Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters

Significance

By determining the structure of a pantothenate energy-coupling factor (ECF) transporter, LbECF-PanT, we revealed the structural basis of how one EcfAA'T module can interact with different S subunits among group II ECF transporters. We also identified the residues that mediate the intermolecular conformational transmission and/or affect the transporter complex stability, and thus are essential for transporter activity. In addition, we identified the pantothenate-binding pocket and the residues constituting the pocket. Last but not least, we found that the structure of EcfT is dynamic and undergoes dramatic changes in the three different transporter complexes, which confer scaffold-mediating complex formations of the ECF module with various EcfS proteins. These findings are incorporated into an updated working model of the ECF transporter.

Abstract

Energy-coupling factor (ECF) transporters are a unique group of ATP-binding cassette (ABC) transporters responsible for micronutrient uptake from the environment. Each ECF transporter is composed of an S component (or EcfS protein) and T/A/A′ components (or EcfT/A/A′ proteins; ECF module). Among the group II ECF transporters, several EcfS proteins share one ECF module; however, the underlying mechanism remains unknown. Here we report the structure of a group II ECF transporter–pantothenate transporter from Lactobacillus brevis (LbECF-PanT), which shares the ECF module with the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT). Structural and mutational analyses revealed the residues constituting the pantothenate-binding pocket. We found that although the three EcfS proteins PanT, FolT, and HmpT are dissimilar in sequence, they share a common surface area composed of the transmembrane helices 1/2/6 (SM1/2/6) to interact with the coupling helices 2/3 (CH2/3) of the same EcfT. CH2 interacts mainly with SM1 via hydrophobic interactions, which may modulate the sliding movement of EcfS. CH3 binds to a hydrophobic surface groove formed by SM1, SM2, and SM6, which may transmit the conformational changes from EcfA/A′ to EcfS. We also found that the residues at the intermolecular surfaces in LbECF-PanT are essential for transporter activity, and that these residues may mediate intermolecular conformational transmission and/or affect transporter complex stability. In addition, we found that the structure of EcfT is conformationally dynamic, which supports its function as a scaffold to mediate the interaction of the ECF module with various EcfS proteins to form different transporter complexes.

In recent years, a family of ATP-binding cassette (ABC) transporters, the energy-coupling factor (ECF) transporters, has been identified in bacteria and archaea. These ECF transporters are responsible for micronutrient uptake from the environment (1–3). Compared with the classical ABC importers, ECF transporters lack periplasmic solute-binding proteins (SBPs) and instead use membrane-embedded EcfS proteins (S components) for specific substrate binding. Besides the EcfS protein, each ECF transporter contains an energy-coupling module (ECF module) comprised of two cytosolic ATPases EcfA and EcfA′ (A/A′ components) and another transmembrane protein, EcfT (T component). Based on the unique features of the ECF modules, ECF transporters have been classified into two groups: group I, in which each S component has a dedicated ECF module, and group II, in which several S components share a common ECF module (4, 5).

In 1970s, several vitamin-associated transporters were found to contain different membrane-embedded substrate-binding proteins but to share a common module for energy coupling, and thus were termed ECF transporters (6). The exact molecular components of these transporters remained unclear, however, until the identification and characterization of the Co²⁺ and Ni²⁺ transporters CbiMNQO and NikMNQO (1). These transporters are now classified as members of group I ECF transporters, and the constituting MN, Q, and O proteins correspond to the EcfS, EcfT, and EcfA/A′ proteins, respectively (3). A subsequent detailed study on another group I ECF transporter, BiMNY (in which the constituting M, N, and Y proteins correspond to the EcfA/A′, EcfT, and EcfS proteins, respectively), identified the tripartite protein complex as a high-efficiency biotin transporter, and found that the solitary BioY can also bind and transport biotin in Escherichia coli (2, 7). In contrast, the EcfS proteins of group II ECF transporters—RibU, ThiT, and FolT—were found to tightly bind the substrate riboflavin, thiamine, and folate, respectively (8-10), but to have no transport activity in their solitary state (3, 11). In addition, for group I ECF transporters, the EcfT protein can form a stable subcomplex with the EcfA protein (2), whereas such a subcomplex can hardly be obtained for group II ECF transporters (11, 12). Different EcfS proteins of group II ECF transporters can form functional complexes with a common ECF module, however (3, 12). These results indicate that group I and II ECF transporters have some distinct features.

