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. Author manuscript; available in PMC: 2017 Aug 30.
Published in final edited form as: Structure. 2017 Feb 2;25(3):446–457. doi: 10.1016/j.str.2017.01.001

NMR structure of the C-terminal transmembrane domain of the HDL receptor, SR-BI, and a functionally-relevant leucine zipper motif

Alexandra C Chadwick 2, Davin R Jensen 2, Paul J Hanson 1, Philip T Lange 1, Sarah C Proudfoot 2, Francis C Peterson 2, Brian F Volkman 2,*, Daisy Sahoo 1,2,3,*
PMCID: PMC5575897  NIHMSID: NIHMS899392  PMID: 28162952

SUMMARY

The interaction of high density lipoprotein (HDL) with its receptor, scavenger receptor BI (SR-BI), is critical for lowering plasma cholesterol levels and reducing the risk for cardiovascular disease. The HDL/SR-BI complex facilitates delivery of cholesterol into cells and is likely mediated by receptor dimerization. This work describes the use of nuclear magnetic resonance (NMR) spectroscopy to generate the first high-resolution structure of the C-terminal transmembrane domain of SR-BI. This region of SR-BI harbors a leucine zipper dimerization motif, that when mutated, impairs the ability of the receptor to bind HDL and mediate cholesterol delivery. These losses in function correlate with the inability of SR-BI to form dimers. We also identify juxtamembrane regions of the extracellular domain of SR-BI that may interact with the lipid surface to facilitate cholesterol transport functions of the receptor.

Keywords: transmembrane domain, SR-BI, dimerization, cholesterol, selective uptake, detergent micelle, juxtamembrane

INTRODUCTION

As more than 600,000 cardiovascular disease-related deaths occur annually in the United States (Mozaffarian et al., 2016), large efforts have been focused on understanding the pathology, progression and prevention of this deadly disease. High density lipoprotein (HDL) prevents cardiovascular disease via multiple mechanisms by virtue of its anti-oxidant and anti-inflammatory properties (reviewed in (Navab et al., 2011)), and most notably, by its ability to promote the excretion of cholesterol via the reverse cholesterol transport pathway. In this pathway, HDL transports cholesterol from peripheral tissues to the liver for net excretion by bile formation (Glomset, 1968). The HDL receptor, scavenger receptor class B type I (SR-BI), facilitates cholesterol flux between HDL and cells, and is therefore an important protein responsible for whole body cholesterol removal. Indeed, hepatic overexpression of SR-BI in mice significantly reduces atherosclerosis (Ji et al., 1999; Kozarsky et al., 2000; Ueda et al., 2000; Ueda et al., 1999; Wang et al., 1998), while disruption of the SR-BI gene leads to a marked acceleration of atherosclerosis (Braun et al., 2002; Rigotti et al., 1997; Varban et al., 1998). Several loss-of-function mutations in the human SR-BI gene, SCARB1, identified in patients with high HDL-cholesterol levels, resulted in impaired HDL binding to the receptor, as well as inefficient delivery of HDL-cholesteryl ester (CE) into cells (Brunham et al., 2011; Chadwick and Sahoo, 2012; Vergeer et al., 2011). Recently, it was discovered that patients harboring a homozygous variant of SR-BI, P376L-SR-BI, have a significantly increased risk of coronary heart disease due to impaired clearance of HDL-CE (Zanoni et al., 2016). This recent work highlights the physiological relevance of the SR-BI receptor and emphasizes the need to understand how SR-BI functions to facilitate net cholesterol excretion.

SR-BI (509 amino acids) consists of a large, heavily glycosylated extracellular domain (408 residues) that is anchored by two transmembrane domains (22 and 23 residues) and two cytoplasmic tails (N-terminal: 9 residues; C-terminal: 47 residues) (Krieger, 1999). In order to facilitate cholesterol transport, HDL and SR-BI must form a “productive complex” whereby HDL binds to the extracellular domain of SR-BI in a precise conformation that allows efficient lipid transport (Liu et al., 2002). Our recent work (Chadwick and Sahoo, 2012; Holme et al., 2016; Kartz et al., 2014; Papale et al., 2011; Papale et al., 2010; Parathath et al., 2004), as well as that by others (Connelly et al., 2003; Guo et al., 2011; Vinals et al., 2003; Yu et al., 2012), has firmly demonstrated that specific regions of the extracellular domain of SR-BI are important for HDL binding, HDL-CE selective uptake, and efflux of free cholesterol (FC) to HDL acceptor particles. The high-resolution crystal structure (Neculai et al., 2013) of the related scavenger receptor family member, lysosome integral membrane protein type 2 (LIMP-2), has provided valuable insight into the structure of the extracellular domain of SR-BI through homology modeling. Although this model has undoubtedly improved our understanding of how SR-BI may perform its cholesterol transport functions, a high-resolution structure of SR-BI, including the tertiary structure of SR-BI’s transmembrane domains and cytoplasmic regions, remains unavailable and hinders a deeper understanding of the HDL/SR-BI interaction.

