Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo

Abstract

Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipidbinding proteins in nematodes have been described, including the fatty acid-and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.Hydrophobic lipophilic molecules such as fatty acids, eicosanoids, retinoids, and steroids have important functions both as energy sources and in metabolic signaling. They affect fundamental cellular processes such as gene transcription, cell development, inflammation, and immune response (1-3). The cellular cytosol is hydrophilic, and lipids need to be solubilized and protected from chemical damage. Their transport and availability are tightly regulated. Proteins that coordinate the lipid traffic include lipoproteins (such as the low density lipoprotein) and carrier proteins, known as lipid-binding proteins (LBPs).2 In vertebrates LBPs belong to the ␤-sheet calycin superfamily (lipocalins and fatty acid-binding proteins (FABPs)) or the ␣-helical serum albumin-like superfamily. Nematodes are one of the most abundant groups of multicellular organisms. Parasitic nematodes cause serious and difficult to treat diseases in humans, animals, and plants affecting human health as well as having a negative impact on agricultural economics. It is estimated that more than one-sixth of the earth's population (mainly in developing countries), suffers from nematode infections, and at least 4 of the 15 neglected tropical diseases listed by the World Health Organization are caused by nematodes. Parasitic worms possess limited lipid metabolism and depend on import of essential lipids from their host (4), which makes the lipid transporters good targets for chemoprophylactic treatments. A 14-kDa FABP (Sm14) has been proposed as a vaccine candidate against Schistosoma mansoni in humans and Fasciola hepatica in cattle and sheep (5, ...

The Potential Role of Binding Proteins in Human Parasitic Infections: An In-Depth Look at the Novel Family of Nematode-Specific Fatty Acid and Retinol Binding Proteins

“…While the results from Ce-FAR-7 suggest that the retinol binding site (P2) is probably regulated by CKII, the fatty acid binding pocket (P1) was not affected by phosphorylation. However, Ce-FAR-7's affinity for retinol is an order of magnitude lower than that for fatty acids, implying that, in contrast to the other FAR family members, transport of retinoids is not the major function of Ce-FAR-7 [15]. Thus, it is still unknown if the FAR protein affinity for signaling lipids, such as retinoids, is regulated in parasitic FARs, and if it is, how phosphorylation impacts the Kds of the bound ligands.…”

“…This process causes changes in the function, activity, localization, and/or stability in about 30 percent of cellular proteins [23]. A preliminary paper investigating the more distantly related Ce-FAR-7 protein presents evidence to suggest that retinol binding is positively regulated by CKII phosphorylation at a conserved site located near the binding pocket associated with retinol binding [15]. Previous research has also demonstrated that this is 100% conserved across all plant, animal and human parasitic nematode FAR proteins known to date [10,14,15,19].…”

“…There is an increasing interest by laboratories in investigating FAR proteins [11,[14][15]30]. Continued studies will provide invaluable information as to exactly which amino acids are key in the binding of fatty acids and retinol, as well as the possible in vivo function(s) of nematode FAR proteins.…”

“…Group A has the highest sequence identity to FARs from parasitic nematodes, such as Hp-FAR-1, Ov-FAR-1 (O. volvulus, human parasite) and Gp-FAR-1 (Globodera pallida, plant parasite) [21]. Although the sequence identity among parasitic FARs is high [14,21], the sequence identity of Ce-FAR-7 (the only FAR protein for which structural information is available) with other FAR proteins is extremely low [15], making data on Ce-FAR-7 more complicated for use in identifying key functional amino acids. In fact, of all of the C. elegans FAR proteins, Ce-FAR-7 is the least representative of known FAR proteins [13,14,21].…”

“…A study conducted in 2009 reported the X-ray crystallographic structure of Ce-FAR-7 from C. elegans, however, this is a non-parasitic nematode belonging to a different evolutionary group [15] Parasitic nematodes possess one or two types of FAR proteins [11,14], but the free-living C. elegans produces eight FAR proteins (Ce-FAR-1 to 8) [13]. They belong to three groups, group A (Ce-FAR-1, 2 and 6), group B (Ce-FAR-1, 4 and 5) and group C (Ce-FAR-7 and 8) [13].…”