Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster

Abstract

We have solved the high-resolution crystal structures of the Drosophila melanogaster alcohol-binding protein LUSH in complex with a series of short-chain n-alcohols. LUSH is the first known nonenzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48, form a network of concerted hydrogen bonds between the protein and the alcohol that provides a structural motif to increase alcohol-binding affinity at this site. This motif seems to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion channels that are known to confer alcohol sensitivity. We suggest that the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.

Figure 1. Ribbon diagrams of LUSH and PBP

(a) Diagram of the LUSH-butanol structure solved to 1.25 Å resolution. Individual helices are labeled α1-α6. α5′ is a stretch of 3₁₀-helix. Butanol is represented as a space-filling model at the center of the protein. The alkyl chain is colore in cyan and the hydroxyl group in red. (b) Diagram of the PBP-bombykol complex from B. mori. The elements of secondary structure are colored as for LUSH. Bombykol is shown in a ball-and-stick representation.

Figure 2. Structure of the LUSH-alcohol binding pocket

(a) Key residues in the alcohol-binding pocket formed by helix-3 (gold), helix-6 (blue) and the C-terminal strand. The network of hydrogen bonds formed by Thr48, Ser52, Thr57 and ethanol are illustrated by red dotted lines. (b) Stereo view of the electron density of residues in the binding pocket of the LUSH-ethanol complex. (c) The same view of the alcohol-binding pocket in the LUSH-butanol complex.

Figure 3. ¹H-¹⁵N HSQC Spectra of LUSH recorded with and without alcohol

¹H-¹⁵N HSQC spectra of (a) LUSH-butanol complex (b) LUSH-ethanol complex and (c) LUSH without alcohol. The alcohol concentration was ~ 40 mM. Without alcohol, a large number of peaks are in intermediate exchange. The presence of ethanol produces a significant improvement in the quality of the spectra but the presence of butanol produces the best appearance of the spectrum.

Figure 4. Comparison of the alcohol-binding site in LUSH with regions of alcohol-sensitive ion channels

Residues that bind alcohol in LUSH and which are potentially conserved in alcohol-sensitive ion-channels are outlined in the black boxes. Residues in the ion channels that have been shown to affect alcohol sensitivity by site-directed mutagenesis are circled.