Structural and functional analysis of regulation of the lipid-transport protein ORP8

This project is supported jointly by the Polish National Science Center and by the Czech Science Foundation within the CEUS-UNISONO international program. It is being carried out in close collaboration with the research group of Evzen Boura at the Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences.

Research background and results

Among the major components of biological cells are semipermeable membranes. The plasma membrane (PM) separates the interior of the cell from its environment. Other membranes enclose intracellular organelles such as the endoplasmic reticulum (ER), the Golgi apparatus, and mitochondria. All of these membranes are composed of diverse macromolecules – mostly lipids and proteins of different types – and the unique lipid composition of a given membrane is essential for its proper functioning within the cell.

Most lipids are synthesized in the ER and must be transported against their concentration gradients to proper target membranes. The intracellular transport of lipids between various organelles is carried out by specialized proteins. Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate lipid transfer at membrane contact sites, such as the ER-PM contact sites, which ensures the transport of specific lipids from the place of their synthesis (ER) to target membranes (PM, Golgi apparatus, mitochondria), and also maintains the proper lipid composition of cellular membranes. As such, the process of lipid transfer by ORPs must be tightly controlled.

The aim of this project was to understand the molecular mechanisms of transport of lipids, namely, phosphatidylserine (PS) and phosphatidylinositol-4 phosphate (PI4P), by the ORP8 protein. The structure of the lipid transport domain of the ORP8 protein was determined and its transport properties were characterized. In these studies, the methods of X-ray crystallography, fluorescence cross-correlation spectroscopy and molecular dynamics simulation were synergistically combined. The results of these studies deepen our knowledge of the structure and mechanisms of lipid transport by the ORP8 protein and also provide a starting point for the design of potential inhibitors of this protein.

In addition, the mechanism of PS and PI4P transport by the yeast Osh6 protein, an ortholog of the human ORP8 protein, was revised. In particular, the role of the PI4P lipid and the importance of the Sac1 phosphatase activity in the transport of PS molecules were clarified. On the basis of molecular dynamics simulations, it was shown how the lipid composition of membranes affects the activity of the Sac1 phosphatase. Moreover, the interplay between the dynamics of the lipid transport protein Osh6 and the physical properties (such as surface charge and of fluidity) of the membranes donating and accepting PS and PI4P molecules were explained. In particular, it was shown that the specific binding of the transported lipid molecule to the transport protein molecule changes the structure and dynamics of the protein, consequently predisposing the protein molecule to unload the lipid cargo in the appropriate target membrane.

The impact of physical properties of lipid membranes (such as their surface charge and fluidity) on the binding of LactC2 and P4M biosensors to their target lipids — PS and PI4P, respectively — was also determined. This achievement allowed us to properly and precisely interpret results of experiments employing the LactC2 and P4M lipid biosensors.

Publications