In the past few years, a number of structural and functional studies have been performed to elucidate the underlying mechanisms of ECF transporters. The crystal structures of three different EcfS proteins of group II ECF transporters—Staphylococcus aureus RibU in complex with riboflavin, and Lactococcus lactis ThiT and BioY in complexes with thiamine and biotin, respectively—have been determined in the substrate-bound state (11, 13, 14). Although the sequences of these EcfS proteins are very dissimilar, they have a common structural fold of a six-helix bundle; however, the structures of the substrate-binding site are quite different. To accommodate for the chemically and structurally different substrates, the orientations of the transmembrane helices of EcfS (SM, especially SM4–6) and the residues constituting the substrate-binding pocket are substantially different, accounting for distinct substrate specificities of different EcfS proteins (15). Based on the biochemical and structural studies of individual EcfS and EcfA/A′ proteins, the conserved AxxxA motif of different EcfS proteins was suggested to form the binding site for the ECF module (13), and several possible working models of transport were proposed (11, 13, 16). The recently determined structures of the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT) revealed the structure of the T component; the interactions among the S, T, and A/A′ components; and the architecture of group II ECF transporter complexes, leading to a proposed more accurate working model of transport (17, 18). The detailed transport mechanism remains illusive, however; in particular, the molecular basis for ECF module sharing, the coupling between EcfT and EcfA/A′, and the conformational transmission between EcfS and EcfT remain unclear (15, 19).

In this paper, we report the crystal structure of a group II pantothenate ECF transporter complex from Lactobacillus brevis (LbECF-PanT) at 3.25-Å resolution. A structural comparison of LbECF-PanT with LbECF-FolT and LbECF-HmpT revealed a common interacting surface of different EcfS proteins with the same EcfT protein. We evaluated the functional roles of the residues involved in the intermolecular interactions by functional studies. Taken together, the structural and functional data provide the molecular basis for how different S components share a common ECF module, and shed new light on the transport mechanism of group II ECF transporters.

Results

Characterization and Structure Determination of LbECF-PanT.

Our sequence alignment results suggests that the L. brevis genome encodes at least seven group II ECF transporters (the putative folate, pantothenate, riboflavin, hydroxymethylpyrimidine, thiazole, biotin, and queuosine transporters), with corresponding specific substrate-binding S components FolT, PanT, RibU, HmpT, ThiW, BioY, and QueT, respectively. As expected, only one set of EcfTAA′ genes encoding the ECF module could be defined. This provides a good system for studying how different S components can use one common ECF module.

To verify the physiological function and activity of the putative pantothenate transporter, we cotransformed the genes encoding PanT, EcfT, EcfA, and EcfA′ (panT-ecfTAA′) into the Escherichia coli DV1 strain, which could not grow in minimum medium without the addition of β-alanine or pantothenate. To analyze the effect of pantothenate on the growth of the E. coli strain, we added a trace amount of calcium pantothenate to the medium. The influence of the pantothenate concentration on the growth of the DV1 strain transformed with empty vector or panT-ecfTAA′ is shown in Fig. 1A. The results indicate that both DV1-derived strains can grow at high pantothenate concentrations of >500 nM, but not at low pantothenate concentrations of <5 nM, and in the range of 20–200 nM pantothenate, the DV1 strain transformed with panT-ecfTAA′ (DV1_LbECF-PanT) can grow well, but the DV1 strain transformed with empty vector cannot. Thus, we added 100 nM pantothenate to the medium for analyze the pantothenate transporter activity of LbECF-PanT in the subsequent growth assays. We then tested the activity of different combinations of the four genes. Our results show that except for panT-ecfTAA′, the DV1 strains transformed with all other tested combinations of the four genes failed to grow (Fig. 1B). These results lead us to conclude that the tetrapartite panT-ecfTAA′ is the functional unit.

Fig. 1.

