Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA

Abstract

(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate–binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo–electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion

“…It is tempting to speculate that c-di-AMP plays a role in the ability of L. monocytogenes to resist osmotic stress as shown here by the relatively high mobility of proteins in the cytoplasm. We note that c-di-AMP is also present in L. lactis but not in E. coli , and thus there is not a simple correlation between resistance to osmotic stress and the regulation of the volume of these cells via the uptake and efflux of potassium ions and compatible solutes ( Commichau et al, 2018 ; Gibhardt et al, 2020 ; Peterson et al, 2020 ; Sikkema et al, 2020 ). Besides, the physiological effect of c-di-AMP on the uptake of potassium in L. monocytogenes is less pronounced than in other Firmicutes ( Gibhardt et al, 2019 ; Stülke and Krüger, 2020 ).…”

In silico method for selecting residue pairs for single-molecule microscopy and spectroscopy

“…Even though the used labelling sites (V360C/N423C) report large differences in distance upon glycine betaine binding and do not affect the binding process, they affect the transfer of substrate from the SBD to the membrane domain of OpuA. In fact, the V360 and N423 are present in the lobes of OpuAC that interact with the transmembrane domain (TMD) of OpuA 19 . Therefore, we aimed to find new residue pairs for smFRET that do not interfere with the docking of OpuAC.…”

“…The results can be inspected manually, for instance by using knowledge of the activity and structure of the entire protein complex. In our case we manually filtered out regions that would affect the interaction of the substrate-binding domain (here OpuAC) with the TMD of the OpuA complex 19 .…”

In Silico Method for Selecting Residue Pairs for Single-Molecule Microscopy and Spectroscopy

“…Even though the used labelling sites (V360C/N423C) report large differences in distance upon glycine betaine binding and do not affect the binding process, they affect the transfer of substrate from the SBD to the membrane domain of OpuA. In fact, the V360 and N423 are present in the lobes of OpuAC that interact with the transmembrane domain (TMD) of OpuA 18 . Therefore, we aimed to find new residue pairs for smFRET that do not interfere with the docking of OpuAC.…”

“…The results can be inspected manually, for instance by using knowledge of the activity and structure of the entire protein complex. In our case we manually filtered out regions that would affect the interaction of the substrate-binding domain (here OpuAC) with the TMD of the OpuA complex 18 .…”