Fig 2: R174C-SR-BI is a rare human variant of SR-BI identified in an individual with high HDL-cholesterol. A: Characteristics and lipid profile of the R174C-SR-BI heterozygous individual. B: A homology model of the extracellular domain of human SR-BI (residues 38–439) was generated using Modeller software, and residues that correspond to sites of known human variants are colored in pink. C: Multiple species sequence alignments of SR-BI were generated by the MUltiple Sequence Comparison by Log-Expectation algorithm. Arg-174 and Asp-185 (possible ion pair) are highlighted in magenta and light green, respectively, and the boxed region represents a conserved N-linked glycosylation sequence (NXT). SR-BI, scavenger receptor class B type I.

Fig 3: Electrostatic surfaces charges are disrupted by R174C-SR-BI. Arg-174 in WT-SR-BI (top, left) was mutated to cysteine (top, middle) and each of the additional mutants using Chimera software. A top down view of the SR-BI homology model is shown with surface electrostatic coloring, ranging from more negative [red, -10 kcal/(mol * e)] to more positive [blue, 10 kcal/(mol * e)]. The region where Arg-174 occupies and the corresponding regions for R174C-SR-BI and the additional mutants are highlighted in yellow circles. SR-BI, scavenger receptor class B type I.

Fig 4: R174C-SR-BI and “salt bridge” mutants form dimers and higher order oligomers. COS-7 cells transiently expressing WT-SR-BI or mutant SR-BI were sonicated, and proteins (10 µg) were separated in the presence of 5% perfluorooctanoic acid under nonreducing conditions for subsequent immunoblot analysis to assess the oligomerization profiles of SR-BI. A representative immunoblot is shown for three independent transfections (n = 3). SR-BI, scavenger receptor class B type I.

Fig 5: Arginine-174 and aspartate-185 may interact through a salt bridge. A: Homology modeling suggests a possible salt bridge interaction between residues Arg-174 (magenta) and Asp-185 (light green) with an approximate distance of 3.2 Å, as calculated by Chimera software. B: Additional mutant constructs were generated to disrupt or retain the putative salt bridge. C: SR-BI immunoblot analysis was performed on cell lysates (10 µg) from COS-7 cells transiently transfected with empty vector or mutant SR-BI. D: SR-BI expression was quantified relative to GAPDH loading control by densitometry. E: Cell surface levels of SR-BI (in 100 µl total lysate), following sulfo-NHS-LC biotinylation of transiently transfected COS-7 cells, were determined by immunoblot analysis. The additional mutants are colored as follows: salt bridge-disrupting mutants (R174D-SR-BI and D185R-SR-BI) in dark red and light red, the charge swap mutant (R174D-SR-BI/D185R-SR-BI) in light teal, and the conservative mutant (R174K-SR-BI) in dark teal. Immunoblots in panels C and E were performed under reducing conditions. Data in panel D represent the mean ± SEM of three independent transfections (n = 3), relative to average WT-SR-BI expression levels (WT = 100%). As determined by one-way ANOVA, no statistical significance was observed between the means for WT-SR-BI and mutant SR-BI receptors. The immunoblots in panels C and E are each representative of three independent transfections (n = 3). SR-BI, scavenger receptor class B type I.