Fig. 4

Securin inhibits binding to the LPE motif of Scc1. a Cartoon of the securin-separase fusion protein containing the full separase-binding region of securin (left) or with securin truncated on the C-terminal side of the pseudosubstrate motif (right). The separase active site (orange) and pseudosubstrate motif (red) are indicated. b Diagram of the human securin sequence, indicating the locations of the pseudosubstrate sequence (EIEKFFP), all LP sites including the ¹³⁰LPE motif, and the positions of the three truncations tested. See Supplementary Figs. 1b and 7 for amino acid sequences. c Michaelis–Menten analysis was performed with the three indicated securinΔ-separase fusion proteins and compared with purified separase lacking securin. Initial velocity was normalized by enzyme concentration. Data points indicate means (±SD) of triplicate samples. See Supplementary Fig. 8 for a control experiment with securin that includes the pseudosubstrate motif. Source data are provided in the Source Data file. d ³⁵S-labeled Scc1 fragments (aa 142–300), with or without mutations in the ²⁵⁵LPE motif, were incubated with the three indicated securinΔ-separase fusion proteins or with purified separase lacking securin. Reaction products were analyzed by SDS-PAGE and Phosphorimaging. e The pseudosubstrate motif in securin was converted to a separase cleavage site using two point mutations (¹¹⁸FP to RE). Separase was incubated with an ³⁵S-labeled securin^RE fragment (aa 93–150) containing these mutations as well as mutations in the indicated LP motifs. Reaction products were analyzed by SDS–PAGE and Phosphorimaging. Uncropped autoradiograph is provided in the Source Data file. f Sequence alignment of securin pseudosubstrate motifs (red), indicating the downstream conserved LPE motifs (gray). g Cartoon of the securin-separase complex, illustrating the pseudosubstrate motif interaction with the active site and the LPE motif interaction with the separase exosite