Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

Abstract

25 proteins, solute translocation, substrate transport 26 27 2 ABSTRACT 28 Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch 29from an open-to a closed-conformation upon substrate binding providing specificity for transport. We 30 investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on 31 transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We 32 show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs 33 sample a range of conformations that activate transport. Certain non-transported ligands leave the 34 structure largely unaltered or trigger a conformation distinct from that of transported substrates. 35Intriguingly, in some cases similar SBP conformations are formed by both transported and non-36 transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the 37 selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP 38 interactions, SBP conformational dynamics and substrate transport. 39 3 INTRODUCTION 40 ATP-binding cassette (ABC) transporters facilitate the unidirectional trans-bilayer movement of a 41 diverse array of molecules using the energy released from ATP hydrolysis 1 . ABC transporters share a 42 common architecture, with the translocator unit comprising two transmembrane domains (TMDs) that 43 form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that bind 44 and hydrolyse ATP. ABC importers require an additional extra-cytoplasmic accessory protein referred 45 to as a substrate-binding protein SBP or domain SBD (hereafter SBDs and SBPs are both termed 46 SBPs) 2-4 . ABC importers that employ SBPs can be subdivided as Type I or Type II based on structural 47 and mechanistic distinctions 5, 6 . A unifying feature of the transport mechanism of Type I and Type II 48 ABC importers is the binding and delivery of substrate from a dedicated SBP to the translocator unit 49 for import into the cytoplasm. 50 Bacterial genomes encode multiple distinct ABC importers to facilitate the acquisition of 51 essential nutrients such as sugars, amino acids, vitamins, compatible solutes, and metal ions 1, 7 . Many 52 ABC importers can transport more than one substrate using high-affinity interactions between SBPs 53 and transported ligands (herein termed cognate substrates) 2 . Despite low sequence similarity between 54 SBPs of different ABC importers, they share a common architecture comprising two structurally 55 conserved rigid lobes connected by a flexible hinge region (Figure 1) 2 . Numerous biophysical 8 and 56 structural analyses 9 indicate that ligand binding at the interface of the two lobes facilitates switching 57 between two conformations, i.e. from an open to a closed conformation. Bending and unbending of the 58 hinge region brings the two lobes together (closed conformation) or apart (open conformation), 59 respec...

Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

“…Sample preparation and labelling of proteins. MalE single and double cysteine variants were obtained and fluorophore-labelled as described previously 69,70 . The cysteine positions for fluorophore attachment were chosen based on the open and closed x-ray crystal structures of MalE (1OMP, 1ANF, respectively).…”

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series

“…MalE single and double cysteine variants were obtained and fluorophore-labelled as described previously. [72,73] The cysteine positions for fluorophore attachment were chosen based on the open and closed x-ray crystal structures of MalE (1OMP, 1ANF, respectively). The double cysteine variants were (i) stochastically labelled with the maleimide derivative of the dyes Alexa Fluor 555 and Alexa Fluor 647 (ThermoFischer Scientific, A20346 & A20347), and AF555, AFD647 & AF647 (Jena Bioscience, APC-007, APC-009 and APC-009) for smFRET experiments.…”