Another important feature of SR-BI that may be critical for mediating HDL-CE delivery into cells is its ability to form homo-oligomers. SR-BI homo-oligomerization has been observed in various cell types (Reaven et al., 2004; Sahoo et al., 2007a; Sahoo et al., 2007b) and is believed to form a non-aqueous “hydrophobic channel” (Rodrigueza et al., 1999) that facilitates the transport of cholesterol between HDL and the plasma membrane. The oligomerization of SR-BI is thought to be mediated by the transmembrane domain regions since they both contain putative dimerization motifs: a glycine dimerization motif (Russ and Engelman, 2000) resides in the N-terminal transmembrane domain of SR-BI (Gaidukov et al., 2011), while a putative leucine zipper domain resides adjacent to and within the C-terminal transmembrane domain. Although both transmembrane domains may contribute to SR-BI homo-oligomerization (Gaidukov et al., 2011), fluorescence resonance energy transfer studies in live cells demonstrated that dimerization occurs through interactions between the C-terminal transmembrane domains (Sahoo et al., 2007b). At this time, the dynamics of SR-BI oligomerization are not understood, and it is also currently unknown how HDL binding may alter this interaction.

In an earlier study, we described the purification strategy for a peptide that encompasses residues 405–475 of SR-BI that includes the C-terminal transmembrane domain (i.e. SR-BI(405–475)), as well as its reconstitution into detergent micelles (Chadwick et al., 2015). In the current report, we present the first high-resolution structural information for SR-BI(405–475) in detergent micelles, as determined by nuclear magnetic resonance (NMR). As this region is believed to be important for homo-dimerization (Sahoo et al., 2007b), we designed a series of mutations that span the putative leucine zipper motif and characterized the functionality of these mutants in vitro, as well as their contribution to receptor conformation by NMR. The novel information obtained from this study improves our knowledge of regions of SR-BI that are necessary for cholesterol transport functions and dimerization, and is undoubtedly an important step in understanding how SR-BI functions to lower plasma cholesterol levels.

RESULTS

SR-BI(405–475) is evolutionarily conserved with a predicted alpha-helical secondary structure

Our study was aimed at investigating the structural properties of the C-terminal transmembrane domain of SR-BI. Bioinformatic analysis of this region of SR-BI indicates that residues 405 to 475 are evolutionarily conserved (~54% identity), similar to full length SR-BI (~57% identity), as illustrated by sequence alignment of SR-BI(405–475) from different species (Figure 1A). Kyte-Doolittle hydropathy analysis (Kyte and Doolittle, 1982) suggests that SR-BI(405–475) is largely hydrophobic, with a predicted transmembrane domain from residues 441–465. This analysis also reveals other, smaller regions with predicted high hydrophobicity (i.e. between residues 409–415 and 434–438) that are located outside of the predicted C-terminal transmembrane domain, and suggest potential membrane-interacting regions.

Figure 1. Sequence alignment, secondary structure prediction, and helical wheel alignment of SR-BI(405–475).

Figure 1

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(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 “*”, while residues with similar properties are denoted with “:”. (B) Prediction software programs were used to predict the secondary structure of SR-BI(405–475). Beta-strands are denoted in blue and alpha-helices in red. (C) Helical wheel alignment of residues 408–419, (D) residues 426–436, and (E) residues 438–465 are shown. Non-polar residues (peach), polar residues (orange), and leucines in the putative leucine zipper (blue) are highlighted.

Transmembrane domains often exhibit alpha-helical structure since the hydrophobic environment elicits strong hydrogen bonding (Donnelly, 1993). We used various secondary structure prediction software programs to better predict the secondary structure of SR-BI(405–475) (Figure 1B). Jpred relies on homology modeling (Drozdetskiy et al., 2015) and predicted a beta-strand near the N-terminal extracellular region of SR-BI(405–475), similar to the homology model generated using the LIMP-2 structure (Neculai et al., 2013). In contrast, both PORTER (Pollastri and McLysaght, 2005) and TALOS (Cornilescu et al., 1999), prediction programs that use the amino acid sequence or NMR chemical shift information, respectively, predicted an N-terminal extracellular alpha-helix of SR-BI(405–475). Unsurprisingly, all three programs predicted a long alpha-helix spanning the anticipated transmembrane domain.

Along with a potential glycine (i.e. GXXXG) dimerization motif, the C-terminal region of SR-BI has a highly conserved putative leucine zipper motif where every seventh residue is a leucine residue. This region spans amino acids 413 to 455 (with the exception of G420 that begins the GXXXG motif, but also sits in place of a leucine residue within the pattern of the motif). Helical wheel analysis of residues 408–419 (Figure 1C), residues 426–436 (Figure 1D) and residues 438–465 (Figure 1E) shows clustering of residues in the putative leucine zipper, as well as amphipathic helical characteristics.

