Lactadherin binds to phosphatidylserine-containing vesicles in a two-step mechanism sensitive to vesicle size and composition

Lactadherin binds to phosphatidylserine (PS) in a stereospecific and calcium independent manner that is promoted by vesicle curvature. Because membrane binding of lactadherin is supported by a PS content of as little as 0.5%, lactadherin is a useful marker for cell stress where limited PS is exposed, as well as for apoptosis where PS freely traverses the plasma membrane. To gain further insight into the membrane-binding mechanism, we have utilized intrinsic lactadherin fluorescence. Our results indicate that intrinsic fluorescence increases and is blue-shifted upon membrane binding. Stopped-flow kinetic experiments confirm the specificity for PS and that the C2 domain contains a PS recognition motif. The stopped-flow kinetic data are consistent with a two-step binding mechanism, in which initial binding is followed by a slower step that involves either a conformational change or an altered degree of membrane insertion. Binding is detected at concentrations down to 0.03% PS and the capacity of binding reaches saturation around 1% PS (midpoint 0.15% PS). Higher concentrations of PS (and also to some extent PE) increase the association kinetics and the affinity. Increasing vesicle curvature promotes association. Remarkably, replacement of vesicles with micelles destroys the specificity for PS lipids. We conclude that the vesicular environment provides optimal conditions for presentation and recognition of PS by lactadherin in a simple binding mechanism. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (122 K)Download as PowerPoint slideHighlights► Lactadherin binds specifically to phosphatidylserine containing membranes. ► This process occurs as a rapid two-step binding mechanism. ► As little as 0.03% PS is detected in the membrane by the kinetic assay. ► Phospatidylserine and increased curvature both promote binding. ► Presentation of PS in surfactant micelles abolishes PS specificity.