Abstract 206: Into The Wormhole: Understanding SR-B1’s Cholesterol Transport Functions Through Structural Studies

Scavenger receptor Class B type 1 (SR-B1) is a glycosylated integral membrane protein that serves as the primary receptor for high density lipoprotein (HDL) and plays a central role in the reverse cholesterol transport pathway (RCT). In RCT, HDL removes cholesterol within atherosclerotic plaques and delivers it to the liver for excretion. HDL-cholesterol is delivered into hepatocytes through interaction with SR-B1, highlighting the need to improve our understanding of SR-B1 structure-function relationships to effectively lower plasma cholesterol levels and cardiovascular disease risk. Currently, structural information about SR-B1 remains limited to a structure of short transmembrane-spanning peptide segment, homology models, and transient transfection studies in cultured cells. Towards our long-term goal of resolving a full-length structure of human SR-B1, we have expressed and purified full-length human SR-B1 from an Sf9 insect cell baculoviral infection system. To verify function, human full-length SR-B1 expressed in plated Sf9 cells displayed a significant increase in DiI-HDL binding and DiI-lipid uptake compared to empty vector controls. Using PFO-PAGE, we demonstrated that SR-B1 forms dimers and higher order oligomers in Sf9 cells. After functional verification, we successfully incorporated purified protein into detergent micelles. Thermal shift assays revealed purified SR-B1 remained stable over a 6-week period. Purified SR-B1 also maintained the ability to bind to apolipoprotein A-I (the main protein component of HDL) and holoparticle HDL with high affinity (K D = 42.77 ± 7.15 μg/mL) by microscale thermophoresis (MST). Additionally, SR-B1 was able to bind oxidized low density lipoprotein (oxLDL). Further, while glycosylation remains important for SR-B1 expression, we are the first to demonstrate by MST that SR-B1’s glycosylation status did not impact HDL binding affinity (K D of 42.77 ± 7.15 μg/mL for glycosylated vs. 41.25 ± 4.92 μg/mL for unglycosylated SR-B1). Availability of functional purified human SR-B1 is a critical step towards a high-resolution structure and these studies lay the foundation for understanding the dynamics of SR-B1-mediated cholesterol transport and novel ways to modulate cardiovascular disease risk.