Mutations of the leucine zipper motif of SR-BI display reduced receptor function(s) and loss of dimerization

In order to test the importance of each residue within the putative leucine zipper region in mediating SR-BI’s cholesterol transport functions, we used site-directed mutagenesis to create single point mutations (L413A-, L427A-, L434A-, L441A-, L448A-, L455A-SR-BI) in the full-length SR-BI receptor. We also created a ΔLZ-SR-BI, where these same leucine residues were replaced with alanine, and the glycine at 420 was replaced with histidine. With the exception of L455A-SR-BI, immunoblot analyses indicated that SR-BI mutant receptors expressed at similar levels as wild-type (WT) SR-BI upon transient transfection in COS-7 cells (Figures 2A and B). L413A- and ΔLZ-SR-BI showed trends for lower levels of expression, although not reaching statistical significance. Levels of cell surface expression were quantified by flow cytometry (Figure 2C).

Figure 2. Expression levels of mutant SR-BI receptors.

Figure 2

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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 control were detected by immunoblot analysis using an anti-SR-BI or anti-GAPDH antibody. Data are representative of 4 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 arbitrary units (AU) represent the mean of 4 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 4 independent transfections. *p<0.05 by one-way ANOVA.

To determine if SR-BI function was compromised upon mutation of the leucine residues, COS-7 cells transiently expressing either empty vector, WT or mutant SR-BI receptors were incubated with [125I]/[3H]COE-HDL and levels of HDL binding (Figure 3A) and COE selective uptake (Figure 3B) were measured. L413A-SR-BI and ΔLZ-SR-BI both displayed a significantly impaired ability to bind HDL and mediate selective uptake of HDL-COE (approximate 65% and 95–98% decreases for both functions respectively, compared to WT SR-BI). Although not significant, L434A-, L441A-, and L455A-SR-BI lost approximately 30–40% of their ability to bind HDL and mediate selective uptake of HDL-COE. Interestingly, both L427A- and L448A-SR-BI displayed WT levels of function.

Figure 3. Mutation of several residues in the putative leucine zipper motif disrupts SR-BI function.

Figure 3

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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 hours. (A) Cell association of [125I]HDL and (B) selective uptake of [3H]COE are shown. 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 hours. 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 hours. Cholesterol species were separated by thin layer chromatography and % cholestenone was determined. Values represent the mean ± SEM of three independent experiments, each performed in quadruplicate. All data sets 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 3012.4 ng HDL-COE/mg cell protein (HDL-COE uptake); 5.5% and 9.2% (cholesterol efflux); 13.1% and 37.9% (cholestenone production). Statistical analyses were determined by one-way ANOVA comparing each mutant to WT SR-BI from respective experiments. *p<0.05, **p<0.01, ***p<0.001

In addition to its ability to deliver CE from HDL into cells, SR-BI also plays a role in stimulating FC efflux from cells to HDL acceptor particles (Acton et al., 1996). We measured the ability of WT or mutant SR-BI receptors to efflux FC to HDL acceptors from pre-labeled COS-7 cells. L413A-, L448A- and ΔLZ-SR-BI displayed significant decreases in FC efflux (approximately 60–100%) as compared to WT SR-BI (Figure 3C). The ability of L455A-SR-BI to efflux FC to HDL was reduced by ~45%, although it did not reach statistical significance. L427A- and L441A-SR-BI did not display any notable changes in function.

SR-BI plays an important role in re-organizing plasma membrane FC, and in the presence of exogenous cholesterol oxidase, this unique function can be studied by quantifying the conversion of cholesterol to cholestenone (Kellner-Weibel et al., 2000). We found that all mutant receptors, with the exception of L434A-SR-BI, had significantly impaired abilities to generate cholestenone as compared to WT SR-BI (Figure 3D).

Next, to test the hypothesis that the leucine zipper was required for oligomerization of SR-BI, WT- or ΔLZ-SR-BI were transiently transfected in COS-7 cells and chemically crosslinked using membrane-impermeable bis(sulfosuccinimidyl)suberate (BS3). Immunoblot analyses of cell lysates revealed the presence of dimers and higher-order oligomers in WT-SR-BI, but only monomeric forms of ΔLZ-SR-BI were observed (Figure 4). Interestingly, oligomeric forms of ΔLZ-SR-BI were more evident at longer exposures (data not shown), suggesting that the receptor takes longer to self-associate in the absence of the leucine zipper motif.

Figure 4. SR-BI self-association is dependent on the leucine zipper motif.

Figure 4

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COS-7 cells transiently expressing WT or LZ-SR-BI were incubated for 1h 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 3 independent transfections.

The NMR structure of SR-BI(405–475) reveals three primary alpha helices

Purified recombinant [U-13C, 15N]-SR-BI(405–475) was reconstituted into 5% (w/v) deuterated-LPPG detergent micelles in H2O at pH 6.8 as previously described (Chadwick et al., 2015). Standard methods were used to assign the 1H, 15N and 13C chemical shifts of SR-BI(405–475) from three-dimensional triple-resonance NMR spectra (Figure 5). The NMR structure of SR-BI(405–475) was solved using distance constraints from 3D NOESY-HSQC spectra and torsion angle constraints produced by TALOS, and reveals three primary alpha helices (Figure 6). Structure statistics are shown in Table 1. As anticipated based on circular dichroism analyses (Chadwick et al., 2015), SR-BI(405–475) is alpha-helical, and NMR structural analyses revealed two shorter alpha-helices in the N-terminal region (residues 409 to 419, and 427 to 436), as well as a long alpha-helix spanning the entire transmembrane domain (residues 438 to 469), similar to the secondary structure prediction analysis.

