Velasco Carneros, LoreaBernardo Seisdedos, GanekoMaréchal, Jean-DidierMillet, ÓscarMoro Pérez, FernandoMuga Villate, Arturo2025-03-112025-03-112024-08Velasco-Carneros, L., Bernardo-Seisdedos, G., Maréchal, J.-D., Millet, O., Moro, F., & Muga, A. (2024). Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system. Protein Science, 33(8). https://doi.org/10.1002/PRO.51050961-836810.1002/PRO.5105https://hdl.handle.net/20.500.14454/2507The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.eng© 2024 The Author(s)ChaperoneDNAJA2Hsc70J-domainPhosphorylationProtein foldingjournal article2025-03-111469-896X