A Three-dimensional Homology Model of Lipid-free Apolipoprotein A-IV Using Cross-linking and Mass Spectrometry

Abstract

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.Apolipoprotein A-IV (apoA-IV) 2 is a 46-kDa plasma protein with myriad proposed functions. In humans, it is primarily produced in the small intestine in response to lipid-rich meals and seems to be involved in the assembly and secretion of chylomicrons (1, 2). Although the apoA-IV knock-out mouse displayed relatively normal lipid absorption on a normal chow diet (3), the importance of apoA-IV during periods of high lipid intake is beginning to become evident. For example, apoA-IV has been shown to facilitate the assembly of very large chylomicrons during high lipid stress in a cell culture model of lipoprotein secretion (4), possibly by regulating the transit rate of the nascent particles through the endoplasmic reticulum (5). The protein has also been postulated to play many roles in the reverse cholesterol transport system (6), a process important for protection from cardiovascular disease whereby excess cholesterol is removed from the vessel wall. ApoA-IV can accept cholesterol similarly to apoA-I via ABCA1 (ATP-binding cassette transporter A1) (7). It can activate lecithin:cholesterol acyltransferase and cholesterol ester transfer protein (8, 9) and bind to hepatocellular membranes (10, 11) possibly to deliver high density lipoprotein cholesterol to the liver. ApoA-IV has also been shown to possess anti-inflammatory and antioxidative properties (12-15) and may even help modulate food intake (16).Like many of the exchangeable apolipoproteins, apoA-IV can exist in at least two states in plasma, lipid-bound and lipid-free. It is associated with newly secreted chylomicrons in the intestinal lymph, but it rapidly disassociates from these upon the initiation of lipolysis in the plasma compartment (17). Thus, the majority of human plasma apoA-IV is found distributed between high density lipoproteins and a lipid-free (or lipidpoor) fraction. The actual di...

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

“…Thus, unlike NMR, de novo structure determination is not possible and external structural information is required for a meaningful model. Previous cross-linking studies inherently suffered from lack of high-resolution knowledge on the subunit structures (25,32), their interfacing modes (23)(24)(25), overall shape of the complex (24,25,33) or combinations of these factors. In the TRiC system all these factors are known to a much higher resolution than the maximum cross-linking length (approximately 28Ǻ).…”

Computational Methods in Mass Spectrometry-Based Protein 3D Studies

“…Advancements in MS3D experimental approaches continuously change the scenarios for computational procedures, by substantially increasing the number of data, as well as the types of crosslinking or labelling agents and proteolytic enzymes. The large number of crosslinks obtained for apolipoproteins (Silva et al, 2005;Tubb et al, 2008) or CopA copper ATPase (Lübben et al, 2009) represent good examples of these trends (Table 1).…”

Improving the diffraction of apoA-IV crystals through extreme dehydration

“…ApoA-IV 64-335 was identified as a core domain through limited proteolysis (Tubb et al, 2008) and selected for crystallization. ApoA-IV 64-335 was expressed in Escherichia coli BL21 (DE3) and purified as described previously (Tubb et al, 2009) with the addition of sizeexclusion chromatography to isolate the dimer species.…”

“…Nascent HDL proceeds through the bloodstream and absorbs excess lipids from peripheral cells for removal. To gain insight into this important mechanism, we pursued the crystal structure of a proteolytically stable fragment of human apoA-IV (residues 64-335) in its dimer configuration (Tubb et al, 2008). In our attempt to determine the structure of apoA-IV , dehydrating the crystals with extreme polyethylene glycol (PEG) concentrations was critical to obtaining well defined diffraction images.…”