Abstract
ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.
Highlights
ABC transporters are essential membrane proteins found in both eukaryotic and bacterial cells that facilitate the uphill transport of ions and chemically diverse compounds in an ATP-dependent manner (Holland et al, 2003)
All ABC transporters share a common architecture consisting of a transmembrane domain (TMD) for substrate recognition and transport, and a nucleotide-binding domain (NBD) that converts the chemical energy of ATP into conformational changes for transport (Beis, 2015)
We demonstrate that the NBDs have intrinsic conformational dynamics in their apo state on the 100 ms timescale, while the TMDs remain static
Summary
ABC transporters are essential membrane proteins found in both eukaryotic and bacterial cells that facilitate the uphill transport of ions and chemically diverse compounds in an ATP-dependent manner (Holland et al, 2003). The structures of several homodimeric (Dawson & Locher, 2006; Ward et al, 2007; Perez et al, 2015) and heterodimeric ABC transporters (Hohl et al, 2012; Noll et al, 2017) revealed distinct conformations and suggest, in combination with biophysical studies (e.g., EPR and NMR; Dong et al, 2005; Zou et al, 2009; Bountra et al, 2017; Timachi et al, 2017; Barth et al, 2018), that they undergo large conformational changes during transport Their complex architecture is, a fundamental hurdle to fully understand the coupling between conformational changes, substrate binding, ATP binding and hydrolysis, and transport. Such detailed mechanistic models of transport would require the knowledge of conformational states from static snapshots, and their interconversion dynamics
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