Abstract 104: NMR Analyses of the Structural and Oligomeric Properties of the C-terminal Transmembrane Domain of SR-BI in Detergent Micelles

Abstract

In the reverse cholesterol transport pathway, high density lipoprotein (HDL) carries cholesterol from peripheral tissues to the liver for excretion via bile formation following interaction of HDL with its receptor, scavenger receptor BI (SR-BI). Due to the absence of a high-resolution structure of SR-BI, our understanding of receptor-ligand interactions between SR-BI and HDL that lead to whole body cholesterol removal remain limited. Our laboratory has demonstrated that oligomerization of SR-BI, likely mediated by its C-terminal transmembrane domain (C-TMD), facilitates delivery of HDL-cholesterol into cells. In this study, we describe the use of novel nuclear magnetic resonance (NMR) strategies to determine the structure and dimerization properties of SR-BI(405-475), a peptide that encompasses residues 405-475 and includes a portion of the extracellular domain, as well as the entire C-TMD of SR-BI. [U-15N,13C]-SR-BI(405-475) was bacterially expressed and purified, and alpha-helical secondary structure was identified by circular dichroism. The ability of SR-BI(405-475) to dimerize was confirmed by chemical crosslinking strategies. Next, shifts in the monomer-dimer equilibrium of [U-15N]-SR-BI(405-475) were assessed by 1H-15N-HSQC analysis upon altering the ratio of peptide to detergent micelles. By comparing the appearance or disappearance of peaks in our NMR spectra, we determined that 1 mM SR-BI(405-475) in 5% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1’-rac-glycerol] (LPPG) micelles in water at pH 6.8 yielded a mixture of monomer and dimer, while 100 μM peptide in 5% LPPG micelles shifted the oligomeric status to primarily monomer. Using 1H-15N-13C 3D NMR residue assignments, we also determined that these appearing/disappearing peaks represented the residues in a GXXXG dimerization motif (G420, A421, M422, G423, and G424), as well as several leucine residues in a putative leucine zipper dimerization motif. Together, we have made significant strides towards acquiring the first high-resolution structural information for SR-BI and have obtained convincing evidence of SR-BI dimerization via its C-TMD. These novel findings may ultimately help in designing therapeutics needed to enhance whole body cholesterol removal.