Proteomics

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Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1


ABSTRACT: Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. Structure determination of the underlying, specific E3-substrate complexes, however, has proven challenging due to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases position substrate proteins for modification. Here we report a cryo-EM structure of the full-length human HECT-type ligase HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We employ mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, visualize its structure by cryo-EM, and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation, and specificity of full-length HECT-type ligases.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Wieland Steinchen  

LAB HEAD: Wieland Steinchen

PROVIDER: PXD045837 | Pride | 2024-02-12

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
230309_HX_475_Ba.fas Other
230309_HX_475_Ba_HACE1_dN21-RAC1_Q61L.zip Other
230309_HX_475_Ba_HACE1_dN21.DnX Other
230309_HX_475_Ba_HACE1_dN21.zip Other
230309_HX_475_Ba_PLGS.zip Other
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