Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich’s ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich’s ataxia therapies.
Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of ‘supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.
A new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electron lasers. These novel sources deliver very intense femtosecond X-ray pulses that promise to yield high-resolution diffraction data of nanocrystals before their destruction by radiation damage. Thus, the concept of `diffraction before destruction' requires rapid replenishment of the sample for each exposure. The chip promotes the self-assembly of an array of protein crystals on a surface. Rough features on the surface cause the crystals to adopt random orientations, allowing efficient sampling of reciprocal space
The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.
Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S] 2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = 164±5 mV, pKox1,27.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 150 mV, pKox1,2=9.8 and 11.5) and cytochrome bc1/b6f Rieske clusters (Em,7 +300 mV, pKox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of [2Fe-2S] cluster containing proteins with bis-histidinyl coordination and proton-coupled electron transfer. Strain Plasmid Gallobenzophenone 25 µM none 50 µM empty Aim32 ∆aim32 Wild type Apd1 empty ∆apd1 empty Aim32 Apd1 a empty ∆sod2 Strain Plasmid ∆sod2/ Δaim32 empty Aim32 Apd1 Pyrogallol 25 µM none 50 µM empty Aim32 ∆aim32 Wild type
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