The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR

Abstract

MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarization (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Protein NMR Spectroscopy at 150 kHz Magic‐Angle Spinning Continues To Improve Resolution and Mass Sensitivity

“…Solid-state NMR plays an important role in de novo 3D structure determination of particular protein classes, such as amyloids. [1][2][3][4][5] The method allows conformational details to be studied with particularly high sensitivity, [6][7][8] and can, for example, identify drug-binding sites or characterize dynamic features. [9][10][11][12][13][14][15] Information content of the NMR spectrum increases with improved spectral resolution.…”

Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters

“…Crosslinking and spectroscopy also identified substrate-induced conformational changes in TMs [65][66][67] . In P-gp, TM4 and TM10 kinks and flexible non-helical regions correlate with ligand presence (Fig.…”

“…Similarly in another study, while the NBDs of both apo and substrate-bound P-gp moved toward each other in simulations, only the substratebound state NBDs aligned with the closed NBD dimer 75 . Spectroscopy studies show that transported substrates cause distortions of TM4 or TM4' to form the NBD dimer 66 . P-gp structures with some competitive inhibitors show analogous TM4 or TM4' kinks ( Fig.…”

Elucidating ligand-bound structures of membrane proteins using solid-state NMR spectroscopy

“…Solid-state NMR has been used to investigate MsbA, a homodimeric bacterial ABC transporter that exports lipid A and other hydrophobic molecules across the bacterial inner membrane. Spadaccini et al measured the chemical shifts of two unique residue pairs in TM helix 4 and helix 6 in the apo state and ATP-hydrolysis transition state in the absence and presence of drug molecules [35]. Due to the large molecular weight of the dimeric protein (130 kDa), 2D 13 C-13 C correlation spectra were measured under DNP conditions.…”