Overall structure of the pantothenate ECF transporter LbECF-PanT. (A) Complementary growth of E. coli DV1, indicating the pantothenate transporter activity of LbECF-PanT. DV1/panT-ecfTA′ is DV1-transformed with PQlink-panT-ecfTAA′ (gray), and DV1/vector is DV1- transformed with PQlink vector (white). (B) The growth curve of DV1 transformed with different LbECF-PanT component combinations (with 100 nM pantothenate in the medium). Blue, red, green, and purple lines indicate the growth curves of DV1 transformed with panT-ecfTAA′, panT-ecfT, panT, and empty vector, respectively. (C) Cylinder cartoon of the structure of LbECF-PanT viewed in parallel to the membrane. PanT is in cyan; EcfT is in orange; EcfA is in gold; and EcfA′ is in gray.

We crystallized the recombinant LbECF-PanT protein complex, and solved the structure at 3.25-Å resolution by molecular replacement using the structure of LbECF-FolT as a template (Table S1). The final structure model contains PanT, EcfT, EcfA, and EcfA′ in a 1:1:1:1 ratio (Fig. 1C). Similar to LbECF-FolT and LbECF-HmpT (17, 18), the overall structure of LbECF-PanT also has a double-cone–like shape; however, the structure of the substrate-binding pocket of PanT differs greatly from that of the substrate-binding pockets of FolT and HmpT. In addition, dramatic conformational differences of the transmembrane helices 3 and 4 (TM3 and TM4) of EcfT are observed in different complexes. As expected, we found no electron density at the pantothenate-binding site of PanT or the ATP-binding sites of EcfA/A′, suggesting that the structure is in the substrate-free conformation or, more specifically, the inward-facing state.

Structure of PanT and the Substrate-Binding Pocket.

The structure of the pantothenate-specific binding protein PanT comprises six transmembrane helices (α1–α6, or SM1–6). As in the other EcfS proteins (15), the periplasmic and cytoplasmic sides of PanT are hydrophilic, whereas the outer surface of the membrane-spanning helices is hydrophobic (Fig. 2 A and B). However, compared with the other group II EcfS proteins with known structures, PanT contains two extra short antiparallel β-strands in the middle of the L1 loop (Fig. 2A), along with a long L3 loop (16 residues) connecting SM3 and SM4. In addition, PanT contains a hydrophobic surface groove formed by SM1, SM2, and SM6 (Fig. 2B).

Fig. 2.

Structure and substrate-binding site of PanT. (A) Cylinder cartoon of the PanT structure. The secondary structure from the N terminal to the C terminal is colored in a rainbow from blue to red. (B) Electrostatic surface model of PanT. The orientation is the same as in A. The hydrophobic surface groove is marked. (C) Top view of PanT diagramed in ribbons (same color code as in A). The residues involved in pantothenate binding are shown as orange sticks. (D) Top view of the PanT surface showing the binding pocket of pantothenate. The L3 loop was removed to show the pantothenate-binding pocket more clearly. (E) (Upper) Results of the growth assay of the DV1_LbECF-PanT mutants are shown. (Lower) Formation of the transporter complex of the mutants verified by SDS/PAGE.

The putative pantothenate-binding pocket in PanT is composed of SM3–6 and is partially sealed by the L3 loop but still accessible to the cytoplasm (Fig. 2 C and D). This feature is slightly different than the LbECF-FolT and LbECF-HmpT structures, in which the substrate-binding pocket is widely open to the cytoplasm owing to a short L3 loop (17, 18). The pantothenate-binding pocket contains eight polar and nonpolar conserved residues, of which residues Thr39, Arg95, Asn131, Thr132, and Val135 are invariant and Tyr30, Trp64, and Phe85 are highly conserved (Fig. 2C and Fig. S1). We postulate that these residues are involved in the specific binding of pantothenate. To verify this speculation, we performed a complementary growth assay using DV1_LbECF-PanT-containing point mutations of the aforementioned residues (Fig. 2E). The results show that residues Arg95 and Trp64 are essential for the growth of DV1_LbECF-PanT, indicating that these residues may play critical roles in pantothenate binding. Because pantothenate is an organic acid, electrostatic or hydrogen-bonding interactions could be involved in coordination. Although the growth of DV1_LbECF-PanT is only slightly affected or unaffected by the single mutations of PanT, T39V, F85A, N131D, T132A, and V135A, it is possible that a combination of these mutations may have more evident effects in coordination with pantothenate. This possibility is suggested by the inability of DV1_LbECF-PanT containing the double mutations of N131D/V135A to grow further. These results imply that these sequentially conserved and structurally gathered residues dictate the specific binding of pantothenate.

