The ATP-binding cassette family: a structural perspective

Abstract

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes, using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes, these transporters reside in the cell's internal membranes as well as in the plasma membrane and are unidirectional-out of the cytoplasm. The range of substances that these proteins can transport is huge, which makes them interesting for structure-function studies. Moreover, their abundance in nature has made them targets for structural proteomics consortia. There are eight independent structures for ATP-binding cassette transporters, making this one of the best characterised membrane protein families. Our understanding of the mechanism of transport across membranes and membrane protein structure in general has been enhanced by recent developments for this family.

Fig. 1

Topology of ABC transporters, which consist of two transmembrane domains (TMDs), typically with six transmembrane spans per domain, and two cytoplasmic nucleotide-binding domains (NBDs) which catalyse nucleotide hydrolysis. The figure shows a version of this arrangement where all four domains are fused together as a single polypeptide, which is typical for eukaryotic ABC proteins. However, apart from the basic requirement for two TMDs and two NBDs, almost any arrangement of these can be found. In bacteria, some ABC transporters are encoded as a single polypeptide, where a TMD and an NBD are fused together, and a homodimer of the protein is the active form. In other cases, bacterial ABC transporters are encoded by two polypeptides—the TMD and NBD subunits—and a homodimer of heterodimers is formed. More complex versions also exist where either the TMD or NBD subunits are non-identical, i.e. three separate polypeptides are expressed to form the active transporter. Similarly, fused TMDs and NBDs are also possible. The sequence of N-terminus–TMD–NBD–C-terminus is the most common for transporters with fused TMD and NBD, but in some cases the sequence is reversed. The topology appears to be universally held, however, with the NBDs located in the cytoplasm. The membrane in which the ABC proteins are found is usually the plasma membrane (inner membrane in bacteria), but in eukaryotes some ABC transporters are found in organellar membranes, in which case the NBDs are still situated on the cytoplasmic side of the organelle membrane. Finally, ABC proteins have evolved with various additions and adaptations. Bacterial transporters in this family often have regulatory domains associated with the NBDs, whilst the TMDs may have several additional transmembrane spans. Fewer than six spans are also predicted for some prokaryotic TMDs, but so far no structures of this type have been solved with fewer than five transmembrane spans per TMD

Fig. 2

Family portrait: structures of ABC transporters. a Sav1866 homodimer [27]. b MalFGK₂ with the periplasmic maltose-binding protein (cyan) [10]. c ModBC, with the periplasmic-binding protein ModA (purple) [28]. d BtuCD [11]. e Putative metal chelate transporter H10796 [12]. f MetNI methionine transporter [29]. g–i MsbA from S. typhimurium, V. cholera and E. coli. respectively [43]. j Murine P-glycoprotein a.k.a. ABCB1 [30]. The approximate location of the lipid bilayer is indicated by the pink band, as estimated by the boundaries of the outward-facing hydrophobic amino acids. Structures a and g–j are thought to be exporters, b–g are thought to be importers. a and g–i are homodimers with one TMD and one NBD fused together. Structures b–e consist of four polypeptide chains, forming the basic domain structure, whilst some have an accessory periplamic substrate-binding protein (b, c). In j, all the domains are fused as a single polypeptide, with the N-terminal half coloured green. The nucleotide-binding domain structures are strongly conserved (with the exception of h and i). Some structures (b, c, f) have an additional extension of the NBD, probably involved in regulation of transport activity. In contrast, the transmembrane domain structures are variable. At least three separate folds are apparent, and primary structure is hardly conserved at all. The unusual structure shown in i displays limited contacts between the two halves of the unit, which may be a product of the purification or crystallisation process. Some of these structures were determined in the presence of ATP (or another nucleotide), some in the absence of nucleotide. However, only two structures so far (b, j) show the allocrite (transported molecule) bound. All are ‘static’ structures that may need to be considered together to gain insights into the transport mechanism

Fig. 3

Conserved regions of the nucleotide-binding domains viewed from the transmembrane region (top) or from the side (parallel to the membrane plane). The signature region (orange atoms) and the Walker A and B regions (yellow and red atoms) all form the ATP-binding pockets, whilst the Q-loop (green atoms), D-loop (purple atoms) and H-loop (blue atoms) also contribute to the binding of two ATP molecules. Residues around the Q-loop may be involved in transmission of conformational changes to and from loops extending down from the TMDs and are placed appropriately for this interaction (top view). The BtuD NBD dimer is shown [11]

