HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain

Abstract

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure where the C-terminal HECT domain heads an extended alpha solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins. c Ub-charged HECT E3 C2476 catalytic site substrate-binding sites UbcH5b-Ub / NEDD4L(HECT) Rsp5 (HECT)-Ub steric clash: C-lobe HECT : ARM8-10

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

“…To further test the utility of LotA N in diagnosing the presence of K6 linkages among a complex mixture, we generated polyUb chains with HUWE1, a human HECT E3 ligase that is reported to assemble a mixture of K6-, K11-, and K48-linked chains (Michel et al , 2017; Jäckl et al , 2018; Grabarczyk et al , 2021). Treatment with the K11-specific DUB Cezanne released the largest amount of monoUb, followed by the K48-specific OTUB1*, then LotA N , and finally the constitutively-activated K63-specific AMSH* (Michel et al , 2015), which had only a marginal amount of activity ( Fig.…”

The giant E3 ligase HUWE1 is linked to tumorigenesis, spermatogenesis, intellectual disability, and inflammatory diseases

“…This study demonstrated that a conformational switch regulates HUWE1 activity (Sander et al, 2017). Recently, cryo-EM structures of Nematocida and human full-length HUWE1 demonstrated that HUWE1 harbors a snake-like and giant substrate-binding ring, which is highly dynamic, enabling engagement with a multitude of diverse substrates for catalysis via the flexible HECT domain (Hunkeler et al, 2021;Grabarczyk et al, 2021). The HUWE1 protein has become an attractive potential therapeutic target for treating cancer and other diseases, and structural analysis will significantly advance the development of activity inhibitors with substrate-selective recruitment.…”

“…Due to its large size, the HUWE1 protein has been identified to mediate the ubiquitination of a multitude of diverse substrates through its giant substrate-binding ring structure, and this ring structure is highly dynamic. The ligase activity of the flexible C-terminal HECT domain is actually determined by its conformational change through dimerization of the C-terminal HECT domain or disruption of the dimer interface (Sander et al, 2017;Grabarczyk et al, 2021;Hunkeler et al, 2021). Furthermore, Huwe1 gene is located on the X chromosome and associated with X chromosome-linked intellectual disability.…”

HUWE1 controls tristetraprolin proteasomal degradation by regulating its phosphorylation

“…Previous studies have indicated that HUWE1 can target broader classes of cellular substrates (Hunkeler et al , 2021; Grabarczyk et al , 2021), but that the targeted proteins may be cell type specific to some degree. Analysis of proteome changes in the three different cell lines identified seven proteins that were consistently increased in all Huwe1 -targeted cell lines (Fig.…”