An Inward-Facing Conformation of a Putative Metal-Chelate-Type ABC Transporter

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Science  19 Jan 2007:
Vol. 315, Issue 5810, pp. 373-377
DOI: 10.1126/science.1133488


The crystal structure of a putative metal-chelate–type adenosine triphosphate (ATP)–binding cassette (ABC) transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.

Transporters catalyze the thermodynamically unfavorable translocation of substrates against a transmembrane concentration gradient through the coupling to a second, energetically favorable process. One of the most widespread families of transporters, the adenosine triphosphate (ATP)–binding cassette (ABC) family (14), uses the binding and hydrolysis of ATP to power substrate translocation. ABC transporters are minimally composed of four domains, with two transmembrane domains (TMDs) and two ABCs or nucleotide-binding domains (NBDs) located in the cytoplasm. Although diverse with respect to physiological function and TMD architecture, ABC transporters are characterized by two highly conserved NBDs that contain critical sequence motifs for ATP binding and hydrolysis, including the P loop present in many nucleotide-binding proteins and the ABC signature or C-loop motif [Leu-Ser-Gly-Gly-Gln (LSGGQ)] that is specific to ABC transporters. These similarities suggest a common mechanism by which ABC transporters orchestrate a sequence of nucleotide- and substrate-dependent conformational changes that translocate the substrate across the membrane through interconversion of outward- and inward-facing conformations; this type of “alternating access” model has been generally found to provide a productive framework for the mechanistic characterization of transporters (5). For prokaryotic ABC transporters functioning as importers, substrate translocation is also dependent on high-affinity periplasmic-binding proteins (6) that deliver the ligand to the outward-facing state of the cognate transporter.

The HI1470/1 transporter from Haemophilus influenzae belongs to the family of binding protein–dependent bacterial ABC transporters that mediate the uptake of metal-chelate species, including heme and vitamin B12 (7). Because iron is often an essential nutrient, members of this family are widely distributed throughout bacteria, including pathogenic organisms such as H. influenzae (8). The molecular architecture for this family of ABC transporters was established by the structure of BtuCD, the B12 importer from Escherichia coli (9). The transporter encoded by genes HI1470 and HI1471 of H. influenzae (10) exhibits 24 and 33% sequence identity to the ABC subunit BtuD and the membrane-spanning subunit BtuC, respectively, and was identified as a promising candidate for structural study during the original screen of homologs explored in the BtuCD analysis (9). After overexpression and purification in decylmaltoside of a histidine-tagged construct, the crystal structure of the intact, nucleotide-free HI1470/1 transporter was phased by isomorphous and multiwavelength anomalous diffraction methods and refined at 2.4 Å resolution (11).

The overall molecular organization of HI1470/1 (Fig. 1A) resembles that observed previously for BtuCD (9), with the functional unit consisting of two copies each of the HI1471 membrane-spanning subunits and of the HI1470 ABC subunits (Fig. 1, B and C). The root mean square deviations (RMSDs) in Cα positions between structurally equivalent residues in the individual subunits of HI1470/1 and BtuCD are ∼1.5 Å; the corresponding RMSD after superposition of equivalent residues in all four subunits of these transporters is 2.4 Å. Each pair of HI1470 or HI1471 subunits in HI1470/1 is closely related (RMSDs ∼1 Å) by a rotational operation that is close to an exact two-fold axis (rotation angle ∼180.5°) passing through the center of the transporter. By means of the program HOLE (12), an evaluation of the permeation pathway that surrounds this axis reveals an important difference related to the detailed arrangement of subunits between HI1470/1 and BtuCD (Fig. 2): Although both transporters maintain a tapered pathway through the membrane-spanning subunits, the pathways open to opposite sides of the membrane, such that HI1470/1 and BtuCD adopt inward- and outward-facing conformations, respectively.

Fig. 1.

