SummaryWe have solved the high-resolution crystal structure of the Drosophila melanogaster alcoholbinding protein LUSH in the complexes it forms with a series of short chain n-alcohols. LUSH is the first known non-enzyme 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 provide a structural motif to increase alcohol binding affinity at this site. This motif appears 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 infer alcohol sensitivity. We suggest the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 Å crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

SummaryIt is now generally accepted that many of the physiological effects of alcohol consumption are a direct result of binding to specific sites in neuronal proteins such as ion channels or other components of neuronal signaling cascades. Binding to these targets generally occurs in water filled pockets and leads to alterations in protein structure and dynamics. However the precise interactions required to confer alcohol sensitivity to a particular protein remains undefined.Using information from the previously solved crystal structures of the Drosophila melanogaster protein LUSH in complexes with short chain alcohols, we have designed and tested the effects of specific amino acid substitutions on alcohol binding. These effects of these substitutions, specifically S52A, T57S and T57A were examined using a combination of molecular dynamics, X-ray crystallography, fluorescence spectroscopy and thermal unfolding. These studies reveal that the binding of ethanol is highly sensitive to small changes in the composition of the alcohol binding site. We find that T57 is the most critical reside for binding alcohols, the T57A substitution completely abolishes binding, while the T57S substitution differentially affects ethanol binding compared to longer chain alcohols. The additional requirement for a potential hydrogen bond acceptor at position 52 suggests that both the presence of multiple hydrogen bonding groups and the identity of the hydrogen bonding residue are critical for defining an ethanol binding site. These results provide new insight into the detailed chemistry of alcohol's interactions with proteins.

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligandfree TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligandindependent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.Orphan nuclear receptors constitute a group of nuclear receptors (NRs) 4 that lack known functional ligands (1). Recent evidence has shown that orphan nuclear receptors play key roles in many essential physiological functions and can be used to identify therapeutic targets for human diseases (2-6). In contrast to those of classic hormone receptors, however, the functions of many orphan receptors remain unclear because the investigation of their physiological functions is limited by the lack of information about their structural features and their functional ligands.Testicular receptors form a subgroup of orphan nuclear receptors that consists of two members, TR2 and TR4 (7,8). TR2 and TR4 are expressed in spermatocytes, erythroid cells, liver, and some regions of the brain and are known to play roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system (7,8). TR4 binds to the direct repeat AGGTCA with a variable number of spacer nucleotides (from 0 to 6) in the promoters of putative target genes (9). TR2 and TR4 homodimerize and heterodimerize with each other but do not heterodimerize with retinoid X receptor (RXR) members. The TR2/TR4 heterodimer binds to direct repeats of DNA elements in the embryonic and fetal globin gene promoter and represses globin gene transcription (10, 11).The domain organizations of both TR2 and TR4 are similar to those of many other NR family members. Located in the middle of the receptor is a DNA binding domain (DBD), and mapped to the C terminus is the ligand binding domain (LBD) with a ligand-inducible activation function region, AF-2. Located at the N terminus and upstream of the DBD is the Nterminal domain (NTD), including one or two activation function regions (AF-1), which is con...