Hierarchical cysteine oxidation controls reversible amyloid formation in an ankyrin repeat protein

Abstract

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.