(A) The ABC transporter HI1470/1 consists of four subunits: two membrane-spanning HI1471 subunits (cyan and blue) and two nucleotide-binding HI1470 subunits (green and pink). The molecular rotation axis is vertical, with the cytoplasmic-facing surface of the transporter toward the bottom. The locations of the N and C termini for one subunit each of HI1470 and HI1471 are indicated. (B) A view of HI1470/1 rotated 90° from that of (A), looking down the molecular two-fold axis toward the membrane-spanning subunits from the periplasmic surface. (C) A view of the HI1470 ABC subunits from the same direction as in (B), looking down the molecular two-fold axis toward the face of the NBDs interacting with the membrane-spanning subunits. The Walker A or P-loop motif (residues 40 to 46) is colored red, the Walker B motif (residues 148 to 154) is colored yellow, and the ABC signature motif (residues 129 to 133) is colored orange. The Walker A or P-loop motif is found at the N-terminal end of helix h1 that is surrounded by the two β sheets of the catalytic core domain. Ribbon diagrams in this report were prepared and rendered with the program PyMOL (32).

Fig. 2.

Visualization of the permeation pathways of HI1470/1 and BtuCD with the program HOLE (12). (A) The permeation pathway generated by the two HI1471 subunits is narrow at the periplasmic surface and open to the cytoplasm, which are located toward the top and bottom of the figure, respectively. (B) In contrast, the pathway for BtuC is closed at the cytoplasm and open to the periplasm. The HOLE representation of the pore surface is shown in a multicolored form that was displayed and rendered with the program VMD (33). Red, green, and blue surfaces designate regions of the permeation pathway with effective radii <0.6, 0.6 to 1.15, and >1.15 Å, respectively. The calculated diameters at the widest part of the pathways illustrated for HI1470/1 and BtuCD are ∼11 and 9 Å, respectively. The permeation pathway in BtuCD is of sufficient size to accommodate a corrin ring but not the entire B12 molecule (9); the ligand for HI1470/1 has not been identified.

Each subunit of the membrane-spanning HI1471 contains 10 transmembrane helices (Fig. 1B), packed in a similar fashion to that observed for BtuC with the N and C termini located in the cytoplasm (9). Two noteworthy aspects of the rather intricate topology of the helical arrangement are the positioning of the helix TM2 through the center of the subunit (which places TM2 in proximity to most of the other membrane-spanning helices) and the similarities in helix packing between the N- and C-terminal halves of HI1471, although with opposite polarities through the membrane. This similarity in packing is particularly evident for the sets of helices (TM2 to TM5 and TM7 to TM10) that are approximately related by a two-fold axis in the plane of the membrane (Fig. 3A). Internal symmetry of this type is rather frequently observed in channels and transporters (13). For HI1471 and BtuC, this internal symmetry extends to the construction of the permeation pathway surrounding the molecular two-fold axis (Fig. 3B). Interactions between transmembrane subunits are dominated by contacts between helices TM5 and TM10 and the extramembrane helix 5a, with residues from TM3 and TM8 lining the permeation pathway. Notably, the regular helical structures of TM3 and TM8 are maintained only to about the center of the TMD and extend in a nonhelical, irregular fashion through the remainder of the membrane (9). Extended polypeptide chain conformations have been previously noted along the permeation pathways of other transporters (1418).

Fig. 3.

(A) Comparison of the homologous membrane-spanning subunits HI1471 and BtuC, after superposition of TM2 in subunit A (to the left) of each structure, as viewed down the molecular two-fold axis from the periplasm. With the exceptions of TM3 to TM5, the helices in the A subunits of HI1471 (cyan) and BtuC (purple) superimpose closely. In contrast, interconversion of the B subunits (to the right) between these two structures (blue and red, respectively) requires an ∼9° twist (indicated by the curved arrow) about an axis oriented in the direction shown to the right, which passes through the helical domain of the ABC subunit. (B) Stereoview of a superposition of helices TM3, TM4, TM5, TM8, TM10, and 5a in subunit A of HI1471 (cyan) and BtuC (purple), as viewed from within the permeation pathway with the molecular two-fold axis vertical. The internal symmetry–relating helices TM3 and TM8 and TM5 and TM10, as well as the irregular structures of TM3 and TM8, may be observed. The extramembrane helix 5a helps restrict the permeation pathway on the periplasmic side of HI1470/1.

