Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study

Abstract

ABC transporters are fascinating examples of fine-tuned molecular machines that use the energy from ATP hydrolysis to translocate a multitude of substrates across biological membranes. While structural details have emerged on many members of this large protein superfamily, a number of functional details are still under debate. High resolution structures yield valuable insights into protein function, but it is the combination of structural, functional and dynamic insights that facilitates a complete understanding of the workings of their complex molecular mechanisms. NMR is a technique well-suited to investigate proteins in atomic resolution while taking their dynamic properties into account. It thus nicely complements other structural techniques, such as X-ray crystallography, that have contributed high-resolution data to the architectural understanding of ABC transporters. Here, we describe the heterologous expression of LmrA, an ABC exporter from Lactococcus lactis, in Escherichia coli. This allows for more flexible isotope labeling for nuclear magnetic resonance (NMR) studies and the easy study of LmrA's multidrug resistance phenotype. We use a combination of solid-state magic angle spinning (MAS) on the reconstituted transporter and solution NMR on its isolated nucleotide binding domain to investigate consequences of nucleotide binding to LmrA. We find that nucleotide binding affects the protein globally, but that NMR is also able to pinpoint local dynamic effects to specific residues, such as the Walker A motif's conserved lysine residue.

Conversion of chemical to mechanical energy by the nucleotide binding domains of ABCB1

“…NBD dissociation of the apo system shows a very weak stabilization by NBD association with a wide (0.2 nm) and essentially flat energy minimum. The extended flat region implies that association of apo NBDs does not result in a single well defined geometry, consistent with experimental data that could not observe NBD dimers in the absence of nucleotides 60,61 . Binding of MgATP to the NBDs leads to marked change in the free energy profile.…”

Coupled ATPase-adenylate kinase activity in ABC transporters

“…As MsbA transports not only lipid A but also the substrate Hoechst-33342 (ref. 52), we were able to utilize a well-established transport assay based on fluorescence spectroscopy52535455.…”

“…The solution after cell disruption was centrifuged at 5,500 g for 15 min to remove whole cells and cells debris. ISOVs were then obtained by a final ultracentrifugation step at 223,000 g for 1 h. The ISOV pellet was resuspended in 50 mM potassium phosphate buffer, pH 7.2, 10% v/v glycerol for the assay546768.…”

“…7a, LmrA was overexpressed in E. coli as previously described54. Briefly, the plasmid was transformed in C43(DE3) cells and expressed with the spin-down protocol as described above for MsbA.…”

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

“…A key alternative for membrane proteins is solid-state NMR, in which a sample is rotated at a so-called magic angle (54.7° in relation to the magnetic field) to average out dipolar couplings [47]; this approach removes traditional size restrictions, and has enabled structural studies in liposomes [e.g. 48, 49]. Still, ion transport proteins can pose distinct challenges in NMR.…”