The Molecular Simulation Group

Structural Changes in Substrate-Binding Component from ECF-Type ABC Transporter

ATP-binding cassette (ABC) transporters of the energy coupling factor (ECF) type are found exclusively in prokaryotes, where they mediate the uptake of vitamins and other trace elements that are vital to the bugs, including numerous human pathogens such as, e.g., Listeria monocytogenes. Since they have no eukaryotic counterparts, they are promising antibiotic targets. This, however, requires a thorough structural and mechanistic understanding.

ECF transporters are membrane proteins that comprise of several components: 1) the energizing module, a soluble dimeric ATPase domain that provides the (free) energy for vitamin uptake through ATP hydrolysis; 2) the S-component, an integral membrane protein that binds the substrate (e.g. the vitamin molecule); and 3) the EcfT component, another integral membrane protein that binds to the S-component. It has been speculated that the S-components can not only bind the substrate, but actually function as transporters themselves. However, since no obvious translocation pathway could be identified in the available high-resolution structures, this remained elusive.

Snapshot from all-atom MD simulation of ThiT in a POPC lipid bilayer. Thiamin (vitamin B1) substrate is shown in yellow, partly covered by the long loop that occludes the substrate binding site. The overall simulation system comprises of about 70.000 atoms in the periodic box.

To address this question, we have studied the S-component ThiT from the thiamin (vitamin B1) uptake system by a combination of all-atom MD simulations, EPR, and fluorescence spectroscopy (carried out in the groups of Dirk Jan Slotboom at the University of Groningen and Heinz-Jürgen Steinhoff at the University of Osnabrück). The simulations, performed in the presence and absence of a vitamin B1 molecule, respectively, guided the atomistic interpretation of the spectroscopic measurements and revealed that substrate-induced structural changes are restricted to a long, partially membrane-embedded loop that in the substrate-bound form occludes the binding pocket like a lid. No large-scale structural transitions required for substrate transport through the membrane were observed. Taken together, these results indicate that solitary ThiT (and presumably also other S-components of similar structure) are high-affinity substrate binding proteins that lack a translocation pathway within the protein. Instead, the substrates can be transported at the interface between the S-component and the EcfT-component. This conjecture is supported by high-resolution X-ray crystal structures published later.

Reference: M. Majsnerowska, I. Hänelt, D. Wunnicke, L. V. Schäfer, H.-J. Steinhoff, D. J. Slotboom. Substrate-induced Conformational Changes in the S-component ThiT from an Energy Coupling Factor Transporter, Structure, 2013, 21 (5), 861-867. (doi)