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.
Narasimharaju Kalidindi, A Leder, Guillaume Mas et al.· Communications Biology· 0 citations
The archive contains the processed ¹⁵N CEST profiles of WT Im7 recorded at 600, 800 and 1200 MHz at 10 °C and at 800 MHz at 15 and 20 °C, the ¹⁵N TROSY-CPMG profiles recorded at 600 and 800 MHz at 10 °C, and the ChemEx experiment, method and parameter files together with the shell scripts that launched every fit reported in the paper. Also included are the scripts that extract the fitted parameters and draw each figure and table, and the self-contained simulations behind Figures 1, S1 and S7.
Ved Prakash Tiwari, Laetitia Rožić, Sebastian Hiller· Zenodo (CERN European Organi...· 0 citations
The archive contains the processed ¹⁵N CEST profiles of WT Im7 recorded at 600, 800 and 1200 MHz at 10 °C and at 800 MHz at 15 and 20 °C, the ¹⁵N TROSY-CPMG profiles recorded at 600 and 800 MHz at 10 °C, and the ChemEx experiment, method and parameter files together with the shell scripts that launched every fit reported in the paper. Also included are the scripts that extract the fitted parameters and draw each figure and table, and the self-contained simulations behind Figures 1, S1 and S7.
Ved Prakash Tiwari, Laetitia Rožić, Sebastian Hiller· Zenodo (CERN European Organi...· 0 citations
“Undruggable” proteins without surface-accessible binding sites pose significant challenges to target-based drug discovery. Innovative approaches are needed to tackle these proteins. One promising strategy is targeting them in their nascent chain form at the ribosome, where they have a different conformation than in the folded form. A systematic approach to screen for such compounds has however been lacking. Here, we present a high-throughput assay to identify small molecules that specifically inhibit a protein of interest in its nascent-chain form. The assay employs a human in vitro transcription/translation system and monitors expression of the protein in real time via fluorescence detection. Specific inhibitors for the nascent chain of interest can then be identified by comparison with a counter screen. The assay was optimized to maximal sensitivity and reaction costs of ∼$0.01 per well, enabling large-scale screens. We validated performance with the reference compound PF846 on the nascent chain of the protein PCSK9. Feasibility for high-throughput screening was demonstrated using a library of 1,760 compounds against the oncogenic KRAS variant A146T and the protein ApoC3, with mean Z′ scores of 0.69 and 0.88, respectively. Sixteen global translation inhibitors were identified in each campaign, while no compounds met the criteria for POI-selective inhibition. The assay thus provides a robust platform for larger-scale screening campaigns.
Patrick D. Fischer, Sebastian Hiller· bioRxiv· 0 citations
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