Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs

Abstract

Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier" nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.

Omnipresent Maxwell's demons orchestrate information management in living cells

“…At this stage, the protein's conformational changes will lower the ATP hydrolysis activation barrier, preparing for the hydrolysis step that will occur later on. Subsequently (possibly simultaneously in some cases), ATP hydrolysis is coupled to mechanical changes, exposing hydrophobic residues to water (or hydrophilic residues in the lipid bilayer, for membrane lipid transporters) and opening up the transporter into the substrate release compartment (Josts et al, 2018). As a consequence, the substrate goes to its relevant compartment, while the transporter's conformation is relaxed, and then reset to its original open state.…”

Toward a structome of Acinetobacter baumannii drug targets

“…However, some slow growing gram‐negative bacteria like Neisseria . meningitidis retain cell viability without expression of this gene . The most similar human protein is the mitochondrial human ABC transporter, with 35.3% amino acid identity.…”

“…meningitidis retain cell viability without expression of this gene. 62,64,65 The most similar human protein is the mitochondrial human ABC transporter, with 35.3% amino acid identity. Comparison of the active site of A. baumannii MsbA and the human ABC transporter shows 87.5% active site amino acid similarity.…”

Elucidating the Structure of Membrane Proteins

“…The technique was first published in 2014 [5]; however, the first structural study of a membrane protein utilizing this method has onlybeen described in 2018 [6]. …”