HDL mimetic peptide ATI-5261 forms an oligomeric assembly in solution that dissociates to monomers upon dilution

Abstract

ATI-5261 is a 26-mer peptide that stimulates cellular cholesterol efflux with high potency. This peptide displays high aqueous solubility, despite having amphipathic α-helix structure and a broad nonpolar surface. These features suggested to us that ATI-5261 may adopt a specific form in solution, having favorable structural characteristics and dynamics. To test this, we subjected ATI-5261 to a series of biophysical studies and correlated self-association with secondary structure and activity. Gel-filtration chromatography and native gel electrophoresis indicated ATI-5261 adopted a discrete self-associated form of low molecular weight at concentrations >1 mg/mL. Formation of a discrete molecular species was verified by small-angle X-ray scattering (SAXS), which further revealed the peptide formed a tetrameric assembly having an elongated shape and hollow central core. This assembly dissociated to individual peptide strands upon dilution to concentrations required for promoting high-affinity cholesterol efflux from cells. Moreover, the α-helical content of ATI-5261 was exceptionally high (74.1 ± 6.8%) regardless of physical form and concentration. Collectively, these results indicate ATI-5261 displays oligomeric behavior generally similar to native apolipoproteins and dissociates to monomers of high α-helical content upon dilution. Optimizing self-association behavior and secondary structure may prove useful for improving the translatability and efficacy of apolipoprotein mimetic peptides.

Figure 1. Helical-wheel projection of ATI-5261

Linear sequence of amino acids is denoted by numbers, beginning with residue 1 in the intended foreground. Cationic residues are found at the polar/non-polar interface and negatively charged amino acids down the center of the polar surface, characteristic of a class A α-helix. A single tryptophan (W) residue is located on the non-polar surface.

Figure 2. ATI-5261 adopts a low molecular weight form in aqueous buffer

Stock solutions of ATI-5261 and peptide 4F were prepared in PBS (pH=7.4). Panel A- Native-PAGE (4–20% Tris-glycine gel) of peptides taken from 1 mg/ml stock solutions: lane 1, mobility of molecular weight (MW) standards including thyroglobulin (669 KD), ferritin (440 KD), catalase (232 KD), lactate dehydrogenase (140 KD) and albumin (66 KD); lane 2, ATI-5261 and lane 3, 4F. Peptide loads were 3 μg per well. Panels B and C- Chromatographic behavior of ATI-5261 in PBS on Superdex 75 columns at a flow rate of 0.5 ml/min. Peptide elution was monitored by absorbance at 280 nm. Representative profiles are shown for stock solution of 2.5 (B) and 20 mg/ml (C).

Figure 3. Small-angle X-ray scattering (SAXS) reveals ATI-5261 forms a tetrameric assembly

Peptide stock solutions were prepared in PBS (pH=7.4). X-ray scattering curves for ATI-5261 at three concentrations are shown. Once scaled for concentration, the X-ray intensity at q = 0 (inset, red line) was determined to be 13.1kDa or 4.1 times the monomeric weight of the peptide when calibrated against molecular weight standards lysozyme, xylanase and BSA. The elongated shape determined from the SAXS curves (teal) has a hollow central core. Picture at bottom right shows hypothetical fit of 4 α-helical strands within volume estimates, illustrating how overlapping segments may account for the elongated shape of the assembly.

Figure 4. The tetrameric assembly of ATI-5261 dissociates to lower molecular weight forms upon dilution

