-pocket. In the “closedapo” structure of MsbA, the “core” half-pockets of both NBDs are approached face to face (“core-approached” case), and this conformation is not necessarily appropriate for ATP binding because it requires the (large) opening of the “core” half-pocket to create space for the entrance of ATP. On the other hand, the “core-exposed” model for the apo state observed in this study is more appropriate for ATP binding (shown in the “apo” structure in Figure 6; the curved arrow indicates the binding of ATP). With regard to heterodimeric ABC transporters, Hohl et al. recently determined the structure of TM287/288 in the “apo” state.75 The obtained “apo” structure was very similar to the “1ATP” structure. In that study, they also measured the distance of the Ser350TM287−Lys475TM288 pair at ABP1 and that of the Asp460TM287−Ser363TM288 pair at ABP2 using the double electron−electron resonance (DEER) method. In the DEER results, the distance distributions of these pairs in the “1ATP” (AMP-PNP) state were similar to those in the “apo” state, though there were slight influences of AMP-PNP addition. These results support the similarity between the “apo” and “1ATP” crystal structures. However, nearly equal distance distributions of a few pairs alone may not be necessarily ascribed to the identity of the overall structures between two protein states. Here we propose another possibility that the “core-exposed” model is one candidate for a structure of the “apo” state. Our simulation data for the core-exposed “apo” model indicated that the distances of the aforementioned two pairs were 28.8 and 29.2 Å and those for the “1ATP” model were 29.9 and 30.8 Å, respectively. The distance differences between the two states are only 1.1 and 1.6 Å, respectively. This could be interpreted as evidence that these pair distances can almost be kept constant in spite of the large domain motion of the NBDs accompanied by an ∼7 Å decrease in the inter-HSD distance from the “apo” to “1ATP” state (Figure 6). The distance data for this “core-exposed” model are in fairly good
observed in the fourth and second runs at 310 K in the 2ATP +S state. The distance in the fourth run case reached ∼4 Å, and Glu517 approached the γ-phosphate of ATP and was close to the position for lytic water binding. In the other runs, the distance found was approximately 7−8 Å, and the Glu517 side chain pointed away from the γ-phosphate of ATP. Subsequently, it was trapped between His548 and Arg549, although it was flipped from the initial position interacting with Gln498. In almost every case, in ABC transporter structures, the catalytic glutamate (or the mutated glutamine) pointed toward lytic water or the (potentially) γ-phosphate position of ATP (ATP analogue).11,42−45 However, in a few cases, the catalytic glutamate pointed away from the catalytic center, as shown in the structure of TM287/288, human ABCB10,70 and ABCB6.71 In such cases, the following scenario for ATP hydrolysis has to be considered. (1) The catalytic glutamate (Glu) is hidden inside the NBD before the occurrence of NBD dimerization. (2) ATP binding induces the conformational change (perturbation) in Glu. (3) NBD dimerization occurs with Glu pointing toward lytic water. (4) Finally, ATP hydrolysis occurs in the consensus ABP. This scenario represents a rare possibility; however, it might take place to avoid futile ATP hydrolysis.
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DISCUSSION A “Core-Exposed” Model for the Apo State in the Transport Cycle of Heterodimeric ABC Transporters. Here, we discuss the “core-exposed” model found in this study. Figure 6 illustrates a hypothetical transport cycle (or an ATPase cycle), including the “apo” structure as a “core-exposed” model, the “1ATP” structure with the one-side-open conformation, and the “2ATP” structure as an ATP-sandwiched dimer. The details of the structural modeling done to draw this figure are given in the Supporting Information. For the NBD structure in the “2ATP” state, the ATP-sandwiched “head-to-tail” NBD dimer has been generally accepted since the Rad50 crystal structure was released.10 However, detailed NBD structures in the “apo” state remain unclear. With regard to homodimeric 6735
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Biochemistry agreement with the data from the DEER experiment within experimental error or fluctuation. Therefore, the presence of the core-exposed “apo” model should not be excluded at least as a transient species. In the study mentioned above,75 it was also shown that the NBDs of TM287/288 remained in contact in the absence of nucleotides (“apo” state). From a structural point of view, a “pivoting motion” around the contacted surface on the Cterminal sides of the NBDs might be important for the conformational transition induced by ATP binding, as discussed in the next sections (Figure S6). Pivoting Motion and Asymmetry of NBD Conformations. Homodimeric transporters show the same ATP affinity at their two consensus ABP sites, whereas heterodimeric transporters tend to have a higher ATP affinity at their degenerate ABP1 