Different EcfS Proteins Use a Common Surface to Interact with the Same EcfT.

Different EcfS proteins of group II ECF transporters can share the same ECF module; however, the sequence alignment of EcfS proteins fails to identify conserved sequence motifs, except for AxxxA in SM1. Because the structures of three group II ECF transporters from the same species, LbECF-PanT, LbECF-FolT, and LbECF-HmpT, are available, we superimposed the three EcfS proteins PanT, FolT, and HmpT together to investigate whether they use a conserved 3D site to interact with the same ECF module. As shown in Fig. 3A, the conformations of SM3–6 of PanT, FolT, and HmpT differ substantially, explaining why these EcfS proteins can accommodate the chemically diverse substrates as has been suggested (15). In contrast, the conformations of SM1 and SM2 show few differences, suggesting a common functional role; thus, we further analyzed the functions of SM1 and SM2.

Fig. 3.

Interactions of transmembrane helix 1 of PanT with EcfT. (A) Superimposition of the PanT, FolT, and HmpT structures. PanT is colored in a rainbow from blue (N terminal) to red (C terminal), and FolT and HmpT are in light blue and gray, respectively. (B) The hydrophobic interactions between transmembrane helix 1 (SM1/α1) of PanT and energy-coupling helices 2/3 (CH2/3) of EcfT. SM1/α1 and SM2/α2 are shown as ribbons color-coded as in A, and the hydrophobic residues of α1 are shown as yellow sticks. CH1/2/3 of EcfT are shown as orange ribbons, and residues in CH2/3 interacting with α1 are colored green. The electrostatic surface of CH1/2/3 is also shown. (C) (Upper) Results of the growth assay of the DV1_LbECF-PanT mutants. (Lower) Formation of the transporter complex of the mutants.

The AxxxA motif in SM1 of the EcfS proteins has been identified as the site involved in the interaction with EcfT, and replacement of either of the two Ala residues with Trp was found to sufficiently disrupt the transporter complex and abolish activity in a thiamine ECF transporter from L. lactis (13). Indeed, we found that these two Ala residues are conserved in SM1 of PanT in LbECF-PanT (corresponding to Ala13 and Ala17) (Fig. 3B); however, replacement of either residue with Trp (A13W or A17W) does not affect the growth of DV1_LbECF-PanT or completely disrupt the interaction of the four components, although the A17W mutation significantly impairs the interaction (Fig. 3C). Structural analyses demonstrated that in addition to Ala13 and Ala17, the interaction surface of PanT with EcfT comprises a number of other hydrophobic residues, including Leu14, Ile18, Leu20, Leu21, Leu24, and Leu28, which protrude from the exterior side of SM1 of PanT. This hydrophobic surface of PanT interacts mainly with several hydrophobic residues of CH2 (Ile163, Ala164, and Val168) and CH3 of EcfT (Ala212 and Ala216) (Fig. 3B).

Our mutational analysis showed that replacement of one of these residues with a charged residue (Asp) or a residue with large side chain (Trp) could not completely disrupt the transporter complex (Fig. 3C, Lower), implying a strong interaction between EcfT and PanT. However, the growth rate of DV1_LbECF-PanT containing the L14D, I18D, or L24D mutation in PanT was significantly reduced, and DV1_LbECF-PanT containing the L20D, L21D, or L28D mutation in PanT or the A216D mutation in EcfT failed to grow (Fig. 3C). Moreover, introducing multiple mutations at the interface, such as double mutations A13D/L14D or A17D/I18D, not only destabilized the complex, but also disrupted transporter activity (Fig. 3 B and C). These results suggest that these hydrophobic residues at the interaction surface play essential roles both in the formation and stabilization of the complex and in the coupling and transmission of the conformational changes between EcfT and PanT. To exert these functions, these residues need to be hydrophobic but not necessarily highly conserved, as further supported by our mutagenesis data showing that the A13W, A17W, L14W, or I18W mutation in PanT and the I163W, A164W, or A212W/A216W mutation in EcfT have minor effects on complex formation and transporter activity and hence the growth of DV1_LbECF-PanT (Fig. 3C). Structural analyses have shown a similar hydrophobic surface in other EcfS proteins as well, explaining why SM1 is chosen by PanT, FolT, and HmpT to interact with EcfT.

