SUMMARY Microbiome-encoded β-glucuronidase (GUS) enzymes play important roles in human health by metabolizing drugs in the gastrointestinal (GI) tract. The numbers, types and diversity of these proteins in the human GI microbiome, however, remain undefined. We present an atlas of GUS enzymes comprehensive for the Human Microbiome Project GI database. We identify 3,013 total and 279 unique microbiome-encoded GUS proteins clustered into six unique structural categories. We assign their taxonomy, assess cellular localization, reveal the inter-individual variability within the 139 individuals sampled, and discover 112 novel microbial GUS enzymes. A representative in vitro panel of the most common GUS proteins by read abundances highlights structural and functional variabilities within the family, including their differential processing of smaller glucuronides and larger carbohydrates. These data provide a sequencing-to-molecular roadmap for examining microbiome-encoded enzymes essential to human health.

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The human NR5A nuclear receptors, steroidogenic factor‐1 (SF‐1, NR5A1) and liver receptor homolog‐1 (LRH‐1, NR5A2), are phospholipid‐sensing regulators of steroidogenesis, development, and metabolism. Their control of such diverse biological processes renders them attractive pharmacological targets for the treatment of several cancers and metabolic diseases such as obesity, type II diabetes, and non‐alcoholic fatty liver disease. However, the evaluation of candidate endogenous ligands and the development of small molecule modulators have been hindered by the lack of a robust direct binding assay. Using a structure‐guided approach, we recently developed a potent NR5A agonist (EC50 of 15 nM) targeting the ligand‐binding pocket. We have leveraged this molecule to create a high‐affinity fluorescent probe that binds SF‐1 and LRH‐1 with affinites of 12 nM and 1 nM, respectively. This probe has enabled the creation of a fluorescence polarization (FP)‐based competition assay suitable for both synthetic small molecules and mammalian phospholipids. FP is solution‐based, allowing proteins to retain their native conformation, and the competition format of the assay ensures that binding will only be detected in the ligand‐binding pocket. We have found that for a set of 25 LRH‐1 agonists derived from the previously‐reported RJW100 scaffold, affinities determined by FP correlate with potencies in a luciferase reporter assay, suggesting that in vitro binding affinity may be predictive of in‐cell activity for this class of molecule. Moreover, binding affinities reported for other NR5A agonists harboring different chemical scaffolds agree with our FP‐based measurements. Finally, we show that the previously‐reported dilauroylphosphatidylcholine agonist binds LRH‐1 with a Ki of 80 nM, suggesting that this assay may be used to evaluate candidate endogenous ligands for these receptors. Support or Funding Information R01 DK115213 02EHD has additional support from the National Science Foundation Graduate Research Fellowship and the Emory University NIH‐5T32GM008367‐27. SGM has additional support from F31 DK111171 03. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The synthetic glucocorticoids (GCs) dexamethasone, mometasone furoate, and triamcinolone acetonide are pharmaceutical mainstays to treat chronic inflammatory diseases. These drugs bind to the glucocorticoid receptor (GR), a ligand-activated transcription factor and member of the nuclear receptor superfamily. The GR is widely recognized as a therapeutic target for its ability to counter proinflammatory signaling. Despite the popularity of GCs in the clinic, long-term use leads to numerous side effects, driving the need for new and improved drugs with less off-target pharmacology. X-ray crystal structures have played an important role in the drug-design process, permitting the characterization of robust structure-function relationships. However, steroid receptor ligand-binding domains (LBDs) are inherently unstable, and their crystallization requires extensive mutagenesis to enhance expression and crystallization. Here, we use an ancestral variant of GR as a tool to generate a high-resolution crystal structure of GR in complex with the potent glucocorticoid triamcinolone acetonide (TA) and a fragment of the small heterodimer partner (SHP). Using structural analysis, molecular dynamics, and biochemistry, we show that TA increases intramolecular contacts within the LBD to drive affinity and enhance stability of the receptor-ligand complex. These data support the emerging theme that ligand-induced receptor conformational dynamics at the mouth of the pocket play a major role in steroid receptor activation. This work also represents the first GR structure in complex with SHP, which has been suggested to play a role in modulating hepatic GR function.