Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

Abstract

ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains a challenge to structural biology, as these proteins are notoriously difficult to study at the molecular level in their active, membrane-inserted form. Here we use solid-state NMR to investigate the multidrug ABC transporter BmrA reconstituted in lipids. We identify the chemical-shift differences between the inward-facing, and outward-facing state induced by ATP:Mg 2+ :Vi addition. Analysis of an X-loop mutant, for which we show that ATPase and transport activities are uncoupled, reveals an incomplete transition to the outward-facing state upon ATP:Mg 2+ :Vi addition, notably lacking the decrease in dynamics of a defined set of residues observed in wild-type BmrA. This suggests that this stiffening is required for an efficient transmission of the conformational changes to allow proper transport of substrate by the pump.

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

“…Therefore, a 4month-old rotor containing the mutant BmrA-E504A apo (in its inward-facing state, see Figure 3A for the spectrum) was opened, and the sediment was unpacked, resuspended in the presence of ATP-Mg, and sedimented a second time into the solid-state NMR rotor. The new spectrum ( Figure 3B, for an overlay with the spectrum obtained directly after rotor filling see Figure 3C) indeed indicated the conformational change to the outwardfacing form as described previously by Lacabanne et al (2019b).…”

“…sedimented sample can still undergo conformational changes, such as those previously detected by solid-state NMR reflecting the transition between the inward-and outward-facing state of the transporter (Lacabanne et al, 2019b). Therefore, a 4month-old rotor containing the mutant BmrA-E504A apo (in its inward-facing state, see Figure 3A for the spectrum) was opened, and the sediment was unpacked, resuspended in the presence of ATP-Mg, and sedimented a second time into the solid-state NMR rotor.…”

“…After 4 years of storage in the NMR rotor, the sample of the membrane protein BmrA-E504A in absence of ligand showed the same DARR spectrum with the same signalto-noise ratio compared to the one measured directly after rotor filling [ Figure 2. Note that LVIKH were selectively unlabeled to reduce spectral overlap in the spectrum (Lacabanne et al, 2018(Lacabanne et al, , 2019b]. An interesting question is whether an aged FIGURE 2 | Comparison of 20 ms 13 C-13 C DARR correlation spectra recorded directly after filling (red) and up to several years later (blue): (A) BmrA E504 [a homology model based on SAV1866 is shown in the last column, PDB ID: 2HYD (Dawson and Locher, 2006), the red spectrum is taken from reference (Lacabanne et al, 2019b)], (B) the pRN1 primase (PDB ID: 6GVT), and (C) 13 C/ 15 N labeled Rpo7 (PDB ID: 1GO3, blue ribbons) in complex with unlabeled Rpo4 [red ribbons, where the red spectrum is taken from reference (Torosyan et al, 2019)].…”

“…Protein-protein complexes were studied by co-sedimentation (Gardiennet et al, 2016;Xiang et al, 2018) and protein-ligand complexes by sedimentation with the respective ligand (e.g. nucleic acids) directly into the NMR rotor (Wiegand et al, 2016a;Kaur et al, 2018;Stöppler et al, 2018;Lacabanne et al, 2019b).…”

“…Here, we describe our experimental observations regarding the stability of four sedimented samples from different protein systems: (i) the bacterial helicase DnaB from Helicobacter pylori in a complex with ADP:AlF 4 − and DNA (Wiegand et al, 2019), (ii) the archaeal pRN1 primase from Sulfolobus islandicus in a complex with DNA and ATP (Boudet et al, 2019), (iii) the membrane protein BmrA, an ABC transporter from Bacillus subtilis (Lacabanne et al, 2019b), and (iv) the two RNA polymerase subunits Rpo4/7 * from Methanocaldococcus jannaschii. We use 2D DARR spectra and 31 P cross polarization (CP) MAS experiments to assess the long-term stability of these sedimented protein samples.…”