ABC transporters: one, two or four extracytoplasmic substrate-binding sites?

Abstract

Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator.

Figure 1

Schematic representation of the architecture of the substrate-binding and translocator moieties of ABC transporters. Cylinders depict transmembrane segments (TMSs). (A) Previously characterized ABC transporter types with independently encoded SBPs. (B–D) Newly discovered chimeric transporters characteristic of the PAO (C) or OTCN (B and D) subfamilies. Chimeric proteins with the substrate-binding domain(s) at the C-terminus (B and D) have an additional TMS (signal anchor), denoted here as VIII. The signal sequence of the chimera with the substrate-binding domain at the N-terminus could potentially be cleaved off as depicted in (C). Since the first two TMSs of OTCN family members are not always present, they are indicated in light grey (A, B and D). The position of the signature motif (EAA) within the different ABC translocator proteins is also depicted. (E) Alignment of the C-terminal signal anchors, which are predicted by the SignalP program (http://www.cbs.dtu.dk/services/SignalP-2.0/); the n-, h- and c-regions characteristic of signal (anchor) sequences are indicated.

Figure 2

Phylogenetic tree of the translocator domains of the OTCN and PAO family members. OpuAB_Bsu, OpuCB_Bsu, OpuCB_Lmo, ProW_Eco, GbuB_Lmo (OTCN) and GlnP_Eco (PAO) are translocators without a substrate-binding domain. The others are the newly-identified chimeric substrate-binding/translocator proteins of which a detailed description is presented in Table 1 (Supplementary data). The programs ClustalX (1.8) and Treeview (1.6.6) were used for sequence alignment and generation of the phylogenetic tree, respectively. The horizontal bar indicates the number of amino acid substitutions per site.

Figure 3

Schematic representation of the domain organization of ABC transporters. A SBP-independent efflux system is shown in (A), and a conventional SBP-dependent uptake system is shown in (B). The newly identified chimeric substrate-binding/translocator systems with two and four substrate-binding sites per functional complex are shown in (C and D), respectively. ATP-binding domains (ABC cassettes) are shown in orange.