Structural and Functional Analyses of a Sterol Carrier Protein in Spodoptera litura

Abstract

BackgroundsIn insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport.ResultsIn this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae.ConclusionsCompounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae.

NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm

“…7 ), which indicated that F53 was an important site involved in the hydrophobic interaction of HaSCP-2 with cholesterol and may be more critical for the ligand binding ability of HaSCP-2 than other sites. This is consistent with the finding in AeSCP-2 with the F32 (corresponding to F53 in HaSCP-2) mutation in mosquito and F53 mutation in SlSCP-2 site 30 31 . Different from AeSCP-2, Y51A mutations decreased the binding affinity of SCP-2 to NBD-cholesterol ( Fig.…”

“…7 and Table 5 ), which suggested that these amino acids may be the important functional sites involved in the intracellular hydrophobic interaction between HaSCP-2 and cholesterol. This was consistent with the mutation analysis in AeSCP-2 and SlSCP-2 30 31 . Especially, the mutation of F53W caused more decreasing in binding affinity than in the other mutations ( Fig.…”

“…4B ) showed that eighteen hydrophobic amino acids such as V25, M29, L32, M36, Y51, F53, V55, I68, F89, I91, V96, L99, I100, L104, P106, I115, I117 and L130 stayed in the inner surface of the cavity, which are likely serving as lipid and/or sterol interaction sites. Besides, two hydrophilic amino acids T128 and Q131, which has been considered as sterol interaction sites 30 , were lying adjacent to the opening of the cavity.…”

“…Recent studies have demonstrated that SCP-2 has cholesterol/lipid binding activities 21 22 23 24 . SCP-2 can bind to cholesterol, palmitic acid, fatty acyl-CoA, acidic phospholipids and bile salts 25 26 27 28 29 30 31 . The binding affinity of SCP-2 to cholesterol is the strongest among the lipids.…”

Transcription factor CAAT/enhancer‐binding protein is involved in regulation of expression of sterol carrier protein x in Spodoptera litura

“…These results suggest that the response of SCPx to cholesterol is complicated, and is perhaps tissue‐ and/or species‐specific. In S. litura , SlSCPx‐2 had high affinity to cholesterol and fatty acids (Zhang et al , ). Over‐expression of SlSCPx and SlSCPx‐2 increased cholesterol uptake into Spli‐221 cells from a sterol‐containing medium (Guo et al , ).…”