The R,R enantiomer of 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC) exerts opposite effects on the transcriptional activity of the two estrogen receptor (ER) subtypes, ER alpha and ER beta. THC acts as an ER alpha agonist and as an ER beta antagonist. We have determined the crystal structures of the ER alpha ligand binding domain (LBD) bound to both THC and a fragment of the transcriptional coactivator GRIP1, and the ER beta LBD bound to THC. THC stabilizes a conformation of the ER alpha LBD that permits coactivator association and a conformation of the ER beta LBD that prevents coactivator association. A comparison of the two structures, taken together with functional data, reveals that THC does not act on ER beta through the same mechanisms used by other known ER antagonists. Instead, THC antagonizes ER beta through a novel mechanism we term 'passive antagonism'.

The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N-and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9-19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9-19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

A novel RNA recognition motif is characterized in an arginine-rich peptide. The motif, derived from lambda transcriptional antitermination protein N, regulates an RNA-directed genetic switch. Its characterization by multidimensional nuclear magnetic resonance (NMR) demonstrates specific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend. The biological importance of the bent alpha-helix is demonstrated by mutagenesis: binding is blocked by substitutions in the N peptide or its target (the boxB RNA hairpin) associated in vivo with loss of transcriptional antitermination activity. Although arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts. An alanine in the N-terminal helix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop. At these positions all 19 possible amino acid substitutions have been constructed by peptide synthesis; each impairs binding to boxB. The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo. The helix-bend-helix motif rationalizes genetic analysis of N-dependent transcriptional antitermination and extends the structural repertoire of arginine-rich domains observed among mammalian immunodeficiency viruses.

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