Figure 5. NMR sequence-specific backbone assignments of SR-BI(405–475).

Figure 5

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The 1H-15N-HSQC NMR spectrum displays the backbone assignments of SR-BI(405–475).

Figure 6. The solution NMR structure of SR-BI(405–475).

Figure 6

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(A) 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 were collected on a Bruker 600mHz 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 to 419), (B) Helix 2 (residues 427 to 436; aligned to residues), and (C) Helix 3 (residues 438 to 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 two-dimensional 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.

Table 1.

NMR refinement statistics of the 20 model ensembles of SR-BI.

Experimental constraints
Distance constraints
 Long 0
 Medium [1<(i-j)≤5] 81
 Sequential [ (i-j)=1] 234
 Intraresidue [i=j] 254
 Total 569
Dihedral angle constraints (ϕ and ψ) 119
Average atomic R.M.S.D. to the mean structure (Å)
SR-BI residues
Helix 1 (Residues 407–419)
 Backbone (Cα, C′, N) 0.56 ± 0.20
 Heavy atoms 1.31 ± 0.26
Helix 2 (Residues 427–416)
 Backbone (Cα, C′, N) 0.48 ± 0.17
 Heavy atoms 1.14 ± 0.25
Helix 3 (Residues 439–468)
 Backbone (Cα, C′, N) 1.33 ± 0.65
 Heavy atoms Helix 1.90 ± 0.71
WHATCHECK quality indicators
 Z-score −1.44 ± 0.21
 RMS Z-score
  Bond lengths 0.61 ± 0.02
  Bond angles 0.61 ± 0.03
 Bumps 0 ± 0
Lennard-Jones energy a (kJ mol−1) −1,076 ± 68
Constraint violations
 NOE distance Number > 0.5 Å 0 ± 0
 NOE distance RMSD (Å) 0.0227 ± 0.0031
 Torsion angle violations Number > 5 ° 0 ± 0
 Torsion angle violations RMSD (°) 0.546 ± 0.128
Ramachandran statistics (% of all residues)
 Most favored 95.3 ± 1.8
 Additionally allowed 2.9 ± 2.1
 Generously allowed 1.3 ± 1.2
 Disallowed 0.5 ± 1.0
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a

Nonbonded energy was calculated in XPLOR-NIH.

In order to understand the overall dynamics of SR-BI(405–475), a heteronuclear two-dimensional 15N-[1H] nuclear Overhauser effect (hetNOE) NMR experiment (Kaiser, 1963) was performed using [U-15N]-SR-BI(405–475) in LPPG detergent micelles (Figure 6E). Our data indicate that the residues with high heteronuclear NOE values correspond to the helical regions of the NMR structure (residues 409–419, 427–435, and 442–463). Residues near the edges of the construct (405–408 and 465–475) exhibit lower heteronuclear NOE values, consistent with greater flexibility and disorder. Additionally, we observed low NOE values for the GXXXG motif region (residues 420–424) that links the two short N-terminal helices and the boundary between the second helix and the predicted transmembrane helix (residues 436–440). Higher and lower heteronuclear NOE values reflect the conformational variability of the NMR ensemble (Figure 6A–C), which consists of three relatively well-defined helical segments connected by flexible linkers.

1H-15N-HSQC analysis of selected leucine zipper mutants confirms residues critical for protein function

Since L413A-, L427A-, L448A-, and ΔLZ-SR-BI displayed decreases in several SR-BI-mediated cholesterol transport functions, we compared the HSQC spectra of L413A-, L427A- L448A-, and ΔLZ-SR-BI(405–475) with the WT SR-BI(405–475) spectrum to assess chemical shift perturbations and determine whether large-scale structural changes contributed to functional impairment (Figure 7). The two mutants that exhibited impaired cholestenone production and/or FC efflux, L427A- and L448A-SR-BI, displayed merely 9 perturbed peaks each, corresponding to nearby residues (Figure 7B, C, F and G), consistent with local effects rather than a major conformational rearrangement. In contrast, mutation of leucine 413 to alanine, that in full-length SR-BI impaired most functions of the receptor, resulted in significant chemical shift changes for at least 18 residues in SR-BI(405–475) spanning the range from 405–423 (Figure 7A and E). Combining these findings with the results of the SR-BI functional assays, it was unsurprising that ΔLZ-SR-BI(405–475) had a very different 1H-15N-HSQC profile, with more than 70% of the residues displaying major chemical shift changes (Figure 7D and H). Despite the numerous changes, the chemical shift dispersion of the ΔLZ-SR-BI(405–475) HSQC spectrum was comparable to the other variants, suggesting that the micelle-bound helical conformation was preserved.

