Cohesin cleavage by separase is enhanced by a substrate motif distinct from the cleavage site

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

StrepII tag (light blue), securin aa 93-202 (purple), Gly-Ser linker (light gray), TEV protease cleavage site (pink), 3x FLAG tag (teal), Separase 1 Met (green), S1126A mutation (red), autocleavage site mutations EXXR à RXXE (dark gray), active site 2029 Cys (orange). Dashed gray lines indicate predicted intrinsically disordered regions. The catalytically dead variant is the same sequence except that autocleavage sites are not mutated and 2029 Cys is mutated to Ser. b. Sequences of the securinΔ-separase constructs from the N-terminus to the beginning of separase, after which the sequences are the same as that of separase in panel a. Annotations are the same as in panel a, except that securin starts at residues 127, 138, or 160. Note that cleavage at the TEV protease site is not necessary for activity toward peptide substrate. For the securinΔ127-separase construct, we were concerned that securin is truncated so close to the pseudosubstrate motif that the adjacent StrepII tag might interfere with the active site; thus, for this construct only we placed the TEV protease site between the 2x StrepII tag and the beginning of securin to allow removal of the tag if necessary. However, activity proved to be normal in the presence of the tag. In these pilot studies, we tested two ClpXP degrons (OmpA and LambdaO). Securin-separase fusion proteins with the indicated ClpXP degron at the N-terminus were incubated with TEV protease followed by ClpXP, or a combination of the two, and subjected to SDS-PAGE and Coomassie Blue staining. TEV protease alone generates a faintly visible securin band. In the absence of TEV protease, ClpXP degrades the securin-separase fusion protein, and greater degradation is seen with the LambdaO degron. After treatment with both TEV protease and ClpXP, ClpXP presumably degrades the cleaved securin, but the securin band, if present, would be obscured by ClpP. Attempts to detect securin by Western blotting were unsuccessful. Based on this and other experiments, LambdaO was found to be the most effective ClpXP degron and was used for subsequent constructs ( Supplementary Fig. 1a).
b. Securin-separase fusion proteins ('Active,' wild-type active site with mutant autocleavage sites; 'Dead,' C2029S mutation with wild-type autocleavage sites) were incubated with TEV protease followed by ClpXP +/-ATP, and subjected to SDS-PAGE and Coomassie Blue staining. Separase activity is apparent as intermolecular cleavage of dead separase in the last lane. The ClpX protein used here undergoes some autodegradation upon activation by ATP.
c. SDS-PAGE and Coomassie Blue staining were used to evaluate the steps in the purification of active separase after TEV protease and ClpXP incubation with the securin-separase fusion protein.

d.
To assess the nature of the separase fragments in the preparations, purified separase was evaluated by alignment of the Coomassie-stained gel (inner lanes) with a Western blot against the N-terminal FLAG tag (outer lanes). These results confirm that the smaller bands in the preparation are N-terminal fragments of separase. Because fragments are seen in the dead separase preparation, we conclude that ClpXP is at least partially responsible for this cleavage. Additionally, the amount of full-length separase is reduced in the active separase preparation, which we suspect is due to separase autocleavage at non-canonical sites, presumably an artifact of high enzyme concentration in vitro. More full-length separase was preserved in shorter reactions (as seen in panel c, lane 7). Despite the extent of separase cleavage into N-and C-terminal fragments as visible by SDS-PAGE, the negative-stain EM results support previous evidence that fragments remain bound together in a structure that is indistinguishable from intact full-length separase at low resolution. Uncropped western blot is provided in the Source Data file.
e. This figure demonstrates that the StrepTrap column separates apo separase from securinbound separase after TEV and ClpXP incubation. Samples from different steps in the purification procedure were subjected to SDS-PAGE and Coomassie-Blue staining (left) and also tested for activity in the cleavage assay with full-length (FL) Scc1 (right). Lanes: 1. Substrate alone (including TEV protease used for elution of Scc1 from beads, plus unidentified contaminants); 2. Untreated securin-separase fusion protein; 3. ClpXP alone; 4. TEV-and ClpXP-treated separase in flowthrough of StrepTrap column, with no securin bound (separase is at low concentration and not clearly visible, but this sample was used for the sizing column whose output is shown in lane 7); 5.
StrepTrap column elution re-treated with ClpXP; 7. Final active separase sample, containing fulllength active separase as well as separase N-and C-terminal fragments. In the Scc1 cleavage assay, cleavage occurs primarily at site 1, producing the top and bottom two cleavage fragments; these fragments are not generated when site 1 is mutated (data not shown). Site 2 cleavage would produce two products of similar sizes, but these are not observed in the site 1 mutant. Instead, the high enzyme concentration in some reactions generates two intermediate fragments that probably represent cleavage at a non-canonical site.