The ABC exporter MsbA probed by solid state NMR – challenges and opportunities

Abstract

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.

Structural basis of MsbA-mediated lipopolysaccharide transport

“…7). This is consistent with a previous finding that LPS co-purifies with MsbA when E. coli membranes are solubilized in DDM for only 1 hour 31 , similar to what we did. To confirm the identity of the assigned LPS density, we purified MsbA from a genetically modified E. coli strain, ClearColi ™ , which only produces lipid IV A , a precursor of LPS with four acyl chains and no glycosylation.…”

Coupled ATPase-adenylate kinase activity in ABC transporters

“…Expression and purification was essentially carried out as described before38. MsbA was cloned into a pET-19b vector containing an N-terminal His 10 -tag connected via an 11 amino acid peptide linker65.…”

“…The dried lipids were resuspended in buffer (50 mM HEPES and 50 mM NaCl, pH 7) and extruded 5 times through membranes with pore diameters of 0.2 μm and 8 times with pore diameters of 0.1 μm. Liposomes were destabilized with 3 mM DDM38. After drop-wise addition of protein in the DDM/liposome solution, the mixture was incubated at room temperature for 30 min.…”

“…Evidence is provided by solid-state NMR based on magic-angle sample spinning (MAS) that allows the structural and mechanistic analysis of full-length membrane proteins directly within proteoliposomes near physiological conditions37. In our previous work, we demonstrated that MsbA is well suited for in-depth MAS-NMR analysis based on 13 C/ 15 N spectra as it shows long-term stability and can be isotope labelled and reconstituted into various lipid bilayers38. Here, for probing its catalytic activity within the lipid bilayer, we utilize real-time 31 P-MAS NMR to monitor substrate turnover by recording progress curves for all compounds involved.…”

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

“…Solid-state NMR is an emerging method to investigate the structure of membrane proteins in a native-like lipid environment (for recent contributions see for example (McDermott 2009;Renault et al 2010;Kaur et al 2015;Hansen et al 2015;Shahid et al 2015;Mehler et al 2015;Mandal et al 2015;Fu et al 2015)). Still, as for other techniques in structure biology, sample preparation of membrane proteins remains challenging, as it is often difficult to express the proteins in a well-folded form, with the isotopic labeling and in the milligram amounts needed for NMR spectroscopy.…”