title: 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.
authors.affiliation: Department of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Germany; Centre for Biomolecular Magnetic Resonance (BMRZ), J.W. Goethe University, Frankfurt, Germany. Electronic address: u.hellmich@uni-mainz.de.
authors.forename: Ute A
authors.initials: UA
authors.lastname: Hellmich
authors.affiliation: Centre for Biomolecular Magnetic Resonance (BMRZ), J.W. Goethe University, Frankfurt, Germany; Department of Biophysical Chemistry, J.W. Goethe University, Frankfurt, Germany.
authors.forename: Leonie
authors.initials: L
authors.lastname: Mönkemeyer
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Saroj
authors.initials: S
authors.lastname: Velamakanni
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
authors.affiliation: Centre for Biomolecular Magnetic Resonance (BMRZ), J.W. Goethe University, Frankfurt, Germany; Department of Biophysical Chemistry, J.W. Goethe University, Frankfurt, Germany; Cluster of Excellence Macromolecular Complexes Frankfurt, Germany. Electronic address: glaubitz@em.uni-frankfurt.de.
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; C101824:LmrA protein, Lactococcus lactis
keywords: ABC transporter; LmrA; MAS NMR; Nucleotide binding; Protein dynamics; Solution NMR