Along with SM1, we also found a surface groove of PanT involved in the interaction with EcfT (Figs. 2B and 4A). This groove runs along the long axis of the transmembrane helices of PanT and is formed by SM1, SM2, the L1 loop, and the C-terminal half of SM6 (SM6C). SM1 and SM6C constitute the walls of the groove, and the L1 loop and SM2 form the base. The interior sides of the groove are hydrophobic and comprise residues Phe7, Val11, Leu14, Leu15, and Ile18 from SM1; Phe27, Leu28, Ile31, Met38, and Leu40 from the L1 loop; Leu43, Thr44, Val47, Ala51, and Leu52 from SM2; and Ile177, Leu181, Leu185, Met188, Pro189, Leu190, Gln193, and Leu197 from SM6C (Fig. S2A). Similar surface grooves are seen in FolT and HmpT (Fig. S3) and in RibU, ThiT and BioY (Fig. S4). Again, the residues constituting this groove are not conserved among different EcfS proteins from the same species, but must be hydrophobic or nonpolar (Fig. S2 B and C).

Fig. 4.

Interactions of CH3 of EcfT with the hydrophobic surface groove of PanT. (A) CH3 binds in the surface groove of PanT. CH3 is shown as a ribbon cartoon and colored orange, PanT is shown with the electrostatic surface, and the residues in CH3 that interact with the hydrophobic surface groove of PanT are shown as sticks. (B) (Upper) Results of the growth assay of the DV1_LbECF-PanT mutants. (Lower) Formation of the transporter complex of the mutants verified by SDS/PAGE.

As clearly evident in the LbECF-PanT, LbECF-FolT, and LbECF-HmpT structures, CH3 of EcfT in each complex binds within the similar surface grooves of PanT, FolT, and HmpT (Fig. 4A and Fig. S3). In particular, the highly conserved residues Leu201 and Met205 and the strictly conserved residues Phe209 and Phe213 protrude from CH3 and interact with the hydrophobic surface groove of EcfS (Fig. S5). We tested the importance of these residues for the transporter activity of LbECF-PanT in a complementary growth assay. The results show that although single mutations L201A, L201R, M205R, F209R, and F213R and double mutations M205A/F209A and F209A/F213A in EcfT have minor effects on the growth rate, double mutations of the foregoing four residues to Arg (M205R/F209R and F209R/F213R) lead to undetectable growth of DV1_LbECF-PanT (Fig. 4B). In addition, double mutations M205R/F209R and F209R/F213R also have significant effects on the complex formation in vitro. These data suggest that the critical roles of these four residues also can be exerted by affecting formation and stabilization of the transporter complex, along with coupling and transmission of the conformational changes between EcfT and PanT.

Together, the hydrophobic surface of SM1 and the hydrophobic groove in the three different EcfS proteins form a common interaction surface to interact with the coupling helices CH2/3 of the same EcfT protein (Fig. 4A and Fig. S3). This explains why different S components can share a common ECF module among group II ECF transporters.

Conformational Differences of EcfT in Different Transporter Complexes.

In addition to the aforementioned common interaction surface that dictates the interaction between EcfT and EcfS (PanT/FolT/HmpT), there is another interaction interface between EcfT and EcfS (Fig. 5A). Specifically, the transmembrane helices TM3 and TM4 of EcfT interact with the L3 loop of PanT (or FolT or HmpT) via hydrophobic interactions (Figs. 1C and 5B). Intriguingly, in the LbECF-PanT structure, the two interaction interfaces between EcfT and PanT bury a total of 6,628 Ų, or 30.8% of the solvent-accessible surface areas; however, our biochemical data show that EcfT cannot form a stable subcomplex with PanT (or FolT) in the absence of EcfA/A′ (Fig. 5C). Our structural analysis data indicate that the EcfS proteins of group II ECF transporters always form a rigid six-helix bundle in the membrane and likely do not have great conformational flexibility, except for the connecting L1, L3, and L5 loops. In contrast, the conformation of the EcfT protein adopts an “L” shape in LbECF-FolT and a “C” shape in LbECF-PanT, suggesting that EcfT has a dynamic conformation that may prevent the formation of a stable subcomplex with EcfS in the absence of EcfA/A′.

