Fig. 4: CUL3 NA motif deletion impairs dimeric assembly of CRL3KLHL22.
From: A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22
a Diagram depicting domain organization of CUL3 and truncation mutants used in subsequent S-tag pulldown, SEC, ITC, and SEC-MLAS assays. CR, Cullin repeats. b–d ITC measurement of binding affinity between KLHL221–178 and CUL3WT (b), CUL3Δ1–13 (c), and CUL3Δ 1–24 (d). The binding constants (KD ± SD) are indicated. This experiment performed once. e Deletion of CUL3 NA motif impairs interaction between CUL3 and KLHL22 in intact cells. KLHL22WT-(S-tag) or KLHL22Δ1–23-(S-tag) were co-expressed with CUL3WT-mCherry, CUL3Δ1–13-mCherry, and CUL3Δ1–24-mCherry, respectively, in HEK 293T cells, and S-tag pulldown assays were performed. This S-tag pulldown assay performed once. f SEC analysis of the effect of CUL3 NA motif deletion on the assembly of the CUL3-RBX1-KLHL221–178 complex. Chromatograms show the elution profiles of CUL3WT-RBX1-KLHL221–178 (blue line), CUL3Δ1–13-RBX1-KLHL221–178 (purple line), CUL3Δ1–24-RBX1-KLHL221–178 (red line), and KLHL221–178 alone (yellow line). Peaks corresponding to dimers and excess unbound CUL3-RBX1 are indicated with dark arrows. The results shown are representative of three biological replicates. g, h SEC-MALS analysis of the effect of CUL3 NA motif deletion on the assembly of CUL3-RBX1-KLHL221–178 complex. g and h Show the elution profiles of CUL3WT-RBX1-KLHL221–178 (g) and CUL3Δ1–24-RBX1-KLHL221–178 (h), respectively. This experiment performed two times. i Cryo-EM density map of the CUL3Δ1–24-RBX1-KLHL221–178 complex. The KLHL221–178 dimer binds only one CUL3Δ1–24-RBX1. The corresponding 2D classification of the CUL3Δ1–24-RBX1-KLHL221–178 complex is shown in the upper right corner. j SEC analysis of the effect of the concentration of CUL3-RBX1 on the assembly of the CUL3Δ1–24-RBX1-KLHL221–178 complex. Chromatograms show the elution profiles of CUL3WT-RBX1-KLHL221–178 (blue line, molar ratios 2:1), CUL3Δ1–24-RBX1-KLHL221–178 (purple line, molar ratios 2:1), CUL3Δ1–24-RBX1-KLHL221–178 (red line, molar ratios 4:1), and KLHL221–178 alone (yellow line). This experiment performed once. For b–h and j, source data are provided as a Source Data file.