HI1470 (Fig. 1C) exhibits the characteristic fold that was first observed for the ABC subunit HisP (19) and subsequently observed for other members of this family. ABC subunits are organized into two domains: a highly conserved catalytic core domain containing the P loop and a structurally more diverse α-helical domain with the ABC signature motif, LSGGQ. The catalytic core domain consists of two β sheets (a predominantly parallel β sheet containing the P loop and a smaller, antiparallel β sheet) that together surround an α helix (h1) extending away from the P loop. Although there are conserved elements of the α-helical domain between different ABC transporters, this region in general is more variable among members of the ABC family (20), and the relative orientation of the helical and catalytic domains is sensitive to the nucleotide state (21). As with BtuCD and the drug exporter Sav1866 (22), the region of the NBD that interacts with the TMD primarily involves the Q loop in the α-helical domain. The Q loop contains a conserved glutamine (Gln73 in HI1470) that participates in the binding of nucleotide to the NBD; the corresponding residue in BtuD (Gln80) was observed to interact with cyclotetravanadate that occupies the nucleotide-binding site. The Q loop has been observed to be conformationally variable, and changes in this region have been proposed to be involved in the coupling of nucleotide hydrolysis to the conformational state of the TMDs (23).

Although the overall architecture of the intact HI1470/1 transporter resembles that of BtuCD, more detailed comparisons highlight differences in tertiary and quaternary arrangements between structures that may be functionally relevant. Relative to a structurally conserved core of seven helices (TM1, TM2, and TM6 to TM10) that is maintained between the TMDs of HI1471 and BtuC (Fig. 3A), three helices (TM3, TM4, and TM5) differ significantly between the two structures (Fig. 3, A and B). These differences are evident in a comparison of HI1470 and BtuC subunits based on superposition of the central TM2 helix (Fig. 3A). Among the more substantial rearrangements between the two structures is a 20° shift in the helix axis of TM5 (Fig. 3B). Because TM5 participates in the subunit-subunit interface surrounding the molecular two-fold axis, these tertiary structure changes are coupled to quaternary changes in the relative positions of the TMDs, as evidenced by the alteration in the crossing angle between helices TM5 and TM10 from –143° in BtuCD to –163° in HI1470/1. When the conserved seven-helical core is used to superimpose one BtuC subunit and one HI1471 subunit, a twist of ∼9° about an axis that is approximately normal to the molecular two-fold axis is required to superimpose the partner TMD subunits (Fig. 3A). The combination of the repositioning of helices TM3 to TM5 with the overall twist motion across the subunit-subunit interface between TMDs has substantial consequences for the permeation pathway. In HI1470/1, the change in orientation of TM5 simultaneously closes access to the periplasm while opening the pathway to the cytoplasm; in contrast, cytoplasmic access to the permeation pathway is closed in BtuCD by residues in the loop between TM4 and TM5. An important additional contribution to the periplasmic restriction in HI1470/1 is provided by the extramembrane helical element 5a immediately following TM5.