Figure 7. SR-BI(405–475) harboring specific Leu mutations displays changes in NMR chemical shifts.

Figure 7

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(A) [U-15N]-L413A-SR-BI(405–475), (B) [U-15N]-L427A-SR-BI(405–475), (C) [U-15N]-L448A-SR-BI(405–475), and (D) [U-15N]-ΔLZ-SR-BI(405–475) were solubilized in water containing 5% LPPG, 10% D2O, and 0.02% sodium azide at pH 6.8, analyzed by 1H-15N-HSQC, and overlayed 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 (E) L413A-SR-BI(405–475) in pink; (F) L427A-SR-BI(405–475) in green; (G) L448A-SR-BI(405–475) in blue; (H) ΔLZ-SR-BI(405–475) in purple. The mutated leucine residues are highlighted in yellow, and all proline residues are highlighted in grey.

SR-BI(405–475) includes regions with possible juxtamembrane interactions

Since Kyte-Doolittle hydropathy analysis suggested possible membrane-interacting segments in the C-terminal extracellular region of SR-BI near residues 409–415 as well as residues 434–438, we used several techniques to further characterize solvent accessibility. MnCl2 titrations were performed to preferentially induce paramagnetic broadening of solvent-exposed residues in SR-BI(405–475). Over increasing concentrations of MnCl2, most of the peaks corresponding to residues 418–429 and 465–475 lost at least 50% of their maximal peak intensity ≤ 50 μM MnCl2 (Figure 8A). Conversely, the majority of residues 409–416 and 425–475 still exhibited at least 50% of their maximal peak intensity at 50 μM MnCl2, suggesting that these residues were embedded within or associated with the micelle.

Figure 8. SR-BI(405–475) has regions with low solvent accessibility.

Figure 8

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(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; ‘U’: 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 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.

Another indicator of high solvent accessibility is the presence of strong exchange cross peaks at the bulk H2O chemical shift (4.63 ppm) in the 3D 15N-edited NOESY-HSQC spectrum. Backbone NH groups for residues that are involved in secondary structure hydrogen bonds or buried within the micelle should exchange with solvent more slowly, and are reflected by the absence of an exchange peak at the H2O chemical shift. Further, when analyzed by 15N-NOESY, water exchange peaks (suggesting solvent accessibility) were absent in NOESY strips for regions of high hydrophobicity such as residues 409 to 415, but present in solvent-exposed regions such as residues 418 to 424 (Figure 8B). Figure 8C highlights residues that demonstrate an absence of water peaks in the 15N-NOESY spectra for two regions of the extracellular domain (residues 409 to 416, and 431 to 440), as well as the entire transmembrane domain.

To further examine the possibility of membrane-interacting regions near SR-BI’s C-terminal transmembrane domain, an SR-BI fragment (residues 405–445) lacking the hydrophobic transmembrane domain was also purified and examined by NMR. Interestingly, no HSQC peaks were observed when SR-BI(405–445) was initially suspended in an aqueous solution. However, addition of increasing amounts of LPPG detergent resulted in the appearance of peaks (Figure 8D) that corresponded closely to many of the resonances for residues 405–445 observed in the SR-BI(405–475) spectrum (Figure S1). This important observation suggests that SR-BI(405–445) requires a hydrophobic environment to fold into its native structural conformation, thereby suggesting that this region harbors a potential membrane-interacting juxtamembrane domain.

DISCUSSION

In the current report, we provide the first high-resolution structural information for residues 405 to 475 of SR-BI that encompass the entire C-terminal transmembrane domain of the receptor embedded in a detergent micelle. We also characterized the functionality of this region of the SR-BI receptor that appears to be critical for receptor function. Our group (Sahoo et al., 2007a; Sahoo et al., 2007b) and others (Azhar et al., 2002; Gaidukov et al., 2011; Landschulz et al., 1996; Reaven et al., 2004) have demonstrated SR-BI oligomerization, a process that is postulated to facilitate HDL-CE delivery into cells, and is most likely mediated, in part, by homo-dimerization between the C-terminal transmembrane domains of SR-BI monomers (Sahoo et al., 2007b).

The C-terminal transmembrane domain of SR-BI possesses a putative leucine zipper motif. We examined the functional importance of each individual leucine within this motif of SR-BI, and used NMR analysis to further understand why several single leucine mutations lead to disruption of different receptor functions. We observed that mutation of L413 significantly disrupts HDL binding, HDL-CE uptake, and cholestenone production and corroborates previous findings (Parathath et al., 2007; Parathath et al., 2004; Sahoo et al., 2007b) that residues in this region are imperative for all of these cholesterol transport functions. Indeed, we recently showed that mutation of tryptophan 415 to phenylalanine disrupted several SR-BI functions (Holme et al., 2016).

