The tertiary structure of apolipoprotein (apo) A-I and the contributions of structural domains to the properties of the protein molecule are not well defined. We used a series of engineered human and mouse apoA-I molecules in a range of physical-biochemical measurements to address this issue. Circular dichroism measurements of alpha-helix thermal unfolding and fluorescence spectroscopy measurements of 8-anilino-1-napthalenesulfonic acid binding indicate that removal of the C-terminal 54 amino acid residues from human and mouse apoA-I has similar effects; the molecules are only slightly destabilized, and there is a decrease in hydrophobic surface exposure. These results are consistent with both human and mouse apoA-I adopting a two-domain tertiary structure, comprising an N-terminal antiparallel helix bundle domain and a separate less ordered C-terminal domain. Mouse apoA-I is significantly less resistant than human apoA-I to thermal and chemical denaturation; the midpoint of thermal unfolding of mouse apoA-I at 45 degrees C is 15 degrees C lower and the midpoint of guanidine hydrochloride denaturation (D1/2) occurs at 0.5 M as compared to 1.0 M for human apoA-I. These differences reflect the overall greater stability of the helix bundle formed by residues 1-189 in human apoA-I. Measurements of the heats of binding to egg phosphatidylcholine (PC) small unilamellar vesicles and the kinetics of solubilization of dimyristoyl PC multilamellar vesicles indicate that the more stable human helix bundle interacts poorly with lipids as compared to the equivalent mouse N-terminal domain. The C-terminal domain of human apoA-I is much more hydrophobic than that of mouse apoA-I; in the lipid-free state the human C-terminal domain (residues 190-243) is partially alpha-helical and undergoes cooperative unfolding (D1/2 = 0.3 M) whereas the isolated mouse C-terminal domain (residues 187-240) is disordered in dilute solution. The human C-terminal domain binds to lipid surfaces much more avidly than the equivalent mouse domain. Human and mouse apoA-I have very different tertiary structure domain contributions for achieving functionality. It is clear that the stability of the N-terminal helix bundle, and the hydrophobicity and alpha-helix content of the C-terminal domain, are critical factors in determining the overall properties of the apoA-I molecule.

The tertiary structure of human and mouse apolipoprotein A–I (apoA-I) are comprised of an N-terminal helix bundle and independently folded C-terminal domain. To define the possible intramolecular interaction between the N- and 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 α-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 lipid 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.