Fig. 5.

Dynamic conformation of EcfT in different transporters. (A) Different conformations of EcfT in LbECF-PanT, LbECF-FolT, and LbECF-HmpT. The EcfTs are shown in cylinders and colored orange (LbECF-PanT), yellow (LbECF-FolT), and green (LbECF-HmpT). PanT is shown with the electrostatic surface. (B) Structures rotated 90° clockwise around the vertical axis relative to A. The arrow indicates the possible movement of PanT after the conformational change in CH2/3 or EcfA/A′. (C) The complex formation of LbECF-PanT. Lane 1, purified with all four components; lane 2, purified after EcfT (N-terminal 6×His tag) and PanT coexpression; lane 3, purified after EcfT and PanT (N-terminal 6×His tag, indicated with an arrowhead) coexpression; lane 4, purified after EcfT (N-terminal 6×His tag) and FolT coexpression. (D) Top view of the different conformations of EcfT in LbECF-PanT, LbECF-FolT, and LbECF-HmpT. The color code is the same as in A. (E) Zoom-in view of CH2/3 in B, showing the possible movement (indicated with arrows) during the transition from the current state to the outward-facing state in the transport process.

Detailed structural comparisons of the same ECF module of LbECF-PanT, LbECF-FolT, and LbECF-HmpT clearly reveal the conformational differences of EcfT (Fig. 5 A, B, and D). In these three complexes, the EcfA/A′ proteins assume almost identical conformations (RMSD = 0.6–0.7 Å); however, superposition of EcfT in the three complexes reveals an RMSD of 1.9–2.2 Å. Specifically, compared with TM3 and TM4 of EcfT in LbECF-FolT and LbECF-HmpT, TM3 and TM4 of EcfT in LbECF-PanT undergo an ∼7° rotation toward PanT and consequently make extensive contacts with the L3 loop (Fig. 5 A, B, and D). TM1, TM2, and TM5 exhibit conformational differences as well. The conformational flexibilities of TM1–5 of EcfT are also demonstrated by their higher average B factor (122.2 Ų) compared with that of EcfT (106.3 Ų), EcfS (91.5 Ų), and EcfA/A′ (62.9/86.6 Ų). The conformational flexibility of EcfT may give it the ability to accommodate different EcfS proteins and also to allow the rigid body movement of the EcfS protein to upload or download the substrate following the scissors-like motion of the coupling helices of EcfT (Fig. 5 B and E).

Interactions Between EcfA/A′ and EcfT Are Essential to the Transport Process.

EcfT forms extensive interactions with EcfA/A′, which have been described in detail in the LbECF-FolT structure (17). Similar interactions are conserved in the LbECF-PanT structure. Among these interactions, residues Arg185 (in XRX motif 1) and Arg226 (in XRX motif 2) of EcfT form salt bridges with residues Asp106 of EcfA and Asp102 of EcfA′ to anchor the coupling helices CH2 and CH3 to the deep surface groove of EcfA/A′, respectively (Fig. 6 A and B). These four residues are strictly conserved among all ECF transporters, indicating that they may have important functional roles (Figs. S5 and S6).

Fig. 6.

Interactions between the XRX motifs of EcfT and the conserved Asp residues in EcfA/A′. (A) Interaction between residue Arg185 (from XRX motif 1 of EcfT) and Asp106 (from EcfA). (B) Interaction between residue Arg226 (from XRX motif 2 of EcfT) and Asp102 (from EcfA′). CH2, CH3, EcfA, and EcfA′ are color-coded as in Fig. 1C. (C) (Upper) Results of the growth assay of the DV1_LbECF-PanT mutants. (Lower) Formation of the transporter complex of the mutants verified by SDS/PAGE.

Our complementary assay results indicate that mutation of any of the four residues to an oppositely charged residue (i.e., D106R, D102R, R185E, and R226E) could abolish pantothenate transporter activity, whereas mutations of the flanking residues A184V, G186A, A225V, G227A, and D106A/D102A had relatively minor effects on transporter activity (Fig. 6C). Nevertheless, all of the foregoing single mutations except D102R had only a minor effect on formation of the transporter complex in vitro. These data suggest that the four residues exert their functions mainly through transmission of the conformational changes between EcfT and EcfA/A′ during the transport process. Our results are consistent with a previous report suggesting that the two conserved Arg residues of EcfT are involved in mediation of the intramolecular signaling (20), and provide further insight into the underlying molecular basis of this function.

