THE LIPID-FREE STRUCTURE OF APOLIPOPROTEIN-A-I - EFFECTS OF AMINO-TERMINAL DELETIONS

Deletion mutants of human apolipoprotein A-I (apo hA-I) have been prod uced 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 Delta(1-43)A-I and apo D elta(187-243)A-I have already been reported. To aid purification of th ese mutants, a histidine-containing N-terminal extension was incorpora ted (+his); in cases where comparison with the (-his) construct was po ssible, little effect on the physical properties due to the (+his) ext ension was found. All mutants have folded structures in their lipid-fr ee state, however these structures differ widely in their relative the rmodynamic stability and extent of secondary structure. The mutant wit h the fewest residues deleted, apo Delta(88-98)A-I(+his), has the leas t secondary structure (only 34% helix) and is also the least stable (D elta G = 2.9 kcal/mol). Determined from sedimentation velocity measure ments on the lipid-free proteins, all but apo Delta(1-65)A-I(+his) exh ibited a range of conformers in solution, which fluctuated around a hi ghly elongated species (dimensions equal to similar to(14-16) x simila r to 2.3 nm). Apo Delta(1-65)A-I(+his) exhibited a discrete species wh ich was less asymmetric (dimensions equal to 9 x 2.9 nm). Apo a(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 car boxy-terminal deletion mutant, apo Delta(187-243)A-I(+/-his) compared to wildtype (wt) apo wtA-I(+/-his), but substantial differences are ob served between wt and the amino-terminal deletion mutants, apo a(1-43) A-I, apo a(1-65)A-I(+his), and apo Delta(88-98)A-I(+his), In contrast, the lipid-binding properties are impaired for apo Delta(187-243)A-I(/-his), as measured by dimyristoyl phosphatidylcholine (DMPC) liposome turbidity clearance kinetics and palmitoyloleoyl phosphatidylcholine (POPC) equilibrium binding. Apo Delta(1-43)A-I, apo Delta(1-65)A-I(+hi s), and apo Delta(88-98)A-I(+his) show lipid affinities statistically similar to apo wtA-I(fhis), but significantly defective DMPC clearance kinetics. Interestingly, lecithin:cholesterol acyltransferase (LCAT) activation results correlate qualitatively with the lipid-binding affi nity for all mutants but apo Delta(88-98)A-I(+his), suggesting that th is mutant has an altered and possibly noncooperative lipid-bound struc ture as well as an altered lipid-free structure, These results suggest helix 1 (residues 44-65) and helix 10 (residues 220-240) are both req uired for native lipid-binding properties, while the presence of inter nal residues, at least helix 3 (residues 88-98), is essential for prop er folding of both the lipid-free and lipid-bound conformations. Impor tantly, studies on apo a(88-98)A-I(+his) provide the first experimenta l evidence that a native-like structure is not necessary for native-li ke lipid affinity, but apparently is necessary for both DMPC solubiliz ation and LCAT activation. These results provide support for a hypothe tical, multistep structure-based mechanism for apo hA-I lipid binding.