Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Abstract

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.

FIGURE 1

A. Far-UV CD spectra (θ₂₂₂(λ))of [198-243]apoA-I: (□) 0.005 mg/mL, (○) 0.01 mg/mL, (△) 0.025 mg/mL, (▽) 0.05 mg/mL, (◇) 0.1 mg/mL, (+) 0.25 mg/mL, and (×) 0.5 mg/mL. The samples were in 0.01M phosphate at pH 7.4 and 25 °C. The insert figure shows the α-helical content of the peptide based on θ₂₂₂, as a function of peptide concentration.

FIGURE 2

Electrophoresis studies on [198-243]apoA-I in solution. A. 1 mg/mL [198-243]apoA-I in solution, separated on SDS PAGE. B. 1 mg/mL [198-243]apoA-I in solution, separated on native 4–15% Phast Gel. C. Glutaraldehyde cross-linked peptides in solution, separated on 10–20% Tricine gradient gel. The concentrations of glutaraldehyde were: (lane1) 0, (lane 2/5) 0.001%, (lane 3/6) 0.01% and (lane 4/7) 0.1%. Peptide concentrations were 2 mg/mL from lane 1 to lane 4 and 1 mg/mL from lane 5 to lane 7.

FIGURE 3

A. Thermal unfolding spectra (θ₂₂₂(T)) of [198-243]apoA-I as a function of peptide concentration. The temperature range was 1°C to 95°C. The two lines show heating and cooling. The scanning rate was 0.0167 K/s (60 s/deg). Four peptide concentrations are shown: (△) 0.025 mg/mL, (▽) 0.05 mg/mL, (□) 0.1 mg/mL and (×)0.5 mg/mL. B. Thermal unfolding spectra (θ₂₂₂(T)) of [198-243]apoA-I, starting from 25 °C to its unfolded state at a lower scan rate of 0.0033 K/s (300 s/deg). Five peptide concentrations are shown: (□) 0.0125 mg/mL, (△) 0.025 mg/mL, (▽) 0.05 mg/mL, (◇) 0.1 mg/mL and (○)0.5 mg/mL. C. Differentiation analysis of data in figure 3A: dθ₂₂₂(T)/dT, as function of temperature. The corresponding peptide concentration and apparent T_m (marked by the arrows) for the five curves from left to right were: (1) 0.0125 mg/mL, ~ 54°C; (2) 0.025 mg/mL, ~ 61°C; (3) 0.05 mg/mL, ~ 70–75°C; (4) 0.1 mg/mL ~ 75–80°C; and (5) 0.5 mg/mL, ~ 80–92°C. D. Differential Scanning Calorimetry (DSC) on 1 mg/mL [198-243]apoA-I with a heating rate of 0.0083 K/s (30 deg/hour). The best fitting with a non-two- state tetramer dissociation model is shown. The symbols are: (—) normalized data from DSC measurement, (…) baseline generated by the fitting model and ( ) fitting curve. Buffer line was subtracted. Only the larger peak at high temperature was analysized. The baseline was initiated according to the shape and further refined by the fitting model.

FIGURE 4

A. The α-helical percentage of [198-243]apoA-I on the basis of θ₂₂₂, as a function of BOG concentration. The peptide concentration was 0.005 mg/mL. The concentrations of BOG from left to right were: 0%, 0.2%, 0.4%, 0.8%, 1.0%, 1.2% and 1.6%. The corresponding BOG:peptide molar ratios were displayed on the top axis. B. The α-helical percentage of [198-243]apoA-I on the basis of θ₂₂₂, as a function of SDS concentration. The peptide concentration was 0.005 mg/mL. The concentrations of SDS from left to right were: 0, 0.0002%, 0.0004%, 0.0008%, 0.001%, 0.0012%, 0.0014%, 0.0018%, 0.002%, 0.0024%, 0.125% and 0.25%. The corresponding SDS:peptide molar ratios are shown on the top axis. C. Cross-linking [198-243]apoA-I in the presence of various amounts of SDS. The peptide concentration was 1 mg/mL. Lane 1: no SDS, no cross-linker as control; lane 2: no SDS and 0.01% glutaraldehyde; lane 3: 0.2% SDS (typical CMC) and 0.01% glutaraldehyde; lane4: 0.02% SDS (~10 fold below its typical CMC but high enough to induce α-helical structure formation in [198-243]apoA-I) and 0.01% glutaraldehyde.

FIGURE 5

Density gradient ultracentrifugation of [198-243]apoA-I/DMPC mixtures at three initial lipid: peptide (L:P) w/w ratios: 1:1, 4:1 and 8:1. A. DMPC distribution in the density gradient. The symbols are: (–▼–) 1:1, (–▲–) 4:1, (^—•^—) 8:1, and (–■–) DMPC alone. B. The corresponding peptide distribution in the density gradient. Symbols are the same as above. The insert figure shows the apparent α-helical percentage of [198-243]apoA-I/DMPC from simple mixtures peptide/DMPC at different DMPC:[198-243]apoA-I w/w ratios with a peptide concentration of 0.005 mg/mL at pH 7.4 and 25°C: (■) no lipid, (▼)1:1, (+) 2:1, (▲) 4:1, (◆)6:1, (●) 8:1, and (×) 10:1. C. The corresponding DMPC: [198-243]apoA-I w/w ratio for each fraction that contains complexes as a function of density. Symbols are the same as in figure 5A. The arrows and boxes marked the peak fraction of each initial ratio: (1) 1:1; (2) 4:1; and (3) 8:1.

FIGURE 6

Negative staining EM images at 45K magnification observation of the peak fractions for two initial DMPC: [198-243]apoA-I w/w samples and their corresponding size distributions. A. 1:1 B. 4:1

FIGURE 7

Glutaraldehyde cross-linked peptide/protein complexes of the protein-DMPC complexes, separated on 10–20% Tricine gradient gel. Panel A. 0.8 mg/mL A-I in solution. The concentrations of glutaraldehyde were: (lane1) 0, (lane2) 0.001% and (lane3) 0.01%. Panel B. 0.8 mg/mL plasma A-I in complex with DMPC from lipid:protein 4:1 w/w simple mixture. The concentrations of cross-linker were the same as panel A. Panel C. 0.8 mg/mL [198-243]apoA-I in complexed with DMPC from lipid:peptide 1:1 w/w simple mixture: (lane1) no cross-linker and (lane2) 0.01% glutaraldehyde. Panel D. 0.8 mg/mL [198-243]apoA-I in the peak fraction of initial DMPC: peptide 4:1 w/w: (lane1) no glutaraldehyde and (lane2) 0.01% glutaraldehyde. Panel C was silver stained to show the protein bands.

FIGURE 8

A. Comparison thermal unfolding (θ₂₂₂(T)) of complexes formed by [1-44]apoA-I, [198-243]apoA-I and plasma A-I with DMPC. The samples were lipid:peptide/protein w/w 4:1 for all, prepared at monomeric peptide or protein concentrations. The scanning rate was 0.0033 K/s (300 s/deg) for all. The top figure shows thermal unfolding heating curves of the three samples; the figure below shows their corresponding differential analysis that yielded the apparent T_m. B. Comparison Temperature-jump data (θ₂₂₂(t)) of complexes formed by [1-44]apoA-I, [198-243]apoA-I and plasma A-I with DMPC. Samples were prepared at monomeric peptide or protein concentrations. The (θ₂₂₂(t)) spectra were recorded every 10 seconds. The top figure shows temperature-jump data of the three samples in full length; the first 2000 seconds of those spectra are amplified in the figure below.