The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-Å diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to ϳ53 and ϳ80 kDa. At high molar ratios, these two products merged, yielding a product of ϳ59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53-and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-Å recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.The structure of apolipoprotein A-I (apoA-I) 2 has been intensely investigated in efforts to understand its highly significant role in protecting against coronary heart disease in humans (1-3). ApoA-I is abundantly found in plasma high density lipoproteins (HDL) and functions as the main carrier of excess cholesterol to the liver in a process termed "reverse cholesterol transport" (4 -6). ApoA-I also plays a significant role in mediating anti-inflammatory/antioxidative processes (7-12) intervening in the escalation of damage to the artery wall. As with many proteins, the functional roles played by apoA-I are tightly coupled to the structure of the apoprotein. Thus, the lack of a detailed lipid-bound apoA-I x-ray crystal structure has seriously hindered our understanding of this apoprotein's unique features (13,14) in its biologically active lipid-bound form. A major advance occurred when the x-ray crystal structure of lipid-free ⌬43 apoA-I was reported (15). This report suggested that apoA-I adopts an antiparallel "belt-like" conformation when bound to a lipid surface. Unfortunately, lipid-bound apoA-I has not yielded crystals of the quality needed to solve its three-dimensional conformation. Instead, a number of alternative and highly innovative approaches have been used to probe the conformation suggested by the lipid-free crystal structure (16 -27). In addition, computer modeling studies suggest that the two molecules of apoA-I wrap in an extended belt completely around the edge of a lipid bilayer, maximizing intermolecular salt bridges, which act to stabilize the protein conformation in an antiparallel arrangement (23). Although all of these studies support the concept of a "belt" model of apoA-I, their inability to distinguish between an "extended belt" or a "hairpin" belt conformation have lead...

The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96Å diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 Å) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoylsn-glycerol-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of 2 apoA-I molecules bound to ~150 molecules of POPC and suggest that the protein hydrophobic interactions at the N-and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N-and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.Apolipoprotein A-I (apoA-I) is a 28 kDa protein synthesized by the liver and intestine and is responsible for modulating the formation, metabolism, and catabolism of high density lipoprotein cholesterol. HDL has been known for decades to be a negative risk factor for predicting the development of coronary artery disease in humans, but the specific mechanism (s) responsible for its protective role in cholesterol metabolism continues to be studied and elucidated (1-3).ApoA-I shares a number of similarities to other members of the apoprotein super gene family, as well as possessing a unique set of properties related to its unique role in lipid metabolism (4). Totally soluble in aqueous solution, apoA-I monomers exist in a lipid-free state, but also avidly bind to each other, as well as lipid surfaces (5). Although apoA-I readily binds lipid surfaces, the formation of small apoA-I containing phospholipid and cholesterol particles, an important step in HDL metabolism, does not occur to a significant extent in the absence of the ABCA1 transporter. During the formation of "nascent" HDL, monomers of lipid-free apoA-I bind to ABCA1 which adds phospholipid and cholesterol to yield a particle containing two 1To whom correspondence should be addressed: Dept. of Pathology, Section on Lipid Sciences, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2147; Fax 336-716-6279; E-mail:msthomas@wfubmc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 25. Published in final edited form as:Biochemistry. -I (3,6,7). In this form, the conformation of ...

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Apolipoprotein AI (apoA-I) is the principal acceptor of lipids from ATP-binding cassette transporter A1, a process that yields nascent high density lipoproteins. Analysis of lipidated apoA-I conformation yields a belt or twisted belt in which two strands of apoA-I lie antiparallel to one another. In contrast, biophysical studies have suggested that a part of lipid-free apoA-I was arranged in a 4-helix bundle. To understand how lipid-free apoA-I opens from a bundle to a belt while accepting lipid it was necessary to have a more refined model for the conformation of lipid-free apoA-I. This study reports the conformation of lipid-free human apoA-I using lysine-to-lysine chemical cross-linking in conjunction with disulfide cross-linking achieved using selective cysteine mutations. After proteolysis cross-linked peptides were verified by sequencing using tandem mass spectrometry. The resulting structure is compact with roughly 4 helical regions, amino acids 44 through 186, bundled together. C- and N-terminal ends, amino acids 1-43 and 187-243, respectively, are folded such that they lie close to one another. An unusual feature of the molecule is the high degree of connectivity of lysine40 with 6 other lysines, lysines that are close, e.g., lysine59, to distant lysines, e.g., lysine239, that are at the opposite end of the primary sequence. These results are compared and contrasted with other reported conformations for lipid-free human apoA-I and an NMR study of mouse apoA-I.