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

Reviewer #1 (Public Review):

Summary:
The type I ABC importer OpuA from Lactococcus lactis is the best-studied transporter involved in osmoprotection. In contrast to most ABC import systems, the substrate binding protein is fused via a short linker to the transmembrane domain of the transporter. Consequently, this moiety is called the substrate binding domain (SBD). OpuA has been studied in the past in great detail and we have a very detailed knowledge about function, mechanisms of activation and deactivation as well as structure.

Strengths:
Application of smFRET to unravel transient interactions of the SBDs. The method is applied at a superb quality and the data evaluation is excellent.

Weaknesses:
The proposed model is not directly supported by experimental data. Rather all alternative models are excluded as they do not fit the obtained data.

Reviewer #2 (Public Review):

Summary:
In this report, the authors used solution-based single-molecule FRET and low-resolution cryo-EM to investigate the interactions between the substrate-binding domains of the ABC-importer OpuA from Lactococcus lactis. Based on their results, the authors suggest that the SBDs interact in an ionic strength-dependent manner.

Strengths:
The strength of this manuscript is the uniqueness and importance of the scientific question, the adequacy of the experimental system (OpuA), and the combination of two very powerful and demanding experimental approaches.

Weaknesses:
A demonstration that the SBDs physically interact with one another and that this interaction is important for the transport mechanism will greatly strengthen the claims of the authors. The relation to cooperativity is also unclear.