Exploring conformational equilibria of a heterodimeric ABC transporter.

M Hadi Timachi,Markus A Seeger,Anshumali Mittal,Tufa Assafa,Enrica Bordignon,Simon Böhm,Cedric Aj Hutter,Michael Hohl

doi:10.7554/elife.20236

M Hadi Timachi, Markus A Seeger + Show 6 more

Open Access

https://doi.org/10.7554/elife.20236

Copy DOI

Abstract

ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.

Highlights

ABC transporters are divided into importers, found exclusively in bacteria, and exporters, present in all phyla of life (Davidson et al, 2008)
Simulations performed with a rotamer library of spin-labeled side chains available in the software MMM (Polyhach et al, 2011) using the apo structure of TM287/288 and a homology model based on Sav1866 indicated that the six pairs allow monitoring of the conformational changes propagated from the nucleotide binding domains (NBDs) to the transmembrane domains (TMDs) (Figure 1 and Figure 1—figure supplement 1)
The maximal ATPase stimulation was less pronounced for the extracellular pairs and for the intracellular pair 131TM288/248TM288, indicating that spin-labeling at these positions changes the properties of TM287/288 in terms of drug binding or communication between the TMDs and the NBDs underlying stimulated ATPase activity in the presence of drugs

Summary

Introduction

ABC transporters are divided into importers, found exclusively in bacteria, and exporters, present in all phyla of life (Davidson et al, 2008). Transport processes mediated by more than forty human ABC exporters fulfil vital functions in our body as they translocate an extraordinarily wide range of cargoes such as lipids, peptides, ions and drugs across lipid bilayers. The same transporters have received increasing attention in past years, playing a central role in the absorption, distribution, metabolism and elimination of pharmaceuticals in the human body (Nigam, 2015). ABC exporters play a major physiological role in the transport of metabolites such as urate, glucuronides and N-lactoyl-amino acids (Jansen et al, 2015; Krumpochova et al, 2012; Woodward et al, 2009)

Methods

Results

Discussion

Conclusion