PT  - JOURNAL ARTICLE
AU  - Takeda, Kazusa
AU  - Muro, Ikumi
AU  - Kobayashi, Fuminori
AU  - Flechsig, Holger
AU  - Kodera, Noriyuki
AU  - Ando, Toshio
AU  - Konno, Hiroki
TI  - Structural dynamics of E6AP E3 ligase HECT domain and involvement of flexible hinge loop in ubiquitin chain synthesis mechanism
AID  - 10.1101/2022.11.18.516873
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.11.18.516873
4099  - http://biorxiv.org/content/early/2022/11/18/2022.11.18.516873.short
4100  - http://biorxiv.org/content/early/2022/11/18/2022.11.18.516873.full
AB  - Ubiquitin (Ub) ligases E3 are an important factor in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe containing the E2-binding site and a C-lobe containing the catalytic Cys residue that forms a thioester bond with Ub. These two lobes are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of this type of E3 ligases remain unclear. Here, we provide the first direct demonstration of structural dynamics of the E6AP HECT domain using high-speed atomic force microscopy. We also investigated structural dynamics of hinge loop flexibility restricted HECT domain, and we found that flexibility of the E6AP hinge loop has a great impact not only on its structural dynamics but also on the formation of free Ub chains mediated by E3-E3 interactions.Competing Interest StatementThe authors have declared no competing interest.