A conserved tyrosine motif encodes a structural vulnerability controlling stability and activity across J-domain proteins

J-domain proteins (JDPs) direct Hsp70 functions through a conserved J-domain that engages and activates Hsp70. JDPs support proteostasis and are linked to development and disease, yet how they are regulated remains unclear. Here, we identify a conserved tyrosine motif outside the canonical four-helix J-domain core, as an unexpected structural control element across the JDP family. NMR shows that phosphomimetic substitutions at these residues disrupt the J-domain fold in DNAJA1 and DNAJB1. Across JDP classes, motif perturbation produces two outcomes: reduced abundance or functional inactivation through impaired Hsp70 engagement without protein loss. Motif disruption also rewires the DNAJA1 interactome and sensitizes yeast expressing the Ydj1 variant to stresses. These findings reveal an unanticipated structural vulnerability coupling J-domain integrity to JDP stability and activity. Recurrent identification of this motif as a phosphorylation site suggests that its modification enables signaling pathways to remodel Hsp70-JDP networks in response to changing growth conditions. NMR spectroscopy and functional assays define a conserved tyrosine motif outside the canonical J-domain core of J-domain proteins. The motif stabilizes the fold, and its phosphomimetic perturbation drives J-domain protein degradation or disables Hsp70 engagement.