The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif–dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

Polyanions provide selective control of APC/C interactions with the activator subunit

“…APC/C substrates interact with the activator WD40 domain via short linear sequence motifs called degrons, of which the D box, KEN box, and ABBA motif are the most common 18,22–29 . Cooperative binding of multiple degron sequences to the activator subunit provides the affinity required for efficient and processive ubiquitylation, and the affinity of degron binding is likely to influence the timing of degradation in vivo 26,30,31 .…”

Revisiting degron motifs in human AURKA required for its targeting by APC/C-FZR1

“…Comparison of the A-box with a panel of D-boxes showed that it docks with an affinity within the range of known D-boxes, although with reduced affinity compared to more canonical D-boxes (Supplementary Figure S4). We note that in silico docking to FZR1 alone ignores any contacts made between substrate and APC10 that probably contribute to the affinity of substrate for the D-box binding pocket (Qin et al 2019) and therefore we may have underestimated the likely preference of Q 45 RVL for the DBR.…”

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C

“…Key factors include the specificity and affinity of individual degrons for the activator and Apc10, as well as the presence of multiple degrons on a substrate. Numerous other factors are also likely to be important, such as the number and positioning of lysines for modification, the distance between degrons, and the orientation of the D box at its bivalent binding site 7,16,18,40 .…”

“…The Nterminal RxxL segment interacts with an acidic patch and aliphatic pocket on the WD40 domain of the activator 10 . The C-terminal residues of the D box interact with the Apc10 subunit [16][17][18] . As a result, the D box helps anchor the activator to the APC/C 19,20 .…”