Mechanisms of autoinhibition and substrate recognition by HACE1. A, Cartoon summarizing the main findings of this study: HACE1 is auto-inhibited by dimerization, with the N helix making critical intermolecular contacts with the HECT domain at the subunit interface. The yin-yang dimer prohibits Ub transfer from E2 and is incompatible with engagement of RAC1 as a substrate. Activation of HACE1 requires release of the N helix, allowing productive interactions with E2 and substrates, such as RAC1. The RAC1 binding mode explains how HACE1 selects GTP-loaded RAC1 rather than GDP-loaded RAC1. We propose that phosphorylation of pivot sites in the N-terminal region of HACE1 may stabilize the monomeric active state. Additionally, interaction partners can regulate the conformation of the N helix and thus HACE1 activity. b, Donor Ub modeled in the cryo-EM structure of the HACE1 ∆N–RAC1 Q61L complex, based on a superposition of the HECT domain with PDB 6XZ1. Credit: Nature Structural and molecular biology (2024). DOI: 10.1038/s41594-023-01203-4
Ubiquitin ligases regulate many processes in living cells. Depending on how many ubiquitin molecules they attach to target proteins and how they control the fate of the protein.
“The ubiquitin code can direct target proteins to specific cellular sites of action, activate them, trigger their incorporation into protein complexes or their degradation,” explains Sonja Lorenz, head of the Ubiquitin Signaling Specificity research group. ubiquitin at MPI. It is therefore crucial that ubiquitin ligases reliably recognize the corresponding proteins among tens of thousands of molecules and transmit the correct code.
How this molecular recognition works is at the heart of the research of Lorenz and his team. “We want to uncover the structural principles of this specific recognition and reveal how they enable different ubiquitin ligases to control specific processes,” explains the biochemist. This knowledge is not only important for understanding the role of ubiquitin ligases in normal or pathologically altered processes in living cells. This also helps make the ubiquitin system more accessible for therapeutic applications.
Dynamic ubiquitin system
However, it is difficult to observe ubiquitin ligases bound to their target proteins. “The ubiquitin system is designed to be highly dynamic,” says Lorenz. “The underlying interactions are therefore very weak and short-lived.” Nevertheless, the group managed to visualize such an interaction between the ubiquitin ligase HACE1 and its main target protein, the signaling mediator RAC1.
The researchers used a chemical trick to achieve this. “Thanks to chemical crosslinking, we were able to stabilize the complex at the right time to make it accessible to structural studies by electron cryomicroscopy,” explains the head of the research group. In collaboration with the MPI Electron Cryomicroscopy Facility, led by Christian Dienemann, the team succeeded, for the first time, in obtaining a “snapshot” of the complete HACE1-RAC1 complex.
Through additional biophysical and functional experiments, Lorenz’s team discovered that the particular architecture of the ubiquitin ligase ensures that HACE1 and its target protein interact with specificity. Their studies show that the ubiquitin ligase forms a platform on which the target protein is positioned. This allows the ligase to reliably recognize its target protein and distinguish it from other proteins. HACE1 can also determine whether RAC1 is in an active or inactive state. This is crucial because RAC1 should only be tagged with ubiquitin in its active form.
However, the HACE1 ubiquitin ligase itself can also adopt an active or inactive state, report Jonas Düring and Madita Wolter, joint first authors of the paper now published in Nature Structural and molecular biology. “Two HACE1 molecules can bind to each other to form a complex, called a dimer, and thus switch off,” explains Düring. Wolter adds: “HACE1 is only active as a single molecule. Dimerization is therefore an important regulatory mechanism. »
“If HACE1 no longer functions properly in the cell or is completely absent, this can lead, for example, to disruptions in nerve development associated with human diseases,” explains Lorenz.
“Through understanding the precise interaction of HACE1 and its target protein RAC1, we will be able to better understand how genetic changes in this molecular labeling machine can disrupt target protein recognition.” The new findings also contribute to the understanding of related ubiquitin ligases, whose target recognition mechanisms are still poorly understood.
More information:
Jonas Düring et al, Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1, Nature Structural and molecular biology (2024). DOI: 10.1038/s41594-023-01203-4
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