Structure of daptomycin and lipids.

Dap depends on PG in the interaction with membrane. (A) DMPG-dependent accumulation of Dap in phospholipid micelles. (B) Kinetics of the fluorescence of Dap in interaction with micelles containing different phospholipids. Micelles contained 20.8 μM various phospholipids (11.5%) and 167 μM DMPC. (C) Plot of the steady-state Kyn fluorescence vs. the content of different negative phospholipids in the micelles. (D) A fast Kyn fluorescence increase within 100 ms in the interaction of Dap with CL-containing micelles. The inset shows the Kyn fluorescence change over 50 s; micelles contained DMPG, POPS or CL at 20.8 μM. All the experiments in (A)-(D) were carried out in 20 mM HEPES (pH 7.57) containing 1.67 mM CaCl2; Dap was always added at last at 15 μM. The micelles were prepared from DMPC at 167 μM alone or together with another lipid at a concentration or content indicated in the plots.

Calcium dependence of the Dap structure and fluorescence in membrane. (A) Calcium sequestration decreases the Dap fluorescence in a way dependent on the phospholipid composition. The kinetic fluorescence measurement was performed under the same conditions as in Figure 1(B). The red dotted line denoted the time point when 10 mM EGTA was added. The controls contained Dap at 15 μM Dap in the same buffer with or without calcium. (B) Circular dichroism spectra of Dap in different membrane environments. The buffer was 20 mM Tris.HCl, pH 7.57; Dap was 180 μM and Ca(CH3COO)2 was 1.0 mM; spectra were recorded 30 min after mixing with micelles contained pure DMPG or CL at 720 μM to maximize the amount of bound Dap; in Ca2+ sequestration experiments, spectra were recorded 30 min after adding 10.0 mM EGTA. The putative PG headgroup, sn-glycerol 3-phosphate (G3P), was 20 mM in attempt to detect its potential interaction with Dap.

The binding affinity of Dap for DMPG by titration. The titration with DMPG was performed in 20 mM HEPES buffer (pH 7.57) containing 15 nM Dap and 1.67 mM CaCl2. The solid line is the fitting curve using the equation F = Fmax × [DMPG]2/(KD + [DMPG]2) where F = fluorescence at 454 nm, which is derived from a binding model in which a fluorescent {Dap·2DMPG} complex is dissociated into non-fluorescent Dap and two DMPG molecules under the condition [DMPG] >> [Dap]. The dashed line is the linear trend line for the control titration of 1.5 μM Dap with POPS in the same buffer containing 1.67 mM CaCl2.

Stable Dap-PG complexes formed in vitro and in Bacillus subtilis. (A) Reverse phase HPLC chromatogram of the complexes. Samples were dissolved in 10% DMSO and 90% water and filtered before injection into a Phenomenex Luna 5 μm C18 column (150 × 4.6 mm). The complexes were eluted by water containing 0.1% trifluoroacetic acid and a linear gradient of acetonitrile from 5-95% over 20 min and detected by ultraviolet absorbance at 254 nm. (B) Fluorescence spectra of the two complexes with excitation at 365 nm. The complexes were dissolved in DMSO and the fluorescence of the Dap-DMPG complex was scaled for easy comparison. (C) Comparative positive-ion MALDI-ToF mass spectrometry of the two complexes. All the peaks with m/z < 500 were from the matrix after comparison with a control. (D) Comparative negative-ion high-resolution ESIMS analysis of the two complexes.

Proposed mechanism for the two-phased uptake of Dap into bacterial membrane. The red dots denote Ca2+.