The ABC subunits of both HI1470/1 and BtuCD pack together such that the P loop of one subunit opposes the signature motif of the other, in a manner originally proposed from modeling studies (24) and subsequently observed for Rad50 (25). The closed state with the most extensive interface between ABC subunits is associated with the ATP-bound form and has been structurally characterized in isolated ABC subunits (2628) and in the intact ABC transporter Sav1866 (22). Relative to this closed state, the dimers of HI1470 and BtuD that are present in the corresponding structures of the nucleotide-free transporters each exhibit more open conformations, because the catalytic domains have opened up by rotations of 20° to 25° relative to the closed dimer of MalK, the ABC subunit of the maltose transporter (27). The HI1470 dimer arrangement most closely resembles the “semi-open” (27) or post-ATP hydrolysis, adenosine diphosphate–bound (29) forms of the MalK dimer, with RMSDs of 2.7 Å between these structures for the conserved structural elements of the catalytic domains in the dimer, as compared to 4.3 Å with the closed state of MalK. The similarity of the BtuD dimer to the semi-open state of MalK has been previously noted (27), although the distinctions between BtuD and the semi-open and closed states of MalK (RMSDs of 2.3 and 2.9 Å) are not as clear for BtuD as for HI1470. Direct comparisons of the BtuD and HI1470 dimers further indicate that the latter has a more open conformation than the former. For example, the distance between the P loop and signature sequence on different subunits is greater in the HI1470 dimer than in BtuD, as is the separation between the signature sequences on different subunits in these two structures (Fig. 4A). Although there are important differences, this comparative analysis suggests that, despite the absence of nucleotides, the arrangement of ABC subunits observed in BtuCD more closely resembles the closed conformation than does HI1470/1.

Fig. 4.

Relationships between dimeric ABC structures. (A) Stereoview of the dimers of HI1470 (green), BtuD (blue; PDB 1L7V), and the ATP-bound state of MalK (orange; PDB 1Q12) based on a superposition of residues in the catalytic core domains of both subunits. At the bottom, one subunit from each structure is indicated by the appropriately colored Cα trace, whereas the trace of the second subunit from HI1470 is indicated in gray at the top. The spheres identify the P-loop and ABC sequence motifs. The closely overlapping spheres labeled with the letter “P” in each image indicate the positions of Gly43,Gly38, and Gly41 used to mark the P loops of HI1470, BtuD, and MalK, respectively; the other spheres designate the locations of Gly131, Gly129, and Gly136 denoting the ABC signature motif of these same structures. Fig. 1C highlights these same elements in the HI1470 dimer (rotated ∼60° about an axis normal to the page). Although the intersubunit spacings between the P loops within all three dimers are similar (∼35 Å; black line), the separations between signature motifs within each dimer are 16, 16, and 24 Å in MalK (orange), BtuD (teal), and HI1470 (green), respectively. The spacings between the P loop and signature motif in different subunits are 11, 14, and 16 Å in MalK, BtuD, and HI1470, respectively. The rotation axes relating the catalytic domains of corresponding BtuD and HI1470 subunits pass near the P loops of these structures and are illustrated as black lines tilted ∼13° from the normal to the viewing direction. The locations of these axes are reflected in the similar distances between P loops in the three dimeric ABC structures, whereas separations involving the signature motifs vary more widely because these regions are farther from the rotation axes. (B) Comparison of the ABC dimers of HI1470/1 (green), BtuD (blue), and ATP-bound MalK (orange) as viewed from the membrane in the same orientation as in (A). The subunits are superimposed onto the conserved regions of the catalytic core domain of one subunit (chain C) of HI1470 (top subunit). The P loops and h1 helices are depicted as ribbons, the signature sequence and associated helix are depicted as a Cα trace, and the main chain atoms in the Q loop are depicted as thick bonds. Although these elements overlap in the top (superimposed) subunits, substantial variation is evident in the lower subunit, particularly the translational shift along the dimer interface between the Q loops and signature motifs of the intact HI1470 and BtuD (blue arrow). In comparison, these elements in MalK are rotated about a hinge axis in a tweezers-type motion (27) to close up the interface relative to BtuD (yellow arrow). (C) A schematic representation illustrating how rotations about local axes in each subunit, parallel to the molecular two-fold axis of the dimer, create a translational shift along the dimer interface. These rotations can consequently be coupled to a twisting motion of the associated membrane-spanning subunits to interconvert inward- and outward-facing conformations.