Stock solution of ATI-5261 prepared in PBS (pH=7.4) were analyzed by gel-filtration chromatography. Panel A. Separation of various physical forms of ATI-5261 on a Superdex 75 column; 0.2 ml of each stock solution (indicated) was injected onto the column; flow-rate was 0.5 ml/min; elution buffer was PBS. Representative chromatographic profiles of ATI-5261 stock solutions: 1 mg/ml (black), 0.4 mg/ml (red), 0.25 mg/ml (blue) and 0.125 mg/ml (green) are shown. Elution was monitored using absorbance at 280 nm. Results are arbitrary absorbance units (AU) scaled to clearly illustrate peak positions; actual AU= 1.0, 0.25, 0.08, and 0.02 for stock solutions of 1, 0.4, 0.25, 0.125 mg ATI-5261/ml, respectively. Panel B- FPLC using Superdex^™ Peptide 10/300 GL columns to separate ATI-5261 forms. Representative profiles of ATI-5261 at 1 mg/ml (black) and 0.1 mg/ml (red) are shown. Calibration curve (inset) shows retention volume of 17.58 ml for (Gly)₆ (0.36 KDa), 12.5 ml for aprotinin (6.5 KDa) and 10.78 ml for ribonuclease A (13.6 KDa). Elution was monitored by absorbance at 215 nm. Panel C- ATI-5261 at various concentrations cross-linked with DMS and separated by SDS-PAGE (10–20% Tricine gel). Cross-linking patterns were visualized by staining with SimplyBlue^™ SafeStain.

Figure 5. ATI-5261 maintains high α-helical content and structural integrity over a wide-range of concentrations

Peptide stock solutions of 2 mg/ml were prepared in 10 mM phosphate buffer (pH=7.4), and diluted with this same buffer as indicated. Panel A. Representative circular dichroism (CD) spectrum of lipid-free ATI-5261 at a concentration of 0.2 mg/ml (62 μM) is shown. Mean α-helicity was calculated from the molar ellipticity at 222 nm (n=4). Panel B. α-helical content of ATI-5261 at various concentrations, ranging from 6 μM to 310 μM (18 – 1000 μg/ml). Panel C. Molar ellipticity ratio ([θ]222/[θ]208nm) plotted against concentration of ATI-5261.

Figure 6. Effect of membrane mimetic trifluoroethanol (TFE) on self-association and secondary structure of ATI-5261

Stock solution of ATI-5261 were prepared in 10 mM phosphate (pH=7.4) and treated with and without TFE for 30 minutes at 25°C before analysis. Panel A. CD spectra of lipid-free ATI-5261 (0.2 mg/ml) in the presence or absence of 50% TFE, showing mean α-helicity calculated from molar ellipticity at 222 nm (n=3). Panel B. [θ]222/[θ]208 ratio of ATI-5261 in the presence and absence of 50% TFE, plotted against concentration of peptide. Panel C. DMS cross-linking/SDS-PAGE of ATI-5261 following treatment (30 min.) with increasing amounts of TFE. A representative stained gel is shown, indicating predominantly monomeric peptide at concentrations > 40% TFE. Panel D. Fluorescence emission spectra of ATI-5261 in the presence or absence of 50% TFE. Results are representative of three separate experiments with different batches of peptide.

Figure 7. Impact of end-group modifications on secondary structure, cholesterol efflux- and lipid-binding- activities of ATI-5261

ATI-5261 was synthesized with and without N-terminal acetyl and C-terminal amide groups and dissolved in 10 mM phosphate buffer (pH=7.4). Panel A. Representative CD scan and mean α-helicity of ATI-5261 (62 μM) lacking end-group modifications. Panel B. Relationship between α-helicity and concentration of ATI-5261 with and without end-group modifications. Panel C. Ability of ATI-5261 to promote cholesterol efflux from J774 macrophages treated with and without cAMP. Cells labeled with [³H]cholesterol were incubated (4 h) with lipid-free ATI-5261 and ATI-5261 lacking end-group modifications (3 μg/ml each) to monitor cholesterol efflux. Values are means ± SD, n=5. Panel D. Dependence of cholesterol efflux on the concentration of lipid-free ATI-5261 with/without end group modifications, determined using J774 cells treated with cAMP. Results are representative of 2 experiments. Panel E. Lipid-binding activity of ATI-5261, prepared with N-terminal acetyl and C-terminal amide groups. Lipid-binding activity was assessed as clearance of turbid solutions of DMPC, using peptide:lipid mass ratios of 1:2 and 1:4 at 25°C and following absorbance at 400 nm. Panel F. Lipid-binding activity of ATI-5261, lacking end-group modifications; conditions identical to panel E. Results are representative of two separate experiments.