The Conformation of Lipid-Free Human Apolipoprotein A-I in Solution

Abstract

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.

Binding of human apoA-I[K107del] variant to TG-rich particles: implications for mechanisms underlying hypertriglyceridemia

“…The secondary structure assignment for residues in the proposed conformation ( Fig. 2B ) is close to that found in hydrogen exchange and mass spectrometry studies ( 40 ), and the tertiary conformation is similar to the four-helical bundle core region of apoA-I inferred from cross-linking studies ( 41,42 ). These observations, together with recent studies that explain conformational and functional features of apoA-I and its natural variants based on the crystal structure and the proposed lipid-free conformation ( 43,44 ), support the view that the proposed conformation for the lipid-free its clearance from the circulation ( 14,21,22 ) and also affect various reverse cholesterol transport pathways that directly modulate plasma HDL cholesterol and apoA-I levels, such as LCAT activation, ABCA-1 mediated effl ux of cholesterol, or the interaction with scavenger receptor class B type I ( 9-11, 15, 16, 50, 51 ), while enhanced binding of the mutated apoA-I to TG-rich lipoproteins may be one of the mechanisms contributing to HTG.…”

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

“…The HX-derived secondary structure for lipid-free apoA-I suggested that the whole C-terminal domain lacks defined structure (12). In addition, lipid-free apoA-I models based on cross-linking experiments suggested that part of the H10 region formed helical structure (37,38). Most recently, MD simulation of full-length apoA-I based on Borhani et al's (35) and our crystal structure suggested that parts of H8 and H10 form helical structure after equilibrium in solution (30).…”

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding

“…Finally, we acknowledge the strong experimental evidence showing that apoA-I exhibits molten globule characteristics in solution (29,30,35,45). apoA-I has a free energy of denaturation that is well below that of most soluble proteins (45,46) with helical segments that are constantly folding and unfolding on a time scale of seconds (44).…”