Reviewer #1 (Public Review):

Summary:

In this manuscript, the molecular mechanism of interaction of daptomycin (DAP) with bacterial membrane phospholipids has been explored by fluorescence and CD spectroscopy, mass spectrometry, and RP-HPLC. The mechanism of binding was found to be a two-step process. A fast reversible step of binding to the surface and a slow irreversible step of membrane insertion. Fluorescence-based titrations were performed and analysed to infer that daptomycin bound simultaneously two molecules of PG with nanomolar affinity in the presence of calcium. Conformational change but not membrane insertion was observed for DAP in the presence of cardiolipin and calcium.

Strengths:

The strength of the study is the skillful execution of biophysical experiments, especially stopped-flow kinetics that capture the first surface binding event, and the careful delineation of the stoichiometry.

Weaknesses:

The weakness of the study is that it does not add substantially to the previously known information and fails to provide additional molecular details. The current study provides incremental information on DAP-PG-calcium association but fails to capture the complex in mass spectrometry. The ITC and NMR studies with G3P are inconclusive There are no structural models presented. Another aspect missing from the study is the reconciliation between PG in the monomer, micellar, and membrane forms.