Identification of structural features of SR-BI that facilitate HDL-cholesterol clearance

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PROJECT SUMMARY High density lipoprotein (HDL) is colloquially known as ‘good cholesterol’ due to its protective effects against cardiovascular disease (CVD). HDL is considered anti-atherogenic due to its ability to remove cholesterol from the periphery and deliver cholesteryl ester to the liver via its receptor, scavenger receptor BI (SR-BI). The interaction between HDL and SR-BI is the most important mechanism that facilitates net removal of cholesterol from the body, and as such, it is imperative to better understand the structural mechanisms that promote the HDL and SR-BI interaction. Structurally, SR-BI consists of two key features that drive receptor function: (i) a large extracellular domain required to bind HDL and mediate cholesterol delivery and (ii) two anchoring transmembrane domains which have been implicated in receptor oligomerization. This proposal is designed to test the central hypothesis that proper SR-BI function is driven by structural features of SR-BI that are important for membrane association and receptor oligomerization. Recently, our lab was successful in solving the high- resolution NMR structure of SR-BI residues 405-475 and this peptide serves as our biggest tool in structural studies. The first Aim of this proposal focuses on the extracellular elements of SR-BI that contribute to binding and delivery of HDL-C. The SR-BI[405-475] peptide encompasses the C-terminal transmembrane domain and also an extracellular region containing a short  helix, referred to in this proposal as Helix 2. Preliminary data suggests Helix 2 is lipid-associated and functional data demonstrate its importance in SR-BI-mediated cholesterol transport. First, we will directly measure plasma membrane association of residues within Helix 2 using innovative electron paramagnetic resonance and tryptophan quenching techniques. Then, to translate the observed in vitro functional changes to an in vivo model, mutants that disrupt the hydrophobicity of Helix 2 will be introduced into SR-BI knockout mice. We will then measure the effect these mutants have on macrophage- to-feces reverse cholesterol transport compared to wildtype mice. The second Aim tackles the role of the transmembrane domains in the formation of SR-BI oligomers and possibly, a hydrophobic tunnel for cholesterol movement. First, the dimerization interface of the C-terminal transmembrane domain will be mapped using novel paramagnetic relaxation enhancement methods. We then plan to resolve a high-resolution structure of the N- terminal transmembrane domain of SR-BI by NMR spectroscopy. These strategies will allow us to build upon currently-existing structural information to form a more complete story of SR-BI’s oligomeric state. SR-BI is physiologically important for maintaining lipid homeostasis, as humans with mutations in SR-BI display impaired cholesterol clearance and, hence, an elevated risk of CVD. Clarifying the mechanisms of productive SR-BI/HDL interactions is vital to understanding HDL-C clearance and ultimately modulating CVD risk. As such, the outcomes of our studies could identify SR-BI as a relevant and attractive target for future therapeutics aimed at activating SR-BI-mediated cholesterol transport and effectively lowering plasma cholesterol levels.