Counting Degrons: Lessons From Multivalent Substrates for Targeted Protein Degradation

Abstract

E3s comprise a structurally diverse group of at least 800 members, most of which target multiple substrates through specific and regulated protein-protein interactions. These interactions typically rely on short linear motifs (SLiMs), called "degrons", in an intrinsically disordered region (IDR) of the substrate, with variable rules of engagement governing different E3-docking events. These rules of engagement are of importance to the field of targeted protein degradation (TPD), where substrate ubiquitination and destruction require tools to effectively harness ubiquitin ligases (E3s). Substrates are often found to contain multiple degrons, or multiple copies of a degron, contributing to the affinity and selectivity of the substrate for its E3. One important paradigm for E3-substrate docking is presented by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ligase that targets hundreds of proteins for destruction during mitotic exit. APC/C substrate targeting takes place in an ordered manner thought to depend on tightly regulated interactions of substrates, with docking sites provided by the substoichiometric APC/C substrate adaptors and coactivators, Cdc20 or Cdh1/FZR1. Both structural and functional studies of individual APC/C substrates indicate that productive ubiquitination usually requires more than one degron, and that degrons are of different types docking to distinct sites on the coactivators. However, the dynamic nature of APC/C substrate recruitment, and the influence of multiple degrons, remains poorly understood. Here we review the significance of multiple degrons in a number of E3-substrate interactions that have been studied in detail, illustrating distinct kinetic effects of multivalency and allovalency, before addressing the role of multiple degrons in APC/C substrates, key to understanding ordered substrate destruction by APC/C. Lastly, we consider how lessons learnt from these studies can be applied in the design of TPD tools.

Keywords: E3-substrate interaction; SLiM; degron; multivalency; targeted protein degradation; ubiquitin ligase.

FIGURE 1

Schematic showing the different modalities of E3-substrate recognition namely: (A) Monovalent, one E3 receptor site and one degron; (B) Allovalent, one E3 receptor site and multiple copies of the same degron; (C) Homo-multivalent, multiple E3 receptor sites as a result of E3 dimerization or oligomerization and multiple copies of the same degron; and (D) Hetero-multivalent, multiple different receptor sites on an E3 subunit and multiple different degrons. Created with BioRender.com.

FIGURE 2

(A) Schematic of the Keap1-Nrf2 interaction described in Section 2.1.1. Created with BioRender.com. (B) Structural model of the full Keap1-CUL3-RBX1 complex bound to Nrf2 assembled as described previously (structural model kindly provided by A. Bullock, University of Oxford) (Canning et al., 2013). A predicted helix in the Neh2 region of Nrf2 is modelled between the bound DLG and ETGE sites.

FIGURE 3

(A) Schematic showing the oligomerization of SPOP via its BACK and BTB domains, described in Section 2.1.2. Created with BioRender.com. (B) Model of SPOP dimer bound to a degron of the substrate Puc. The proteins are coloured: Cul3, blue; Rbx1, green, and Puc in black. SPOP domains are coloured: MATH degron-binding domain in purple; BTB domain in pink, and BACK domain in magenta. The model was made in Chimera (https://www.rbvi.ucsf.edu/chimera) using the following PDBs, 3IVV, 4EOZ, 3HQI and 1LDJ. (Zheng et al., 2002; Zhuang et al., 2009; Ji and Privé, 2013).

FIGURE 4

(A) Illustration of the cross section of the APC/C showing key subunits including the coactivator subunit (CDC20/FZR1), D-box co-receptor APC10, ubiquitin-bound E2s (UBE2C and UBE2S), catalytic subunits (APC2 and APC11) and a bound model substrate containing a KEN box and a D-box. (B) Structure of the three degrons (KEN (yellow), D-box (green) and ABBA (pink)) from APC/C-Cdh1 modulator 1 (Acm1) bound to S. cerevisiae APC/C activator protein Cdh1 (blue). The model was made in Chimera (https://www.rbvi.ucsf.edu/chimera) using PDB:4BH6 (He et al., 2013). (C) Schematic showing key APC/C subunits including coactivator subunit, E2 binding positions, and a bound model multivalent substrate containing a KEN box and a D-box Schematic showing proposed changes in substrate valencies during mitotic exit. D-boxes indicated in green, KEN motifs in yellow, Schematics were created with BioRender.com.

FIGURE 5

(A) Schematic showing the allovalent mechanism of interaction between the multiple phosphodegrons of the intrinsically disordered Sic1 and the E3 Cdc4, described in Section 2.2.1. Created with BioRender.com. (B) Structure of a Sic1 phosphodegron bound to Cdc4. Cdc4 is in blue and Sic1 in tan, with the phosphate groups shown in red. The model was made in Chimera (https://www.rbvi.ucsf.edu/chimera) using PDB:3V7D (Tang et al., 2012).