No abstract

Hypoxia‐inducible factor‐2 (HIF‐2) is a heterodimeric transcription factor formed through dimerization between an oxygen‐sensitive subunit HIF‐2α subunit and its obligate partner subunit ARNT. Enhanced HIF‐2 activity drives some cancers, while reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF‐2 activity via direct‐binding ligands could provide many new therapeutic benefits. Here, we explored HIF‐2α chemical ligands using combined crystallographic, biophysical, and cell‐based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF‐2α PAS‐B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first‐in‐class HIF‐2α agonists and found they significantly displaced pocket residue Y281. Its dramatic side‐chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF‐2α PAS‐B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF‐2α‐ARNT heterodimerization. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTE Psy ) from Pseudomonas syringae pv. syringae B728a. Compared with RecTE Psy , the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas . Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P . aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P . syringae . Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E . coli . The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas .

Hypoxia-inducible factors (HIFs) are α/β heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/β heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.