Substrate-Induced Conformational Changes in the S-Component ThiT from an Energy Coupling Factor Transporter

Abstract

Energy coupling factor (ECF) transporters are a recently discovered class of ABC transporters that mediate vitamin uptake in prokaryotes. Characteristic for ECF-type ABC transporters are small integral membrane proteins (S-components) that bind the transported substrates with high affinity. S-components associate with a second membrane protein (EcfT) and two peripheral ATPases to form a complete ATP-dependent transporter. Here, we have used EPR spectroscopy, stopped-flow fluorescence spectroscopy, and molecular dynamics simulations to determine the structural rearrangements that take place in the S-component ThiT from Lactococcus lactis upon binding of thiamin. Thiamin-induced conformational changes were confined to the long and partially membrane-embedded loop between transmembrane helices 1 and 2 that acts as a lid to occlude the binding site. The results indicate that solitary ThiT functions as a bona fide high-affinity substrate binding protein, which lacks a translocation pathway within the protein.

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…Molecular dynamics simulations of the solitary S component for thiamin ThiT, of which a crystal structure had been determined before the structures of the complete ECF transporters, are consistent with these generic features of membrane proteins 25,39 (FIG. 2b).…”

“…Spectroscopic techniques, such as electron paramagnetic resonance (EPR) 39,54 and Förster resonance energy transfer (FRET) 55 , can be used to assay the dynamics of proteins during catalysis and seem to be suitable for studying the proposed toppling of the S component during the translocation cycle. In addition, crystal structures of ECF complexes in the presence of nucleotides and transported substrates, are likely to provide further mechanistic insight, and may reveal, for example, whether the proposed toppling event is specifically coupled to ATP binding or hydrolysis.…”

“…3a), the substrate binds to the transporter at a site that is located roughly at the centre of the membrane (midway between the extracellular and cytosolic surfaces of the bilayer), which it reaches by diffusing through an aqueous cavity in the protein that is only accessible from one side of the membrane 39 and shows that the substrate-binding site is located close to the extracellular side of the membrane, whereas in the ECF-FolT complex, this site is accessible from the cytosolic side of the membrane. The loop between transmembrane helices 1 and 2 in ThiT (loop 1-2) functions as a gate over the substrate-binding site.…”

“…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 . When this gate is closed, the substrate-binding site is occluded.…”

Structural basis for a homodimeric ATPase subunit of an ECF transporter

“…Similar to other typical ABC transporters, the new class of transporters contains two homologous or identical ATPases (A component), which associate with the transmembrane domains (T components), forming an ECF module (Zhang et al, 2010;. In contrast with the canonical ABC transporters that employ soluble substrate binding proteins (SBPs), membrane proteins in ECF transporters is exclusively responsible for substrate binding (therefore termed S component) (Berntsson et al, 2012;Majsnerowska et al, 2013). An ECF module can be shared by highly diverse S components, thus allowing the transport of chemically different substrates via the same complex (Fisher et al, 2012).…”