The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

The E3 ubiquitin ligase CHIP (C‐terminus of Hsc70‐interacting protein) is believed to be a central player in the cellular triage decision, as it links the molecular chaperones Hsp70/Hsc70 and Hsp90 to the ubiquitin proteasomal degradation pathway. To better understand the decision process, we determined the affinity of CHIP for Hsp70 and Hsp90 using isothermal titration calorimetry. We analyzed the influence of CHIP on the ATPase cycles of both chaperones in the presence of co‐chaperones and a substrate, and determined the ubiquitination efficacy of CHIP in the presence of the chaperones. We found that CHIP has a sixfold higher affinity for Hsp90 compared with Hsc70. CHIP had no influence on ADP dissociation or ATP association, but reduced the Hsp70 cochaperone Hdj1‐stimulated single‐turnover ATPase rates of Hsc70 and Hsp70. CHIP did not influence the ATPase cycle of Hsp90 in the absence of co‐chaperones or in the presence of the Hsp90 cochaperones Aha1 or p23. Polyubiquitination of heat‐denatured luciferase and the native substrate p53 was much more efficient in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicating that CHIP preferentially ubiquitinates Hsp70‐bound substrates. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904367: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904785: HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904047: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7), HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903424: Alpha‐lactalbumin (uniprotkb:http://www.uniprot.org/uniprot/P00711), HSP70 (uniprotkb:http://www.uniprot.org/uniprot/P08107) and CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903354: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSC70 (uniprotkb:http://www.uniprot.org/uniprot/P11142) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by isothermal titration calorimetry (h...

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.