Interaction between the N- and C-terminal domains modulates the stability and lipid binding of apolipoprotein A-I

Abstract

The tertiary structures of human and mouse apolipoprotein A-I (apoA-I) are comprised of an N-terminal helix bundle and a separate C-terminal domain. To define the possible intramolecular interaction between the N- and the C-terminal domains, we examined the effects on protein stability and lipid-binding properties of exchanging either the C-terminal domain or helix between human and mouse apoA-I. Chemical denaturation experiments demonstrated that replacement of the C-terminal domain or helical segment in human apoA-I with the mouse counterparts largely destabilizes the N-terminal helix bundle. Removal of the C-terminal domain or alpha-helix in human apoA-I had a similar effect on the destabilization of the helix bundle against urea denaturation, indicating that the C-terminal helical segment mainly contributes to stabilizing the N-terminal helix bundle structure in the apoA-I molecule. Consistent with this, KI quenching experiments indicated that removal or replacement of the C-terminal domain or helix in human apoA-I causes Trp residues in the N-terminal domain to become exposed to solvent. Measurements of the heats of binding to egg phosphatidylcholine (PC) vesicles and the kinetics of solubilization of dimyristoyl PC vesicles demonstrated that the destabilized human N-terminal helix bundle can strongly interact with lipids without the hydrophobic C-terminal helix. In addition, site-specific labeling of the N- and C-terminal helices by acrylodan to probe the conformational stability and the spatial proximity of the two domains indicated that the C-terminal helix is located near the N-terminal helix bundle, leading to a relatively less solvent-exposed, more organized conformation of the C-terminal domain. Taken together, these results suggest that interaction between the N- and C-terminal tertiary structure domains in apoA-I modulates the stability and lipid-binding properties of the N-terminal helix bundle.

Figure 1

Urea-induced denaturation of human and mouse hybrid apoA-I monitored by molar ellipticity (A) or Trp fluorescence (B). ●, human WT; ▲, apoA-I h(1–189)/m(187–240); △, apoA-I h(1–220)/m(218–240). Protein concentration was 50 µg/ml.

Figure 2

Chemical denaturation of the C-terminal-truncated human apoA-I mutants monitored by Trp fluorescence. (A) Urea-induced denaturation of human WT (dotted line), human 1–222 (△), human 1–189 (▲), and human L230P/L233P/Y236P (●). Protein concentration was 25 µg/ml. (B) Comparison of the free energies of denaturation induced by GdnHCl and urea. **P<0.01 compared to human WT apoA-I (ΔG_D° in GdnHCl and urea denaturation are 4.6 ± 0.3 and 6.2 ± 0.2 kcal/mol, respectively).

Figure 3

Solubilization of DMPC vesicles (A) and binding enthalpies to egg PC SUV (B) for human and mouse hybrid apoA-I. (A) Increase in fraction cleared of turbidity in 10 min after incubation of DMPC vesicles (0.25 mg/mL) with increasing concentration of protein at 24.6 °C. ●, human WT; ○, human 1–189; ▲, apoA-I h(1–189)/m(187–240); Δ, apoA-I h(1–220)/m(218–240); ∇, human 190–243; ▼, mouse 187–240. (B) Protein solutions were injected into excess SUV in an isothermal titration calorimeter at a PC-to-protein molar ratio of > 10,000.

Figure 4

Acrylodan fluorescence emission spectra monitored at different concentrations of urea (from 0 to 6.4 M) for apoA-I V53C-Ac. The inset shows the change in acrylodan GP for apoA-I V53C-Ac (●) and F229C-Ac (○) as a function of urea concentration. Protein concentration was 25 µg/ml.

Figure 5

FRET between Trp residues in the N-terminal domain and acrylodan for apoA-I V53C-Ac (A and B) and F229C-Ac (C and D). Fluorescence emission spectra excited at 295 nm for unlabeled (solid lines) and acrylodan-labeled (dashed lines) apoA-I variants were recorded in the absence (A and C) or presence (B and D) of 3M GdnHCl. Protein concentration was 25 µg/ml.