Volume 25, Issue 3, Pages 446-457 (March 2017) NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif  Alexandra C. Chadwick, Davin R. Jensen, Paul J. Hanson, Philip T. Lange, Sarah C. Proudfoot, Francis C. Peterson, Brian F. Volkman, Daisy Sahoo  Structure  Volume 25, Issue 3, Pages 446-457 (March 2017) DOI: 10.1016/j.str.2017.01.001 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Sequence Alignment, Secondary Structure Prediction, and Helical Wheel Alignment of SR-BI(405–475) (A) The sequence of SR-BI(405–475) was aligned across species. The putative leucine zipper (blue) and the GXXXG motif (green) are highlighted. Identical residues across all species are denoted below the sequence with asterisks, residues with strong similar properties are denoted with double dots, and residues with weak similar properties are denoted with a single dot. (B) Prediction software programs were used to predict the secondary structure of SR-BI(405–475). β Strands are denoted in blue and α helices in red. (C–E) Helical wheel alignment of residues 408–419 (C), residues 426–436 (D), and residues 438–465 (E). Non-polar residues (peach), polar residues (orange), and leucines in the putative leucine zipper (blue) are highlighted. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Expression Levels of Mutant SR-BI Receptors WT or mutant SR-BI receptors were transiently expressed in COS-7 cells. (A) Total cell lysates were separated by 8% SDS-PAGE and SR-BI, and loading controls were detected by immunoblot analysis using an anti-SR-BI or anti-GAPDH antibody. Data are representative of four independent transfections. (B) Densitometry was performed with ImageJ software, and relative SR-BI band intensity was normalized to GAPDH and quantified with respect to WT SR-BI control (set to 100%). Densitometric values in a.u. represent the mean of four independent transfections ± SEM. (C) Cell-surface expression was analyzed by flow cytometry using an antibody directed against the extracellular domain of SR-BI. Data represent the mean ± SEM from four independent transfections. ∗p < 0.05 by one-way ANOVA. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Mutation of Several Residues in the Putative Leucine Zipper Motif Disrupts SR-BI Function WT or mutant SR-BI receptors were transiently expressed in COS-7 cells. Cells were incubated with [125I]/[3H]COE-labeled HDL (10 μg/mL) at 37°C for 1.5 hr. (A and B) Cell association of [125I]HDL (A) and selective uptake of [3H]COE (B). Values represent the mean ± SEM of at least six independent experiments, each performed in triplicate. (C) Cells pre-labeled with [3H]cholesterol were incubated with HDL (50 μg/mL) for 4 hr. Radioactivity associated with the media and cells was counted and % efflux was calculated. Values represent the mean ± SEM of three independent experiments, each performed in quadruplicate. (D) Cells pre-labeled with [3H]cholesterol were incubated with exogenous cholesterol oxidase (0.5 U/mL) for 4 hr. Cholesterol species were separated by thin-layer chromatography and percentage of cholestenone was determined. Values represent the mean ± SEM of three independent experiments, each performed in quadruplicate. All datasets are presented following subtraction of empty vector values and were normalized to respective WT SR-BI (normalized value = 100%). Before normalization, the mean experimental values for vector and WT SR-BI, respectively, were 71.1 and 305.3 ng HDL/mg cell protein (HDL binding); 817.8 and 3,012.4 ng HDL-COE/mg cell protein (HDL-COE uptake); 5.5% and 9.2% (cholesterol efflux); and 13.1% and 37.9% (cholestenone production). Statistical analyses were determined by one-way ANOVA comparing each mutant with WT SR-BI from respective experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 SR-BI Self-Association Is Dependent on the Leucine Zipper Motif COS-7 cells transiently expressing WT or LZ-SR-BI were incubated for 1 hr at 4°C with 0, 0.1, 0.3, or 1 mM BS3 amine-reactive crosslinker. Cell lysates were separated by 8% SDS-PAGE, and SR-BI and GAPDH levels were detected by immunoblot analyses. Data shown are representative of immunoblots from three independent transfections. