Abstract:

Degradation by the ubiquitin proteasome system rests on two decisions: which ubiquitin chain is built on a substrate, and how that chain is read. We approach both ends with cryo-EM and biochemical reconstitution.

On the writing side, our structures of large HECT ligases converge on one picture: they are oligomeric machines rather than isolated catalytic domains. UBR5 is a 1.2 MDa tetramer that works as a chain elongator rather than a conventional initiator, placing it as a general regulator of protein lifetime in the nucleus. HERC1, the largest HECT ligase, is a 1.6 MDa trimer carrying ten interaction platforms per protomer. It engages the orphaned proteasome subunit PSMC5 across two protomers, with trimerisation required for multi-monoubiquitination. Both ligases contrast in their ubiquitination activity, while HERC1 is a great chain initiator, UBR5 is a chain elongator.

On the reading side, we used a time-resolved cryo-EM and synthetic ubiquitin chains, catching recognition before the chain is stripped. Structures of the human 26S proteasome show K48-linked chains wrapping around the RPN10 ubiquitin-interacting motif in an unexpected spiral, while K11/K48-branched chains engage a distinct cleft between RPN2 and RPN10. Structure-guided mutagenesis and cellular assays show that both binding modes are required for efficient degradation and cell cycle progression, explaining why branched substrates are cleared preferentially.

Bio:

David Haselbach is a group leader and Head of the EM Technology Platform at the Research Institute of Molecular Pathology (IMP) in Vienna, where he develops and applies advanced cryo-electron microscopy approaches to understand how molecular machines assemble and function. His research primarily focuses on molecular mechanisms underlying protein degradation and other essential cellular processes. He received his PhD in Holger Stark’s lab at the Max Planck Institute for Biophysical Chemistry in Göttingen in 2014, where he subsequently continued as a postdoctoral researcher. In 2017, he joined the IMP as a fellow and was promoted to group leader in 2020. His work combines structural biology, biophysics and innovative cryo-EM methods to capture molecular machines in action and has contributed to understanding complexes including the proteasome and spliceosome. He was selected as an EMBO Young Investigator in 2023.