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1.  The role of multiple hydrogen bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH 
Journal of molecular biology  2008;376(5):1360-1376.
Summary
It 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.
doi:10.1016/j.jmb.2007.12.063
PMCID: PMC2293277  PMID: 18234222
Alcohol; Hydrogen Bonding; Binding affinities; LUSH; X-ray crystallography
2.  Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor 
PLoS Biology  2008;6(9):e227.
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.
Author Summary
Unlike other classes of receptors, nuclear receptors can bind directly to DNA and act as transcription factors, playing key roles in embryonic development and cellular metabolism. Most nuclear receptors are activated by signal-triggering molecules (ligands) and can regulate their activity by recruiting coactivator proteins. However, the ligands are unknown for a subset of “orphan” nuclear receptors, including the chicken ovalbumin promoter-transcription factors (COUP-TFI and II, and EAR2). COUP-TFs are the most conserved nuclear receptors, with roles in angiogenesis, neuronal development, organogenesis, and metabolic homeostasis. Here we demonstrate that COUP-TFII is a ligand-regulated nuclear receptor that can be activated by unphysiological micromolar concentrations of retinoic acids. We determined the structure of the ligand-free ligand-binding domain of the human COUP-TFII, revealing the 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. These results suggest a mechanism where ligands activate COUP-TFII by releasing the receptor from the autorepressed conformation. The identification of COUP-TFII as a low-affinity retinoic acid receptor suggests ways of searching for the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
Structural and functional studies reveal that the orphan nuclear receptor COUP-TFII is a low-affinity receptor for retinoic acids. paving the way to finding the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
doi:10.1371/journal.pbio.0060227
PMCID: PMC2535662  PMID: 18798693

Results 1-2 (2)