Abstract
The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.
Highlights
Biological membranes contain hundreds of lipid types, which differ in acyl chain length and degree of unsaturation, and head group polarity, size, and charge [1]
In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide microcin J25 (MccJ25)
Mass Spectrometry of McjD Reveals Co-purified Phospholipids and Lipopolysaccharides—Following expression and purification of McjD from E. coli, we used non-denaturing mass spectrometry to determine the oligomeric state of the protein and presence of any bound lipids
Summary
Biological membranes contain hundreds of lipid types, which differ in acyl chain length and degree of unsaturation, and head group polarity, size, and charge [1] Such a complex lipid environment is essential to maintain the structural integrity and. There has been an increase in the numbers of membrane protein structures determined at medium to high resolution. Some of these structures show resolved lipids, which most probably are involved in specific interactions because they have survived the purification and crystallization processes. In the low resolution crystal structure of the eukaryotic P-glycoprotein, no lipids were observed [10, 11], but non-denaturing mass spectrometry identified tightly associated lipids and a clear dependence of ligand and lipid binding [12]
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