NMR analysis of the SR-BI(405–475) variants revealed spectral perturbations that were largely consistent with the observed functional effects. For example, residues 405–422, 427, and 428 of L413A-SR-BI(405–475) exhibited dramatic chemical shift differences relative to SR-BI(405–475). As residues 415–419 have significantly altered chemical shifts in L413A-SR-BI(405–475), it is not surprising that this mutant has disrupted function in our in vitro assays, and lends support to the notion that this region is critical for protein function. Conversely, mutation of L427 did not impair most SR-BI functions and only caused significant local NMR chemical shift changes for residues 424–432. Interestingly, the L448A-SR-BI mutant, which revealed chemical shift changes for the majority of residues 441–450, is still able to bind and deliver HDL-CE efficiently. However, previous work has demonstrated that glutamine 445 is a critical plasma membrane cholesterol sensor (Saddar et al., 2013), and that mutation of this residue attenuates the receptor’s ability to interact with plasma free cholesterol, albeit while still maintaining normal cholesterol flux functions; perhaps the L448A-SR-BI mutation alters the local chemical environment enough to impair the plasma membrane cholesterol sensing capability of Q445. Lastly, the ΔLZ-SR-BI mutant had a near-complete impairment in all cholesterol flux functions, and showed significant chemical shift changes by NMR for the majority of residues 405–464. In addition, mutation of the leucine zipper also appears to disrupt the receptor’s ability to form dimers and higher-order oligomers as evidenced by chemical crosslinking studies in cultured cells. Given that ΔLZ-SR-BI oligomers were observed at longer exposures (albeit to a lower extent than WT-SR-BI), one must consider the possibility that the leucine zipper is one of many domains that could mediate self-association of SR-BI. Indeed, previous studies have shown that other regions of SR-BI are implicated in receptor oligomerization (Gaidukov et al., 2011; Reaven et al., 2004).

Based on hydropathy analysis, secondary structure prediction, and homology modeling of residues 405–435 with the LIMP-2 structure (Neculai et al., 2013), we anticipated that SR-BI(405–475) would have two or three alpha helical segments that comprised segments of the extracellular domain (residues 408–419 and 426–436) and the entire transmembrane domain (residues 438–465). A portion of the solved SR-BI(405–475) structure aligns similarly to the homology model generated from the LIMP-2 structure (Neculai et al., 2013), with an alpha helix near the C-terminal transmembrane domain region (residues 426–436 compared to residues 423–432 reported previously (Neculai et al., 2013)). Of note, the N-terminal region of SR-BI (405–475) contains an alpha-helix in the NMR structure (residues 409–419) that is a beta-strand in the previous SR-BI homology model (Neculai et al., 2013).

The NMR structure of SR-BI(405–475) also highlights several key structural features, such as a kink in the alpha-helical transmembrane domain induced by proline 459. Proline residues residing in transmembrane alpha-helices play a critical role in protein signaling, structural flexibility, and oligomerization (reviewed in (Cordes et al., 2002; von Heijne, 1991)). We predict the kink at P459 may be functionally important for facilitating movement in membrane channels (Bright et al., 2002; Jin et al., 2002; Tieleman et al., 2001) necessary for cholesterol transport. Alternatively, this kink may act as a “hinge and/or swivel” that is necessary for binding of PDZK1 to the C-terminal cytoplasmic tail of SR-BI (Kocher and Krieger, 2009), and/or SR-BI-mediated signaling (Saddar et al., 2010). These possibilities are currently being explored.

Secondary structure prediction, as well as the solved NMR structure, suggest that the GXXXG region (G420-A421-M422-G423-G424) is an unstructured loop. Although not predicted to lie within the helix itself, G420 and L413 are situated facing the same side of the protein. Based on our 3D NMR chemical shift information, as well as backbone 15N relaxation rates of SR-BI(405–475) by [1H,15N] hetNOE, there was a marked flexibility observed in the GXXXG region. Flexibility of GXXXG motifs has been previously shown for a number of other proteins (Rogne et al., 2008), and has been implicated in the dimerization of proteins (reviewed in (Cymer et al., 2012)). It is possible this flexibility is required to facilitate the transition of SR-BI between monomeric and dimeric states and upon ligand binding, may adopt a more rigid secondary structure to help accommodate the delivery of cholesterol. Based on previous work, this GXXXG motif is critical for protein function (Parathath et al., 2004), but its potential relevance for protein oligomerization remains unknown.

Through the analysis of 1H-15N-HSQCs and paramagnetic broadening NMR experiments, we explored the solvent accessibility of SR-BI(405–475) residues. Analysis of the presence of water peaks in the 15N-NOESY spectrum revealed a large portion of extracellular residues that did not appear solvent-exposed. Through paramagnetic broadening NMR experiments, our data revealed that residues located in the extracellular region of SR-BI(405–475) displayed significant NMR peak broadening as compared to residues in the predicted transmembrane domain region. Several residues in an extracellular hydrophobic region (409 to 416), as well as residues located in the amphipathic helix region near the transmembrane domain (residues 426–439) were protected against broadening similar to residues located in the transmembrane domain (residues 440–465), suggesting potential extracellular contact site(s) that may interact with the lipid environment. Indeed, impairment of receptor function in vitro occurs upon mutation of W415 (Holme et al., 2016) and L413 that reside within this extracellular hydrophobic region, and may be the direct consequence of disruption of possible protein/membrane interactions. Further, SR-BI(405–445), a shorter peptide consisting of only the extracellular region of SR-BI, formed soluble aggregates in an aqueous solution that were undetectable by NMR until addition of LPPG detergent. These important observations suggest that there are one or two hydrophobic regions of the extracellular portion of SR-BI(405–475) that either interact with the lipid environment as a juxtamembrane domain, or alternatively, are buried in the structure of the full length SR-BI.