Discussion

Based on our structural and functional analyses, we can deduce the coupling events occurring in the transport process of ECF transporters. Charge interactions between residues Asp106-Arg185 and Asp102-Arg226 are involved in transmission of the conformational change from EcfA/A′ to EcfT. The coupling helix CH3 of EcfT connecting with the surface groove of EcfS may work as a transmission gear, through which the movement of EcfT is translated to the movement of EcfS. The SM1–CH2 interaction area may provide a hydrophobic sliding surface for the relative movement of EcfS against EcfT. In group II ECF transporters, different EcfS proteins from the same species have similar transmission gear parts and sliding surfaces, which are key elements in modulation of the conformational transmission between the EcfT and EcfS proteins, and thus can share one common ECF module.

It is widely accepted that ATP binding, hydrolysis, and product release can induce conformational changes in the nucleotide-binding domain (NBD) proteins, which are transmitted to the transmembrane domain (TMD) proteins in the ABC transporters (21–25). Based on a wealth of studies, researchers are beginning to elucidate the details of the transport process of canonical ABC importers (26–31). The process may be described in four continuous steps (beginning from the inward-facing conformation): (i) Driven by the ATP binding, the open NBD dimers use a tweezers-like motion to approach one another; (ii) following the conformational changes in the NBDs, the two separated coupling helices each extending from one TMD and connecting to the NBD come in close proximity; (iii) the coupling helices transform the conformational changes from the NBDs to the TMDs, leading to the clothespeg-like motion of the TMDs from an inward-facing conformation to an outward-facing conformation to accept the substrate from the periplasmic SBP; and (iv) ATP hydrolysis and product release reset the NBDs to the open conformation and the TMDs to the inward-facing conformation to release the substrate to the cytoplasm. This model has been widely accepted in the research field of ABC importers, although exceptions may exist.

Enlightened by the results for canonical ABC importers and the present study, we now have a better understanding of the transport process of ECF transporters (also starting from the inward-facing conformation). Powered by ATP hydrolysis, EcfA/A′ undergo an open to closed conformational change. This movement can be translated into a “closing scissors”-like motion of the coupling helices CH2/CH3 of EcfT, because the XRX motifs (residues Arg185 and Arg226) are anchored into the deep groove of EcfA/A′ (residues Asp106 and Asp102). Thus, the two “blades,” CH2 and CH3, adopt a more upright conformation. CH3 binding within the surface groove of EcfS could be the key factor mediating the conformational transmission from EcfT to EcfS, whereas the other interaction surface represented by SM1 of EcfS with CH2 of EcfT may function as a sliding surface. Following the movement of CH2/CH3, the EcfS may be forced to assume an upright rotation through the sliding surface. As the rotation of EcfS continues, the orientational changes of the transmembrane helices TM3 and TM4 of EcfT, which interact with the L3 and L5 loops of EcfS, will probably occur; thus, the conformation of the L3 and L5 loops of EcfS likely will undergo dramatic changes. These conformational changes will surely alter the substrate-binding pocket of EcfS (i.e., the shape, properties, and conformation of the lid-L1loop) to allow tight binding of the substrate to the outward-facing “apo” EcfS, and likely will alter the interaction strength between EcfS and EcfT. In short, EcfT’s dynamic properties make it an essential scaffold in mediation of the conformational transmission from EcfA/A′ to EcfS (model shown in refs. 15 and 19).

Materials and Methods

Detailed information on protein expression, crystallization, data collection, and the complementary assay are provided in SI Materials and Methods. In general, the LbECF-FolT protein complex was expressed in E. coli BL21 (DE3) and purified to homogeneity for crystallization. All data were collected at beamline BL17U of the Shanghai Synchrotron Radiation Facility and processed with HKL2000 (32). The structure was determined with molecular replacement using the LbFolT-ECF transporter as a template (PDB ID code 4HUQ). Data collection and model refinement statistics are summarized in Table S1. The transporter activity was tested using an E. coli DV1 (panF panD) strain.