site and a lower ATP affinity at their consensus ABP2 site (TM287/288,7 CFTR,76 and MRP177). It was found in the apo state simulations that “core” motifs in degenerate ABP1 were exposed to the solvent, whereas “core” motifs in the consensus ABP2 were slightly covered by their counterpart in the HSD in both runs at the high temperature (323 K) (Figure 2C). This ABP1-exposed and ABP2-covered conformation might be responsible for the different affinities of degenerate and consensus ABPs described above. Moreover, two NBDs make contact at the C-terminal side and allow a (fluctuating) pivoting motion around the contact surface (Figure S6). It is likely that the residues at the degenerate ABP1 site have a slightly repulsive nature, while those at the consensus ABP2 site are slightly attractive on each subdomain surface in the “apo” state. Thus, these interactions are related to the difference in the affinities of ABP1 and ABP2. The pivoting motion would not be restricted to heterodimeric ABC transporters. The asymmetric nature of NBD opening and closing was observed in computational and experimental studies.7,25,28,33,36−39,41,48,50,70,75 Moreover, crystal structures of ABC exporter ABCB10 and ABC importers such as MalFGK2-E and BtuCD-F show C-terminal contacts (some of them have regulatory domains). Therefore, the pivoting motion and the conformational asymmetry in the structural dynamics of the NBDs may be common properties among ABC transporters. Recently, the structure of the heterodimeric ABC transporter TmrAB in the “apo” state has been revealed,78 which shows a small contact between the NBDs. This result also supports our finding of the “core-exposed” model in the “apo” state. In addition, for CFTR, the difference between the “C1 (apo)” and “C2 (1ATP)” states remains unclear because of a lack of available structural data.51,52 Therefore, the results obtained in this study provide important insights into the structural dynamics of CFTR and other heterodimeric ABC proteins.
with two ATP molecules (2ATP or 2ATP+S states) induced dimerization of the NBDs or formation of the consensus ABP2. The conformation in the apo state is one of the major topics of investigation related to heterodimeric ABC transporters such as CFTR. Our simulation results suggest that the conformation of the NBDs acquires a “core-exposed” structure, in which the “core” half-pockets, including the Walker A and B motifs, are exposed to the solvent, and the α-helical subdomains of the NBDs approach each other. This “core-exposed” model is appropriate for ATP binding because ATP molecules are able to bind freely to the “core” half-pocket without large conformational changes. Using Hoechst 33342 as a substrate, we conducted multiple MD simulations (2ATP+S state). We found that binding of the substrate to the DBP in the TMDs moderately shifts the probability distribution for the formation of ABP2 to the shorter side compared with the results obtained from our substrate-free simulation (2ATP state); i.e., binding of the substrate to the TMDs slightly enhanced the formation of ABP2 through allosteric TMD−NBD communication. With regard to ATP hydrolysis, the rotation of the catalytic residue Glu517 on ABP2 was observed in MD simulations for the substrate-bound state. Binding of ATP to the NBDs and binding of the substrate to the TMDs are important events in the transport cycle of ABC transporters. Our findings in this study provide important insights into the structural dynamics of ABC transporters.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00947. Motif sequences in heterodimeric and homodimeric ABC transporters, structure of the NBDs and inter-HSD distances, final WA−C distances on ABP1, drug-binding residues in P-gp and corresponding residues in TM287/ 288 and substrate position in the simulation, initial and final conformations of the substrate in the TMDs in simulations of the 2ATP+S state, substrate-interacting residues in simulations, distances between ATP and Glu517 and conformations of Glu517 in simulations of the 2ATP+S state, sructural modeling of NBD dimer conformations in the transport cycle, and pivoting motion of the NBDs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
CONCLUSION Understanding the structural dynamics of heterodimeric ABC transporters leads to drug discovery or progress in molecular biology and medical sciences. In this work, we investigated the structural dynamics of the heterodimeric ABC transporter TM287/288 through multiple 100 ns MD simulations in different ATP-bound and substrate-bound states. As a result, binding of an ATP molecule to the degenerate ABP1 (1ATP state) stabilized the one-side-open conformation of the NBDs, which corresponds to the TM287/288 structure determined with the ATP analogue AMP-PNP. On the other hand, systems
ORCID
Tadaomi Furuta: 0000-0003-4568-9043 Funding
This work was supported by JSPS KAKENHI Grants JP16H00825 (to M.S.) and JP15K00400 (to T.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Notes
The authors declare no competing financial interest. 6736
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