Despite the breadth of new structural knowledge garnered from these studies, numerous questions still remain as to how the structural features of SR-BI support efficient cholesterol transport functions. In particular, the forces that drive SR-BI homo-oligomerization remain unanswered. Chemical crosslinkers demonstrated homo-dimerization of recombinant SR-BI(405–475) in LPPG detergent micelles (Chadwick et al., 2015), yet the lack of dimerization of ΔLZ-SR-BI in cultured cells. Indeed, previous studies demonstrate that nearly 50% of oligomeric SR-BI receptors can be targeted for crosslinking (Gaidukov et al., 2011), yet it is difficult to ascertain whether this number under- or over-estimates SR-BI’s oligomeric status, as crosslinker targeting efficiencies, as well as the dynamics of the receptor, remain unknown. Further, the proportion of SR-BI that is oligomeric can also vary by cell and tissue type, as well as by hormonal stimuli (Azhar et al., 2002; Reaven et al., 2004). Importantly, the role of HDL in promoting receptor oligomerization has not been fully explored. Sophisticated techniques like fluorescence recovery after photobleaching (FRAP) (Dorsch et al., 2009) can potentially address the transient and dynamic nature of SR-BI oligomerization. While we continue our efforts to conclusively detect oligomerization by NMR, data interpretation must take into account our use of detergent micelles as a membrane mimetic, rather than the use of bicelles or nanodiscs as our membrane system (reviewed in (Borch and Hamann, 2009)). Further, while the C-terminal transmembrane domain region of SR-BI has been shown to be important for receptor oligomerization, it may not be completely sufficient for this process.

It has been proposed that SR-BI oligomers form a hydrophobic channel that facilitates cholesterol transport into the membrane (Rodrigueza et al., 1999), perhaps via homo-dimerization mediated by C-C and N-N transmembrane domain interactions. However, as the SR-BI homology model, based on the solved LIMP-2 structure, showed the presence of a hydrophobic tunnel in the monomeric protein, it is possible that the purpose of SR-BI oligomerization is to cluster multiple receptors that bind HDL for subsequent CE transfer through each individual, monomeric hydrophobic channel. However, it is also conceivable that CE can be delivered into cells using channels within each monomer and/or via a larger hydrophobic channel formed by receptor oligomerization. It also remains unknown whether the FC efflux function of SR-BI (i.e. the movement of cholesterol out of cells to HDL) requires the formation of an oligomeric hydrophobic channel.

In summary, we present the first solved structure of a portion of SR-BI spanning the C-terminal transmembrane domain region and report several key structural regions imperative for protein function. This novel structural information will undoubtedly further our understanding SR-BI function, and importantly, help us elucidate SR-BI/HDL interactions as we develop therapeutics to increase cholesterol flux out of the body and prevent atherosclerosis

EXPERIMENTAL PROCEDURES

Materials

The materials used are described in Supplemental Experimental Procedures.

Plasmids for mammalian cell transfection

Site-directed mutagenesis was used to generate six different leucine-to-alanine single point mutations at residues 413, 427, 434, 441, 448, or 455 in the murine SR-BI coding region cloned into a pSG5 vector (Stratagene, Inc; La Jolla, CA; (Connelly et al., 1999)). A single construct where all six of these leucine residues were mutated to alanine was also generated (herein referred to as ΔLZ-SR-BI) and included a glycine-to-histidine mutation at residue 420 (Parathath et al., 2004). Cloning, mutagenesis and sequencing were performed by Top Gene Technologies (Pointe-Claire, Quebec, Canada).

Cell culture and transfection

COS-7 cells were transiently transfected as previously described (Connelly et al., 1999), and cellular assays were performed 48 hours post-transfection, unless otherwise noted. Cell maintenance is described in Supplemental Experimental Procedures.

Cell surface expression

Cell surface expression of SR-BI was verified by flow cytometry (Papale et al., 2011) using a FACS Calibur or an Accuri C6, with additional details provided in Supplemental Experimental Procedures.

HDL labeling, cell association of [125I]HDL and uptake of [3H]HDL-COE

Non-hydrolyzable [3H]COE and [125I]dilactitol tyramine were used to double-label HDL as previously described (Connelly et al., 1999). Radiolabeled HDL preparations had average specific activities of 36.7 dpm/ng of protein for [3H] and 208.0 dpm/ng of protein for [125I]. COS-7 cells were transfected with empty pSG5 vector, WT or mutant SR-BI and cell association of [125I]-HDL and selective uptake of non-hydrolyzable [3H]-COE were assayed simultaneously as previously described (Connelly et al., 2003).

