Structural divergence of paralogous S components from ECF-type ABC transporters

Abstract

Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiaminspecific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd ¼ 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains how the S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane. membrane transport | biotin transport | vitamine uptake E nergy coupling factor (ECF) proteins are an abundant class of ATP-binding cassette (ABC) transporters involved in the import of vitamins and transition metal ions in prokaryotes (1-4). Like all ABC transporters, ECF transporters consist of two cytosolic nucleotide-binding domains (NBDs), which are associated with integral membrane subunits that form the translocation pore. In ECF transporters the two NBDs (EcfA and EcfA', which may be identical or homologous) and a single membrane subunit (EcfT) form a so-called energizing or ECF module. A second integral membrane protein (the S component) binds the substrate and forms a complex with the ECF module to create a functional transporter. This organization is typical for ECF transporters (3-5), because other ABC importers utilize a soluble substrate-binding protein to capture ligands (6, 7). In many ECF transporters multiple S components (specific for different substrates) can interact with the same energizing module (3, 5). Strikingly, S components from a single organism, which interact with the same ECF module, are generally not homologous at the sequence level.To gain insight in the characteristic modularity of ECF transporters, one needs to compare crystal structures of different S components that interact with the same ECF module (i.e., S components from a single organism). Crystal structures of the S components ThiT from Lactococcus lactis (thiamin-specific) and RibU from Staphylococcus aureus (riboflavin-spe...

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…Both of these metal-binding S components seem to require auxiliary small integral membrane proteins for substrate binding -CbiN for CbiM, and either NikN or NikKL (which is a complex of two proteins) for NikM 6,69,70 -but the exact role of these additional proteins remains to be elucidated. CbiM and NikM have seven predicted membrane-spanning α-helices, in contrast to the common six helices of other S components [23][24][25] . Whether CbiM and NikM share the conserved structural core of the other S components is not clear.…”

“…By contrast, the two membrane proteins in ECF transporters (the S component and EcfT) are unrelated; thus, these proteins have an asymmetrical conformation. to have the same orientation in the membrane as ThiT 23,24 .…”

“…So far, 21 different S component families have been identified, each of which is specific for a distinct substrate 3,4,6 (TABLE 1). Although S components for different substrates lack a high degree of sequence similarity (they have on average 10-20% sequence identity), crystal structures of isolated S components have shown that their global structures are conserved [23][24][25] . It is likely that this fold is shared by all S components (with the possible exception of the S components for Co 2+ and Ni 2+ ; BOX 1).…”

“…It is likely that this fold is shared by all S components (with the possible exception of the S components for Co 2+ and Ni 2+ ; BOX 1). A conserved functional feature of S components is their high affinity for substrates, with dissociation constants in the low to sub-nanomolar range 9,11,24,26 .…”

“…Whether such transport is physiologically relevant is unclear as all group I transporters have a dedicated ECF module. In addition, although the R. capsulatus BioY transporter was shown to be active in recombinant Escherichia coli cells, it has not been possible to confirm this transport activity using the purified protein reconstituted in liposomes 24 .…”