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 NMR Sequence-Specific Backbone Assignments of SR-BI(405–475) The 1H,15N-HSQC NMR spectrum displays the backbone assignments of SR-BI(405–475) at 40°C in 5% (w/v) deuterated LPPG. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 The Solution NMR Structure of SR-BI(405–475) (A–C) Purified [U-13C,15N]-SR-BI(405–475) was solubilized in water containing 5% deuterated LPPG detergent, 10% D2O, and 0.02% sodium azide at pH 6.8. 3D NMR spectra including HNCO, HNCOCA, HNCOCACB, HNCACB, CCONH, HBHACONH, and HCCH-TOCSY (total correlation spectroscopy) were collected on a Bruker 600-MHz spectrometer at 40°C. The structure was calculated by GeNMR (Berjanskii et al., 2009) and CYANA (Guntert, 2004), and further refined manually. The overlay of 20 calculated structures of SR-BI(405–475) are shown for (A) helix 1 (residues 409–419), (B) helix 2 (residues 427–436; aligned to residues), and (C) helix 3 (residues 438–469). Alignment was performed for each helix individually. (D) A single calculated structure of SR-BI(405–475) is shown for simplicity. (E) The heteronuclear 2D 15N-[1H] nuclear Overhauser effect (hetNOE) NMR experiment was performed using [U-15N]SR-BI(405–475) in 5% (w/v) LPPG detergent micelles in H2O, at pH 6.8. The transmembrane domain is overlined in black. Values represent the mean ± SD of two independent experiments. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 SR-BI(405–475) Harboring Specific Leu Mutations Displays Changes in NMR Chemical Shifts [U-15N]L413A-SR-BI(405–475) (A), [U-15N]L427A-SR-BI(405–475) (B), [U-15N]L448A-SR-BI(405–475) (C), and [U-15N]ΔLZ-SR-BI(405–475) (D) were solubilized in water containing 5% LPPG, 10% D2O, and 0.02% sodium azide (pH 6.8), analyzed by 1H,15N-HSQC, and overlaid with [U-15N]SR-BI(405–475) in black. Residues with significant chemical-shift changes are mapped on the SR-BI(405–475) structure and are highlighted as L413A-SR-BI(405–475) in pink (E); L427A-SR-BI(405–475) in green (F); L448A-SR-BI(405–475) in blue (G); and ΔLZ-SR-BI(405–475) in purple (H). The mutated leucine residues are highlighted in yellow, and all proline residues are highlighted in gray. (I) The residues with significant chemical shift changes mapped to the structures in (E–H) are highlighted in blue. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 8 SR-BI(405–475) Has Regions with Low Solvent Accessibility (A) SR-BI(405–475) was prepared in water containing 5% (w/v) LPPG and 10% (v/v) D2O at pH 6.8. Increasing concentrations of MnCl2 were titrated into the NMR sample (0–200 μM MnCl2) and 1H,15N-HSQC spectra were acquired. Peak intensities from the resulting 1H,15N-HSQC spectra were recorded. The resulting peak intensity at MnCl2 concentration of 50 μM is plotted for each residue as a percentage of its original peak intensity. P, proline residues; D, residues corresponding to unassigned peaks (due to degeneracy or peak shifting). (B) 15N-NOESY spectra collected during structure calculation were investigated for the presence of water peaks for each residue. Representative strips are shown with water peaks at 4.63 ppm highlighted in blue. (C) Residues in the SR-BI(405–475) structure that exhibit the presence of water peaks are highlighted in blue, as determined by the 15N-NOESY spectrum. (D) [U-15N]SR-BI(405–445), a construct consisting primarily of the extracellular region, was suspended in water with 10% D2O at pH 6.8. Increasing percentages of LPPG were titrated into the sample and analyzed by 1H,15N-HSQC. See also Figure S1. Structure 2017 25, 446-457DOI: (10.1016/j.str.2017.01.001) Copyright © 2017 Elsevier Ltd Terms and Conditions