Fig 2: Sequence alignment of HECT domains of human E6AP, HUWE1, NEDD4, UBE3C, HECTD1, and UBR5. Residues strictly conserved (red boxes) and partially conserved (yellow boxes) are shown. Blue circles indicate residues mediating E2 binding in the structure of E6AP:UBCH7;10 the conserved catalytic cysteine is shown as a green circle, and the flexible hinge loop connecting the N and C lobes is shown as an orange box.

Fig 3: Compatibility of E2s to support HECT ligase activity. A) Sequence alignment of UBE2L3 (UBCH7) and UBE2D family members UBE2D1 (UBCH5A), UBE2D2 (UBCH5B), and UBE2D3 (UBCH5C). Residue F63 (UBE2L3), critical for binding to the HECT domain of E6AP and conserved between UBE2L3 and UBE2Ds (F62), is shown as a purple circle. Noncatalytic Cys residues are shown as black circles; catalytic Cys is shown as a yellow circle. Auto-ubiquitylation assays were carried out with WT ubiquitin, His-E1, E2, and tagged HECT catalytic domains: B) GST-NEDD4, C) GST-UBE3C, D) GST-HUWE1, E) GST-HECTD1, and F) GST-UBR5. After 3 h at 30 °C, reactions were terminated by addition of 4×LDS/DTT and resolved on 4–12 % SDS PAGE gels. Auto-ubiquitylation was detected with an anti-GST antibody followed by LI-COR infrared detection (top panels) or with an anti-ubiquitin antibody followed by ECL detection (lower panels). Reactions lacking E2 (-E2) were negative controls. Asterisks indicate unmodified GST-HECT domains.

Fig 4: In vitro labelling of GST-HECT domains with Ub-based ABPs, A) Ub-VME,25 or B) Biotin-Ub-PA (also referred to as Ub-PA).26 C) GST-tagged NEDD4, UBE3C, HUWE1, HECTD1, or UBR5 was incubated for 8 h at 30 °C with either Ub-VME (V) or Ub-PA (PA). Reactions were resolved on a 4–12 % PAGE and detected by Coomassie (left) or silver staining (right). Asterisks indicate unlabelled GST-HECT; arrows indicate GST-HECT domains labelled by Ub-ABP. D) Time-course [hours] of labelling of GST-NEDD4 at 30 °C with Ub-ABPs and E2–Ub-ABP.