Fig. 4

Secreted substrates: buried co-factors and allocrites in ABC transporters. a The Sav1866 transporter (blue and yellow atoms representing the two polypeptides) in the presence of nucleotide (green atoms, AMP-PNP). The nucleotide-binding site is almost buried in the structure, as revealed when the front half of the structure is removed [91]. There is no pathway large enough to allow entrance/exit of nucleotide without significant conformational movements allowing the opening up of the NBD dimer. Note how the two polypeptides in Sav1866 both contribute to each nucleotide-binding pocket, a characteristic feature of the nucleotide-binding domains of ABC proteins. b The maltose transporter (MalFGK2) is the sole ABC protein structure where maltose, the allocrite (red atoms), is present. The maltose-binding pocket in the transmembrane region, formed by the F (blue) and G (yellow) TMD polypeptides, is completely occluded by the periplasmic maltose-binding protein (MBP, orange), and there is no obvious pathway to the cytoplasmic region, as revealed when the front half of the structure is removed [91]. In this view of the MalK dimer, there is a glimpse of the ATP-binding pocket from the outside, but this window is too narrow to allow exit/entrance of co-factor. The impression (perhaps false, see Table 1) gained from such images is of a very tight dimer of NBDs that concertedly bind the nucleotides

Fig. 5

Dance of the TMDs. Comparison of the transmembrane regions of the MalFGK2 (left) and the ModBC [91] transporters, which share the same basic topological fold. Alignment of MalG (grey) and ModB (black) using the ‘EAA’ loop (dashed ellipse) which connects to the NBDs shows that the ModBC structure is more open to the cytoplasmic side due to an increased tilting of two alpha helices indicated by the white arrows which splay out the TMDs. Since the MalFGK2 structure is for the nucleotide-bound state, whilst the ModBC structure lacks nucleotide, this has led to the suggestion [92] that these structures could represent outward and inward facing conformations, i.e. snapshots of crucial stages in the transport cycle of ABC transporters [57]

Fig. 6

Working model: schematic representation of the inward- and outward-facing conformations proposed for the transport mechanism of ABC proteins. The left panel presents the inward-facing conformation, typified by the ModABC structure. The NBDs (red) have moved apart in the nucleotide-free state, and the TMDs (blue) are open to the cytoplasm. The right panel shows the outward-facing configuration typified by the structure of MalFGK2. The NBDs have moved together and concertedly bind ATP (black pentagons). The TMDs are more open to the extracellular medium, and a channel communicating between the periplasmic-binding protein (MBP) and the TMDs is partly occupied by the transported allocrite (maltose, hexagon)

Fig. 7

Family resemblances. Green netting shows the Coulomb density map of the eukaryotic ABC transporter P-glycoprotein (ABCB1) obtained in the presence of nucleotide using ~8-Å resolution electron crystallography data which have recently been combined with small-angle X-ray scattering data [50, 55]. The front of the map is removed in the right panel, and the netting has been made semi-transparent, allowing the interior and back of the map to be observed. When compared with the closest current homologous structure—that of Sav1866 ([27], yellow ribbon trace)—there is a striking similarity in overall shape and size as well as in the opposing tilts of the helices on the front and back sides of the map. The interior view (right panel) also shows some density matching with the intracytoplasmic loop II of Sav1866 that crosses over from one TMD to the opposing NBD (as indicated by the white dashed ellipse). These structural data therefore imply that ABCB1 probably possesses a similar overall architecture to the Sav1866 bacterial ABC exporter with ‘domain-swapping’ within the TMDs and NBDs. Tracing the polypeptide chain is not possible at the current resolution of the ABCB1 map

Fig. 8

Dance of the TMDs, part 2. Conformational changes associated with allocrite-binding and nucleotide-binding, as suggested by comparison of the structures of ABCB1 (P-glycoprotein) in the absence of nucleotide and Sav1866 in the presence of nucleotide [91]. One half of each transporter is highlighted in black for clarity. The positions and identities of the intracytoplasmic loops of the TMDs are indicated by the dashed ellipses and numbers. The approximate orientations of transmembrane helices 4 and 5 (TM4, TM5) versus the rest of the TMD in each transporter are indicated by the double arrows. As in Fig. 5, nucleotide binding is apparently giving rise to a change in the angle between transmembrane α-helices in the TMD. TM helices 4 and 5, which are linked via intracytoplasmic loop 2, subtend a wider angle with the rest of the TMD in the nucleotide-free state. Because the intracytoplasmic loop 2 crosses over to connect to the opposing NBD, the change in angle of the transmembrane helices readily suggests a mechanism for controlling the formation of the sandwich dimer of NBDs as well as linking this to the generation of an outward-facing conformation of the transporter

Fig. 9

Mind the gap. Structures of Sav1866 (left) and MetNI (right) [91] using space-filling representations for the non-hydrogen protein atoms. Hydrophobic amino acid residues are coloured white and polar residues are coloured black. A clear delineation between the transmembrane region and the extramembraneous regions is evident for each protein. A ‘V’-shaped gap extending towards the putative lipid bilayer (grey region) is present for each protein (arrows). For Sav1866, the gap would contact the outer leaflet of the lipid bilayer, whilst for MetNI [91] it would be the inner leaflet