The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact

Abstract

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that physical interactions between SBDs and cooperativity in substrate delivery could be more widespread than recognized thus far.

Probing the stability and interdomain interactions in the ABC transporter OpuA, using single-molecule optical tweezers

“…The force required to break this interaction was higher at low salt (buffer only) than in the presence of buffer plus 600 mM KCl. This increase in force matches the observation of a larger high FRET population in the low salt condition from smFRET data 28 . Additionally, the experimental values for the change in length 11 ± 5 nm and 15 ± 6 nm in buffer without and with salt, respectively, are in the same order of magnitude as expected from an interaction ( Materials and Methods, Figure 6 ).…”

“…OpuA-K521C has been purified and reconstituted in MSP1E3D1 lipid nanodiscs formed from a synthetic lipid mixture consisting of a weight ratio of 38:12:50 DOPE:DOPG:DOPC. It has previously been shown that the salt-, glycine betaine-, ATP- and c-di-AMP-dependent ATPase activities of the mutant are similar to those of the wild-type 28 .…”

“…The transmembrane ABC-transporter OpuA from Lactococcus lactis has been successfully expressed and purified as described previously. The protein retains its substrate-dependent ATPase activity and ionic strength-dependent activation when reconstituted in lipid nanodiscs 21,24,28 . Therefore, OpuA is an ideal candidate for single-molecule studies on the impact of substrate and ionic strength on the conformational states and dynamics of a membrane protein.…”

“…Note that the numbers shown in the figure are approximations and possible flexibility of the anchoring helix has not been taken into account (bottom right, see also Materials and Methods). ( 1 ) Interaction with the TMD as resolved in the low-salt cryo-EM structure as reported by van den Noort et al 28 , ( 2 ) the lobes interact on the side of the substrate-binding pocket, ( 3 ) each SBD interacts with the transmembrane domain where the maximum distance has been calculated based on the length of the linkers, and ( 4 ) docking of one of the SBDs where the second SBD is parallel to the pulling direction.…”

“…An increase in ionic strength screens the electrostatic interactions of the cationic patch with the membrane, which activates the transport. Single-molecule Förster resonance energy transfer microscopy (smFRET) and cryo-electron microscopy (cryo-EM) results have implied an interaction between the two SBDs, which is especially prominent in low ionic strength conditions 28 ( Figure 1C ). The relative orientation of the interacting SBDs could not be deduced based on smFRET and cryo-EM results on their own.…”