Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5

Hehl, L. A.; Horn-Ghetko, D.; Prabu, J. R.; Vollrath, R.; Vu, D. T.; Perez Berrocal, D. A.; Mulder, M. P. C.; van der Heden van Noort, G. J.; Schulman, B. A.

doi:10.1101/2023.06.06.543850

bioRxiv · 10.1101/2023.06.06.543850

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5

Hehl, L. A.·Horn-Ghetko, D.·Prabu, J. R.·Vollrath, R.·Vu, D. T.·Perez Berrocal, D. A.·Mulder, M. P. C.·van der Heden van Noort, G. J.·Schulman, B. A.

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Abstract

Ubiquitin chain formation by HECT catalytic domain-containing E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We employed chemistry and cryo-EM to visualize stable mimics of the intermediates along K48-linked ubiquitin chain formation by the human E3, UBR5. The structural data reveal a {approx}620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly-tethered ubiquitin-binding UBA domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor ubiquitin, with its K48 lured into the active site by numerous interactions between the acceptor ubiquitin, manifold UBR5 elements, and the donor ubiquitin. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing ubiquitin transfer from E2 to E3, and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2, and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked ubiquitin chain forging machine.

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Hehl, L. A., Horn-Ghetko, D., Prabu, J. R., Vollrath, R., Vu, D. T., Perez Berrocal, D. A., Mulder, M. P. C., van der Heden van Noort, G. J., Schulman, B. A.. 2023-06-07. Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5. https://doi.org/10.1101/2023.06.06.543850

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