Assays to measure free cholesterol efflux and sensitivity to cholesterol oxidase

COS-7 cells transiently transfected with empty vector, WT, or mutant SR-BI were pre-labeled with [3H]cholesterol. Free cholesterol release from cells to HDL was assessed as described (Connelly et al., 2003) at 72 h post-transfection, whereas cells were assayed for sensitivity to exogenous cholesterol oxidase as previously described (Connelly et al., 2003) at 48 h post-transfection. Empty vector values were subtracted from WT SR-BI values and statistical comparisons were calculated by one-way ANOVA with Bonferroni post-tests for all groups.

Chemical crosslinking of SR-BI

Forty-eight hours post-transfection, COS-7 cells transiently expressing WT or LZ-SR-BI were incubated for 1h at 4°C with 0, 0.1, 0.3, or 1 mM BS3 (bis(sulfosuccinimidyl)suberate), a water-soluble, membrane-impermeable, amine-reactive crosslinker (Thermo Fisher Scientific). Cells were washed twice with ice-cold PBS and lysed in 1% NP-40 lysis buffer. Cell lysates were separated by 8% SDS-PAGE and SR-BI levels were detected by immunoblot analyses,

Plasmids for bacterial expression

Generation of plasmids for bacterial expression of SR-BI peptides is described in Supplemental Experimental Procedures (Chadwick et al., 2015). The two peptides used in these studies correspond to SR-BI residues 405–475 and residues 405–445, herein referred to as SR-BI(405–475) and SR-BI(405–445), respectively.

Purification of bacterially-expressed SR-BI constructs

Purification of SR-BI constructs was performed as previously described (Chadwick et al., 2015) and detailed in Supplemental Experimental Procedures. Briefly, bacterially-expressed SR-BI(405–475) was grown in M9 medium supplemented with either 15NH4Cl or 15NH4Cl and [U-13C]-glucose to produce [U-15N]- or [U-15N,13C]-labeled peptides. Cell pellets were solubilized with 10% empigen detergent and purified using a gravity flow column containing nickel (His60 Ni Superflow) resin. Further purification by reversed phase HPLC was performed. Peptides were lyophilized and stored at −80°C until used for NMR analysis.

NMR Analyses

Details of sample preparation and NMR analyses are described in Supplemental Experimental Procedures. Briefly, NMR samples (0.5–1 mM SR-BI protein) were prepared in water containing 5% (w/v) LPPG, 10% (v/v) D2O, and 0.02% (v/v) sodium azide at pH 6.8, unless otherwise specified. Resonance assignments and distance constraints were obtained for SR-BI(405–475) at 40 ˚C on either a Bruker Avance III 500 MHz or Bruker 600 MHz spectrometer. NMRPipe (Delaglio et al., 1995) was used for processing and data was analyzed using Xeasy (Bartels et al., 1995). TALOS was used for determining Φ and Ψ dihedral angle constraints (Cornilescu et al., 1999). CYANA was used (Herrmann et al., 2002) to generate the initial structures of SR-BI(405–475) and structures were further refined manually. Protein structure coordinates, NMR constraints and chemical shifts were deposited in the RCSB protein data bank and the BioMagResBank (PDB ID 5KTF; BMRB ID 30137).

NMR analysis of SR-BI(405–445) upon titration of LPPG detergent

SR-BI(405–445) (500 μM) was suspended in water containing 10% (v/v) D2O at pH 6.8. Increasing amounts of LPPG detergent (0–1% (w/v)) were titrated into the NMR sample and analyzed by 1H-15N-HSQC.

MnCl2-induced paramagnetic broadening measured by NMR

SR-BI(405–475) (1 mM) 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 peak intensities at the 50 μM MnCl2 titration were divided by the original peak intensities (0 μM MnCl2) and plotted.

Supplementary Material

Supplemental files

Acknowledgments

We thank Kay Nicholson for excellent technical assistance. This work was supported by National Institutes of Health grants R01HL58012 (D.S.), R01AI058072 (B.F.V.), and AHA14PRE185800221 (A.C.C.).

Footnotes

AUTHOR CONTRIBUTIONS

Conceptualization, A.C.C, D.R.J, F.C.P, B.F.V, and D.S; Methodology, A.C.C, D.R.J, F.C.P, B.F.V, and D.S; Validation, F.C.P.; Formal Analysis, A.C.C. and F.C.P.; Investigation, A.C.C, D.R.J, P.J.H, P.T.L, S.C.P. and F.C.P.; Resources, B.F.V. and D.S.; Writing – Original, A.C.C. and D.S.; Writing – Review & Editing, A.C.C., F.C.P, B.F.V. and D.S; Visualization, A.C.C. and D.S.; Supervision, F.C.P., B.F.V. and D.S.; Funding Acquisition, A.C.C., B.F.V. and D.S

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