A Mechanistic Model for the Release of Ceramide from the CERT START Domain

Abstract

Ceramide transfer protein CERT is the mediator of non-vesicular transfer of ceramide from ER to Golgi. In CERT, START is the domain responsible for the binding and transport of ceramide. A wealth of structural data has revealed a helix-grip fold surrounding a large hydrophobic holding the ceramide. Yet little is known about the mechanisms by which START releases the ceramide through the polar region and into the packed environment of cellular membranes. As such events do not lend themselves easily to experimental investigations we used multiple unbiased microsecond-long molecular simulations. We propose a membrane-assisted mechanism in which the passage of the ceramide acyl chains is facilitated by the intercalation of a single phosphatidylcholine lipid in the cavity, practically greasing the ceramide way out. We verify using experimental lipidomics data that CERT forms stable complexes with phosphatidylcholine lipids, in addition to ceramide, thus providing a validation for the proposed computational model.

A system-wide analysis of lipid transfer proteins delineates lipid mobility in human cells

“…3a). Binding of phosphatidylcholine (palmitoyl-oleoyl-phosphatidylcholine, POPC) to CERT through insertion of one fatty acid tail into its lipid binding pocket was also observed in molecular dynamics simulations of the CERT-START domain with membranes using an all-atoms model 66 . Simulations of CERT-phosphatidylcholine adducts in an aqueous environment in the presence and absence of the cargo ceramide resulted in both cases in the formation of stable complexes.…”

Model mechanism for lipid uptake by the human STARD2/PC-TP phosphatidylcholine transfer protein

“…In addition, the intercalation of a single phosphatidylcholine lipid in the cavity disrupts the LTP-cargo interactions, and facilitates the release of the ceramide cargo through the polar membrane interface. 12 It is worth noting that in STARD11, like for STARD2 but unlike for CPTP, the lipid cargo is loaded head first.…”

“…Some are highly sensitive to lipid composition, such as Osh4 33,34 and STARD4 which show specificity toward phosphatidylinositol bisphosphate (PIP2) lipids, 35,36 while other LTPs show low or no sensitivity to lipid compositions such as the CERT START domain (STARD11). 12 We performed simulations of apo STARD2 in the presence of an ER bilayer model, and of its holo form in the presence of the outer mitochondrial membrane (OMM) (see SI for the lipid composition). To correct for the change of orientation of W101 observed in the apo-water simulation, we applied a dihedral restraint on the W101 χ1 and χ2 angles to maintain its experimental conformation (see Methods section for detailed protocols).…”