Christoph Göbl

Glutathione is synthesised across all kingdoms of life, but certain bacteria, including the major pathogen Streptococcus pneumoniae, import host glutathione instead. Glutathione import is known to involve the substrate‐binding protein GshT, but the molecular basis underlying glutathione scavenging by pneumococcal GshT remains incompletely understood. We used isothermal titration calorimetry to demonstrate that pneumococcal GshT binds glutathione with low micromolar affinity. Thermal shift assays with glutathione derivatives demonstrated that two adequately spaced carboxylates in glutathione are essential for binding. Monitoring NMR chemical shift perturbations of mutated GshT variants upon glutathione addition revealed that two conserved positively charged residues lining the ligand‐binding pocket determine the ability to bind glutathione. Mutagenesis of these residues in S. pneumoniae abolished GSH uptake, impaired growth and lowered oxidant tolerance. Together, our data demonstrate that a lock‐and‐key charge complementarity drives the GshT‐glutathione interaction. Our findings provide insights into pathogen survival mechanisms that may inform strategies to combat infectious disease.

The formation of amyloids, including functional amyloids, is observed for an increasing number of proteins but the molecular mechanisms that control this structural transition remain poorly understood. Here we report that the kinase inhibitor protein P18 (drP18) from Danio rerio (zebrafish), which contains two cysteine residues, undergoes a complex and hierarchical redox switch that strictly governs reversible amyloid formation. We identify cysteine 50 (C50) acting as a regulatory residue. Upon oxidation, C50 forms an intramolecular disulfide bond with the executioner cysteine 128 (C128), thereby blocking it. C50 can become S-glutathionylated, and upon oxidation, C128 then forms intermolecular disulfides that lead to rapid transition into amyloid fibrils. S -glutathionylation of C50 therefore enables amyloid formation of drP18 and the outcome is oxidant-dependent with diamide, hydrogen peroxide, peroxymonocarbonate and hypothiocyanous acid each leading to amyloid assembly with distinct kinetics and morphologies. These amyloids are fully reversible, where disulfide reduction is leading to disassembly. Whereas monomeric drP18 inhibits CDK4-mediated retinoblastoma phosphorylation, the amyloid conformation abolishes this inhibition, and reduction restores both structure and function. Expression of drP18 in zebrafish embryos yields Congo red-positive, oxidation-dependent aggregates in vivo . Together, our findings show that a regulatory cysteine controls an executioner cysteine to induce reversible, functional amyloid formation, revealing that proteins can encode sophisticated mechanisms to control amyloid assembly. Significance: The functions of proteins are based on their three-dimensional shapes, and some can switch into alternative structures called amyloids, some of which are disease-related and others appear to have biological functions. We show that a zebrafish protein, drP18, can transition into an amyloid fold, driven by a sophisticated two-cysteine oxidation mechanism. drP18 carries two distant cysteine residues that respond differently to oxidative conditions, producing a locked form or amyloid-like aggregates depending on the conditions. Unlike disease-associated amyloids, this transition is fast, chemically controlled, and fully reversible. These results reveal how two distant cysteine residues can work together as a chemical switch to determine protein structure, suggesting that oxidation-driven amyloid formation can act as a more general mechanism of protein regulation.

The tumor suppressor protein p16 ^INK4a plays a key role in cell cycle control and is frequently inactivated in human cancers. We previously discovered that oxidation of its single cysteine residue induces a structural transition from the functional monomeric form into inactive amyloid fibrils. Here, we investigate whether five physiologically relevant oxidants can trigger the conformational switch of the protein. Using time‐resolved SDS‐PAGE, thioflavin‐T fluorescence assays, mass spectrometry, and electron microscopy, we show that peroxymonocarbonate and hypothiocyanous acid (HOSCN) efficiently induce cysteine‐dependent dimerization with subsequent amyloid formation of recombinant p16. In contrast, hydrogen peroxide, hypochlorous acid, and taurine chloramine are less efficient and predominantly lead to the formation of amorphous aggregates. These findings demonstrate that physiological oxidants differ in their ability to induce amyloid transitions in p16, with potential implications for oxidative stress‐mediated inactivation of this cell‐cycle regulator protein.

The INK4 family of proteins restricts uncontrolled cell cycle progression by inhibiting cyclin-dependent kinases 4 and 6. The family consists of small, monomeric and mainly alpha-helical proteins that are conserved across all vertebrate species. We recently discovered that the human INK4 protein p16 converts into amyloid structures upon oxidation of the single cysteine residue present. Here we investigate the Danio rerio (zebrafish) orthologue P18 protein. The 170-residue protein contains two cysteines which may similarly mediate transition into amyloids upon oxidation. We present the near complete backbone assignment of the reduced P18 protein in solution. These chemical shift data provide the foundation for studying oxidation-induced structural changes and protein interactions.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16^INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16^INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.