Structure-function relationship of exchangeable apolipoproteins

Exchangeable apolipoproteins play a vital role in lipid and lipoprotein homeostasis. To understand the lipid-binding mechanism of exchangeable apolipoproteins in this study the structure-function relationship of insect exchangeable apolipoprotein apolipophorin-III (apoLp-III) has been investigated by utilizing disulfide mutants of locust apoLp-III. Based on the ability of the disulfide mutants to interact with various lipid-surfaces the role of apoLp-III helices and loops during the lipid-binding process of apoLp-III has been elucidated. In the second part of the study the organization of two adjacent human apolipoprotein AI molecules in reconstituted discoidal lipoproteins was investigated by chemical cross-linking and mass spectrometry. The difficulties associated with the chemical cross-linking coupled with mass spectrometry approach are discussed in the second part

Helical structure and stability in human apolipoprotein A-I by hydrogen exchange and mass spectrometry

Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) in the circulation and governs their biogenesis, metabolism, and functional interactions. To decipher these important structure–function relationships, it will be necessary to understand the structure, stability, and plasticity of the apoA-I molecule. Biophysical studies show that lipid-free apoA-I contains a large amount of α-helical structure but the location of this structure and its properties are not established. We used hydrogen-deuterium exchange coupled with a fragmentation-separation method and mass spectrometric analysis to study human lipid-free apoA-I in its physiologically pertinent monomeric form. The acquisition of ≈100 overlapping peptide fragments that redundantly cover the 243-residue apoA-I polypeptide made it possible to define the positions and stabilities of helical segments and to draw inferences about their interactions and dynamic properties. Residues 7–44, 54–65, 70–78, 81–115, and 147–178 form α-helices, accounting for a helical content of 48 ± 3%, in agreement with circular dichroism measurements (49%). At 3 to 5 kcal/mol in free energy of stabilization, the helices are far more stable than could be achieved in isolation, indicating mutually stabilizing helix bundle interactions. However the helical structure is dynamic, unfolding and refolding in seconds, allowing facile apoA-I reorganization during HDL particle formation and remodeling

Effects of the Iowa and Milano Mutations on Apolipoprotein A‑I Structure and Dynamics Determined by Hydrogen Exchange and Mass Spectrometry

The Iowa point mutation in apolipoprotein A-I (G26R) leads to a systemic amyloidosis condition, and the Milano mutation (R173C) is associated with hypoalphalipoproteinemia, a reduced plasma level of high-density lipoprotein. To probe the structural effects that lead to these outcomes, we used amide hydrogen–deuterium exchange coupled with a fragment separation/mass spectrometry analysis (HX MS). The Iowa mutation inserts an arginine residue into the nonpolar face of an α-helix that spans residues 7–44 and causes changes in structure and structural dynamics. This helix unfolds, and other helices in the N-terminal helix bundle domain are destabilized. The segment encompassing residues 116–158, largely unstructured in wild-type apolipoprotein A-I, becomes helical. The helix spanning residues 81–115 is destabilized by 2 kcal/mol, increasing the small fraction of time it is transiently unfolded to ≥1%, which allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming peptide. The Milano mutation situated on the polar face of the helix spanning residues 147–178 destabilizes the helix bundle domain only moderately, but enough to allow cysteine-mediated dimerization that leads to the altered functionality of this variant. These results show how the HX MS approach can provide a powerful means of monitoring, in a nonperturbing way and at close to amino acid resolution, the structural, dynamic, and energetic consequences of biologically interesting point mutations

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution*

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1–189 and 190–243) and mouse apoA-I (residues 1–186 and 187–240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation