A thermoacidophile-specific protein family, DUF3211, functions as a fatty acid carrier with novel binding mode

Abstract

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/β structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C14) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.

Fig 1

Crystal structure of STK_08120. (A) The structure is colored blue and yellow for the β-sheet and loops, respectively. Among the three α-helices, the α3-helix and the other helices (α1 and α2) are shown in purple and green, respectively. Dashed curves indicate kinked regions around Lys109 and Ile123. (B) Stick-and-sphere models and dashed lines represent hydrophobic contacts among the residues on the three α-helices and a salt bridge between Asp23 and Arg134, respectively. The purple region corresponds to the C-terminal half (residues Leu116 to Arg134) of the α3-helix. (C) Elution patterns of STK_08120 (blue) and standard proteins (orange) on a Superdex 75 HR 10/30 gel filtration column. The absorption wavelength was 280 nm for detecting protein peaks. Values in parentheses are molecular masses estimated from the amino acid sequence for STK_08120.

Fig 2

Cavities of STK_08120 and START domain proteins. (A) STK_08120. (B) ZhuI polyketide aromatase/cyclase (PDB identifier, 3TFZ [33]). (C) ABA receptor PYL2 (PDB identifier, 3NS2 [34]). (D) Phenolic oxidative coupling protein Hyp-1 (PDB identifier, 3IE5 [35]). Cavities are represented by the yellow mesh model, and arrowheads indicate the entrances to the cavities. Gate loops are shown by orange color in the ZhuI, PYL2, and Hyp-1 structures. Purple regions correspond to the α3-helix of STK_08120 and the C-terminal long helices of ZhuI, PYL2, and Hyp-1.

Fig 3

Residues composing a hydrophobic cavity of STK_08120. Nonpolar (A) and polar (B) residues are represented by stick models. Dashed lines and an arrowhead indicate a hydrogen bond and the cavity entrance, respectively.

Fig 4

Myristic acid-bound structure of STK_08120. (A) Myristic acid in the hydrophobic cavity is represented by a stick model along with the F_o-F_c electron density colored in violet (1.5σ). The light blue surface shows the amino group of Lys109. (B) Myristic acid is represented by a stick-and-sphere model. The other stick models indicate the residues surrounding the myristic acid within 4 Å. (C) Hydrophobic contacts are illustrated using a LIGPLOT diagram. Yellow lines surrounding the model of myristic acid represent solvent accessibility: dark thick line, low (tight hydrophobic contact between protein and ligand); light thin line, high (loose hydrophobic contact). (D) Superposed structures of STK_08120 are colored pink and cyan for apo and myristic acid-bound states, respectively. The arrowhead indicates a flexible loop (I41-E42) with comparatively high B factors (>50 Ų for apo state and >30 Ų for myristic acid-bound state). Myristic acid is represented by a yellow stick model.

Fig 5

Binding modes for long-chain fatty acids. (A) STK_08120-myristic acid complex. (B) FABP-palmitic acid complex (PDB identifier, 2IFB [44]). (C) LTP-palmitic acid complex (PDB identifier, 1FK3 [45]). Green sticks are the interaction residues with the carboxyl group of myristic acid or palmitic acid.