A MAS NMR Study of the Bacterial ABC Transporter ArtMP

Abstract

ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively (13)C,(15)N-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

Solid‐State NMR Spectroscopy on Complex Biomolecules

“…[54,96,121] Further studies on interactions between ligands and membrane proteins involved other ion channels [122] and G-proteincoupled receptors (GPCRs). [123] Many other membrane proteins have also been investigated using ssNMR spectroscopy, including membraneembedded enzymes, [49,98,124] histidine kinases, [29] ABC transporters, [125] and bacterial outer-membrane proteins. [126] Again, an appropriate discussion on this research area is beyond the scope of this Review, and the reader is referred to recent reviews on this subject.…”

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

“…Fast sample rotation about an axis inclined to the magnetic field at 54.7° (the 'magic angle'), averages all anisotropic interactions leading to well-resolved spectra (Figure 1). The first solid state NMR experiments on an ABC importer were described for ArtMP in 2D-crystals (Akbey et al, 2014;Lange et al, 2010). The studies were later complemented by EPR spectroscopy (Hellmich et al, 2012b) and by solution state NMR on the isolated NBDs (Hellmich et al, 2012a).…”

Festkörper‐NMR‐Spektroskopie an komplexen Biomolekülen

“…[54,96,121] Weitere Untersuchungen von Ligand-MP-Wechselwirkungen wurden an anderen Ionenkanälen [122] und G-Protein-gekoppelten Rezeptoren (GPCR) durchgeführt. [123] Schließlich wurde eine ganze Reihe weiterer Membranproteine mit FK-NMR-Spektroskopie untersucht, darunter membrangebundene Enzyme, [49,98,124] Histidin-Kinasen, [29] ABC-Transporter [125] und Proteine der bakteriellen Außenmembran. [126] Eine ausführliche Diskussion würde über den Umfang dieses Aufsatzes hinausgehen, wir verweisen deshalb auf aktuelle Übersichtsartikel zu diesen Themen.…”