Daptomycin–Phosphatidylglycerol Domains in Lipid Membranes

Daptomycin is an acidic, 13-amino acid, cyclic polypeptide that contains a number of nonproteinogenic residues and is modified at its N-terminus with a decanoyl chain. It has been in clinical use since 2003 against selected drug-resistant <i>Staphylococcus aureus</i> and <i>Enterococcus spp</i> infections. In vitro, daptomycin is active against Gram-positive pathogens at low concentrations but its antibiotic activity depends critically on the presence of calcium ions. This dependence has been thought to arise from binding of one or two Ca²⁺ ions to daptomycin as a required step in its interaction with the bacterial membrane. Here, we investigated the interaction of daptomycin with giant unilamellar vesicles (GUVs) composed 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). We used fluorescence confocal microscopy to monitor binding of the peptide to GUVs and follow its effect on the membrane of the vesicle. We found that in the absence of POPG or Ca²⁺ daptomycin does not bind measurably to the lipid membrane. In the presence of 20–30% PG in the membrane and 2 mM Ca²⁺, daptomycin induces the formation of membrane domains rich in acidic lipids. This effect is not induced by Ca²⁺ alone. In addition, daptomycin causes GUV collapse, but it does not translocate across the membrane to the inside of intact POPC/POPG vesicles. We conclude that pore formation is probably not the mechanism by which the peptide functions. On the other hand, we found that daptomycin coclusters with the anionic phospholipid POPG and the fluorescent probes used, leading to extensive formation of daptomycin–POPG domains in the membrane

Sorting of Lipidated Peptides in Fluid Bilayers: A Molecular-Level Investigation

Nearest-neighbor recognition (NNR) measurements have been made for two lipidated forms of GlyCys, interacting with analogues of cholesterol and 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC) in the liquid-ordered (<i>l</i>_o) and liquid-disordered (<i>l</i>_d) phases. Interaction free energies that have been determined from these measurements have been used in Monte Carlo simulations to quantify the distribution of the peptides between liquid-ordered and liquid-disordered regions. These simulations have shown that significant differences in the lipid chains have a very weak influence on the partitioning of the peptide between these two phases. They have also revealed an insensitivity of the peptide partition coefficient, <i>K</i>_p, to the size of the <i>l</i>_o and <i>l</i>_d domains that are present. In a broader context, these findings strongly suggest that the sorting of peripheral proteins in cellular membranes via differential lipidation may be more subtle than previously thought

Push–Pull Mechanism for Lipid Raft Formation

A quantitative assessment has been made of the interaction between exchangeable mimics of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and cholesterol in the liquid-ordered (<i>l</i>₀) and the liquid-disordered (<i>l</i>_d) states using the nearest-neighbor recognition (NNR) method. This assessment has established that these lipids mix ideally in the <i>l</i>₀ phase (i.e., they show no net attraction or repulsion toward each other) but exhibit repulsive interactions in the <i>l</i>_d phase. The implications of these findings for the interactions between unsaturated phospholipids and cholesterol in eukaryotic cell membranes are briefly discussed

Eliminating the Roughness in Cholesterol’s β‑Face: Does it Matter?

One of the long-standing issues surrounding cholesterol (Chol) relates to its two-faced character. In particular, the consequences of its having a rough β-face and a smooth α-face on its structural influence in cell membranes has remained elusive. In this study, direct comparisons have been made between cholesterol and a “smoothened” analog, DChol (i.e., 18,19-dinorcholesterol) using model membranes and a combination of nearest-neighbor recognition, differential scanning calorimetry, fluorescence, and monolayer measurements. Taken together, these results indicate that subtle differences exist between the interaction of these two sterols with the different states of 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC). Chol has a greater condensing power than DChol, but only slightly so, i.e., on the order of a few tens of calories per mole