The lipid-free structure of apolipoprotein A-I: Effects of amino- terminal deletions

Abstract

Deletion mutants of human apolipoprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the function of the specific domains comprising residues 1-43, 1-65, 88-98, and 187-243, respectively, in the lipid-free conformation and in the lipid-binding mechanism of apo hA-I. Initial studies on apo Δ(1-43)A-I and apo Δ(187- 243)A-I have already been reported. To aid purification of these mutants, a histidine-containing N-terminal extension was incorporated (+his); in cases where comparison with the (-his) construct was possible, little effect on the physical properties due to the (+his) extension was found. All mutants have folded structures in their lipid-free state, however these structures differ widely in their relative thermodynamic stability and extent of secondary structure. The mutant with the fewest residues deleted, apo Δ(88-98)A- I(+his), has the least secondary structure (only 34% helix) and is also the least stable (ΔG = 2.9 kcal/mol). Determined from sedimentation velocity measurements on the lipid-free proteins, all but apo Δ(1-65)A-I(+his) exhibited a range of conformers in solution, which fluctuated around a highly elongated species (dimensions equal to ~(14-16) x ~2.3 nm). Apo Δ(1-65)A- I(+his) exhibited a discrete species which was less asymmetric (dimensions equal to 9 x 2.9 nm). Apo Δ(88-98)A-I(+his) showed extreme heterogeneity with no predominating conformer. Spectroscopic studies (ANS binding and circular dichroism) indicate that there is little difference in the lipid- free structure of the carboxy-terminal deletion mutant, apo Δ(187-243)A- I(±his) compared to wild-type (wt) apo wtA-I(±his), but substantial differences are observed between wt and the amino-terminal deletion mutants, apo Δ(1-43)A-I, apo Δ(1-65)A-I(+his), and apo Δ(88-98)A-I(+his). In contrast, the lipid-binding properties are impaired for apo Δ(187-243)A- I(±his), as measured by dimyristoyl phosphatidylcholine (DMPC) liposome turbidity clearance kinetics and palmitoyloleoyl phosphatidylcholine (POPC) equilibrium binding. Apo Δ(1-43)A-I, apo Δ(1-65)A-I(+his), and apo Δ(88- 98)A-I(+his) show lipid affinities statistically similar to apo wtA-I(+his), but significantly defective DMPC clearance kinetics. Interestingly, lecithin:cholesterol acyltransferase (LCAT) activation results correlate qualitatively with the lipid-binding affinity for all mutants but apo Δ(88- 98)A-I(+his), suggesting that this mutant has an altered and possibly noncooperative lipid-bound structure as well as an altered lipid-free structure. These results suggest helix 1 (residues 44-65) and helix 10 (residues 220-240) are both required for native lipid-binding properties, while the presence of internal residues, at least helix 3 (residues 88-98), is essential for proper folding of both the lipid-free and lipid-bound conformations. Importantly, studies on apo Δ(88-98)A-I(+his) provide the first experimental evidence that a native-like structure is not necessary for native-like lipid affinity, but apparently is necessary for both DMPC solubilization and LCAT activation. These results provide support for a hypothetical, multistep structure-based mechanism for apo hA-I lipid binding.