The disordered negatively charged <i>C</i>‐terminus of the large <scp>HECT E3</scp> ubiquitin ligase <scp>HERC2</scp> provides structural and thermal stability to the <scp>HECT</scp><i>C</i>‐lobe

Homologous to the C-terminus of E6AP (HECT) and RCC1-like domain (RLD)-containing protein 2 (HERC2) is a large, 528 kDa E3 ubiquitin ligase that is associated with cancer, oculocutaneous albanism type 2, Prader-Willi syndrome, and other neurological diseases. HERC2 has been found to contribute to double-stranded DNA break repairs, tumor suppression, maintaining centrosome architecture, and ubiquitylation. The C-terminal portion of the HECT domain (C-lobe) of HERC2 is responsible for transferring ubiquitin to a substrate but the precise function of the other eight domains in HERC2 are unknown. Interestingly, HERC2 contains a unique and negatively charged C-terminal tail adjoined to the C-lobe that is predicted to act as a linker to promote interactions between HERC2 and its binding partners. This study aims to better understand the function and relevance of HERC2 in disease by investigating the structural aspects of the HERC2 C-lobe and HERC2 C-terminal tail using AlphaFold followed by molecular dynamics (MD) simulations, multidimensional nuclear magnetic resonance (NMR), and circular dichroism (CD). Secondary structure content analysis from MD simulations and the fully resonance assigned ¹H-¹⁵N HSQC spectra of the HERC2 C-lobe and the isolated C-terminal tail confirm that the C-lobe is well-folded but the C-terminal tail is disordered. CD melting curves indicate that the flexible C-terminal tail provides improved stability to the C-lobe. Additionally, MD simulations have identified that the interaction between residues D4829 and R4728 is prevalent among the non-bonded contacts between the tail and the C-lobe. Overall, our results demonstrate that the negatively charged C-terminal tail is disordered, provides stability to the C-lobe, and may act as a flexible scaffold for protein-protein interactions.