Structure and mechanism of the S component of a bacterial ECF transporter

Abstract

The energy-coupling factor (ECF) transporters, responsible for vitamin uptake in prokaryotes, are a unique family of membrane transporters. Each ECF transporter contains a membrane-embedded, substrate-binding protein (known as the S component), an energy-coupling module that comprises two ATP-binding proteins (known as the A and A' components) and a transmembrane protein (known as the T component). The structure and transport mechanism of the ECF family remain unknown. Here we report the crystal structure of RibU, the S component of the ECF-type riboflavin transporter from Staphylococcus aureus at 3.6-Å resolution. RibU contains six transmembrane segments, adopts a previously unreported transporter fold and contains a riboflavin molecule bound to the L1 loop and the periplasmic portion of transmembrane segments 4-6. Structural analysis reveals the essential ligand-binding residues, identifies the putative transport path and, with sequence alignment, uncovers conserved structural features and suggests potential mechanisms of action among the ECF transporters.

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 .…”

“…Unexpectedly, the orientation of the S components FolT and HmpT in the complete ECF transporters is entirely different, even though their global folds are the same as RibU, ThiT and BioY [21][22][23][24][25] (FIG. 2c).…”

“…The structures of the isolated S components BioY, RibU and ThiT indicate that substrate binding takes place at a site that is close to the extracellular side of the membrane [23][24][25] (FIG. 2b), and the loop that connects membrane-spanning segments 1 and 2 (known as Loop 1-2) functions as a gate that controls access 39 .…”

“…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).…”