Although a detailed mechanistic description will clearly require biochemical and structural characterization of multiple states of an ABC transporter system (including bound nucleotides, substrate, and binding protein), the observation that the permeation pathways of HI1470/1 and BtuCD are oriented in opposite directions can help identify structural elements underlying this transition. The conformational transformations relating HI1470/1 and BtuCD, although maintaining the overall two-fold molecular symmetry, do not exclusively involve rigid body movements of individual subunits (Fig. 3A). Still, the rigid body description provides a useful reference framework for this analysis; for example, when the entire HI1470/1 and BtuCD transporters are superimposed so that the two-fold axes coincide, the transformation that is calculated for the catalytic core domain of individual ABC subunits between the two structures corresponds to a rotation of ∼10° about an axis tilted 13° from the molecular two-fold axis. The rotation axis corresponding to this transformation passes near the P loop (Fig. 4A), with the consequence that the structural adjustments are relatively modest in the catalytic core domain between structures but increase with increasing distance from this region, as is particularly evident for the ABC signature sequence. When the catalytic domains of one NBD in the intact HI1470/1 transporter and one NBD in the intact BtuCD transporter are superimposed, the relative positions of the partner NBDs observed in these structures are shifted by a translation or screw component of ∼4.5 Å along an axis parallel to the interface between NBDs (Fig. 4B); this translational component repositions the two NBDs in a direction perpendicular to that generated by the tweezers-type motion observed between different nucleotide states of MalK (27), which also corresponds to the hinge motion between BtuD and the closed form of MalK. Notably, the direction of this translational shift coincides with the direction of the twist motion observed between the TMDs of HI1470/1 and BtuCD (Fig. 3A). This screw component arises from the coupling of the local rotation axes relating individual NBDs in different structures to the molecular two-fold rotation, which generates a displacement along the subunit-subunit interface as the separation between NBDs varies (Fig. 4C). The linkage between NBD positioning and the twist between TMDs supports a coupling mechanism connecting the permeation pathway and nucleotide state of the transporter, where the ABCs can remain juxtaposed during the transport cycle.

The structures of HI1470/1 and BtuCD demonstrate that inward- and outward-facing conformations of an importer-type ABC transporter may be accommodated with relatively little change in overall architecture. Because neither HI1470/1 nor BtuCD were crystallized in the presence of nucleotide, binding protein, or ligand, the energetic basis of the differential stabilization of alternate conformations is not obvious; one possibility is that the substitution of the native bilayer with detergent has shifted the equilibrium between inward- and outward-facing conformations. A comparable phenomenon has recently been discussed for the conformation of the voltage sensor in potassium channels (30). Lattice contacts overlapping the molecular two-fold axis of HI1470 and the periphery of BtuD could also play a role in stabilizing the observed conformations of the ABCs. Consequently, despite the differences in structures of HI1470/1 and BtuCD, it is not possible to establish the correspondence between nucleotide state and transporter conformation with certainty; however, the closer juxtaposition of ABC subunits in BtuCD relative to HI1470/1 suggests that the outward-facing conformation of the transporter corresponds to the closed (ATP) state of the NBDs, as suggested by Chen and Davidson (27) and as observed for Sav1866 by Dawson and Locher (22). A notable aspect of the switch in translocation pathways between inward- and outward-facing conformation is the packing rearrangement of helices TM3 to TM5 with respect to the remainder of the TMD. In view of the internal duplication evident in the helix packing arrangements of HI1470/1 and BtuCD, as well as other channels and transporters (13), this suggests the possibility that the internal symmetry is inherent in the mechanistic transition between inward- and outward-facing conformations. The roles of binding protein, ligand, and particularly nucleotide binding and hydrolysis in driving these conformational transitions remain crucial mechanistic issues.

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