The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter

Maria E Zoghbi,Rebecca S Cooper,Guillermo A Altenberg

doi:10.1074/jbc.m115.698498

Abstract

ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward- and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37 °C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Highlights

ATP-binding cassette (ABC)2 transporters constitute one of the largest families of membrane proteins and are found in all domains of life [1,2,3]
MsbA Reconstitution and ATPase Activity—To study the functioning of an ABC transporter in a lipid bilayer and at physiological temperature, we reconstituted detergent-solubilized MsbA in nanodiscs, self-assembled complexes that consist of a lipid bilayer encased within a membrane scaffold protein (MSP) [27]
In an attempt to study the structure and activity of a membrane protein in native-like conditions, we have used LRET to measure distance changes between the nucleotide-binding domains (NBDs) of the ABC transporter MsbA incorporated into a nanodisc lipid bilayer at 37 °C

Summary

Experimental Procedures

Protein Expression and Purification—A Cys-less Salmonella typhimurium MsbA with an N-terminal decahistidine tag (MsbA CL, the designation for MsbA with mutations C88A and C315A) was used as background to generate the single Cys mutant T561C. For experiments of MsbA in detergent micelles, the labeled protein was run through a Superdex 200 10/300 gel filtration column (GE Healthcare) equilibrated with 100 mM NaCl, 20 mM Tris/HCl, pH 7.5, 0.065% dodecylmaltoside, 0.04% sodium cholate, and 0.2 mM TCEP) to remove free unreacted LRET probes. For experiments of MsbA in a lipid bilayer the free unreacted LRET probes were removed after labeled MsbA had been reconstituted, as described below. The sample was run through a Superdex 200 10/300 gel filtration column (GE Healthcare) equilibrated in nanodisc buffer to remove free unreacted LRET probes and to collect the fraction enriched in MsbA-containing nanodiscs that was used for the experiments. Control experiments mixing equal proportions of nanodiscs containing MsbA labeled with Tb3ϩ-only and Bodipy FL-only showed essentially not intermolecular LRET. The number of experiments (n) corresponds to independent measurements from at least three different protein preparations

Results

93 Ϯ 1 37 Ϯ 4 59 Ϯ 7 39 Ϯ 3

Discussion