Cullin-RING Ubiquitin Ligase Regulatory Circuits: A Quarter Century Beyond the F-Box Hypothesis

Abstract

Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.

Keywords: E3 ligase; F-box protein; NEDD8; cullin; cullin-RING ligase; ubiquitin.

Figure 1

CRL assembly and regulatory circuits. (a) Key components of the CRL system and their interactions with a cullin–RBX core complex. Linkage of NEDD8 (yellow) to the cullin (green) WHB domain induces CRL conformational dynamics, as indicated by the dashed brackets, and stimulates CRL binding to E2 or E3 ubiquitin carrying enzymes (brick). RBX1/2 is shown in red, and ubiquitin in orange. CSN (pink) removes NEDD8 but is obstructed by substrate (purple) bound to SR (grey). CAND1 (navy) binds unneddylated CRLs and expunges and replaces SR bound to cullin–RBX core. (b) Organization of the CRL system as a regulatory circuit in which a stimulus induces substrate binding to an SR, the CSN serves as a sensor for a substrate-bound SR, and the signal from the stimulus is thus transduced depending on whether or not a cullin is deneddylated. Neddylation interacts with the ubiquitylation machinery to promote the effector response: ubiquitylation, which, in turn, often drives protein degradation. The CSN detects the absence of a substrate bound to an SR. Lack of SR-bound substrate enables deneddylation and feedback control through SR exchange catalyzed by CAND proteins. (c) Cullin proteins comprise multiple domains, including CRs, a 4HB, an intermolecular C/R domain involving coassembly with RBX1 or RBX2, and a WHB domain that is neddylated. RBX proteins contribute to the C/R domain and have a flexibly tethered E3 ligase RING. The CR1 domain binds an SR module. Neddylated cullin WHB and RBX RING domains both rearrange to bind various ubiquitin-carrying enzymes. CSN primarily binds the 4HB, C/R, WHB, and RING domains. CAND antagonizes many other partner proteins by interacting across virtually an entire unneddylated cullin–RBX complex. Abbreviations: 4HB, 4-helix bundle; CAND, cullin-associated NEDD8-dissociated; C/R, intermolecular cullin/RBX; CR, cullin repeat; CSN, COP9 Signalosome; CUL, cullin; PROTAC, protein-targeting chimeric molecule; S, substrate; SR, substrate receptor; Ub, ubiquitin; WHB, winged-helix B.

Figure 2

The CRL regulatory circuit depends on interchangeable SR modules binding to specific cullins. SR modules typically display one or more domains or subunits that bind to substrates and one domain that binds to a cullin’s CR1 domain. CUL1’s CR1 domain binds SKP1, which in turn binds the substrate-binding F-box protein, with SKP1–F-box protein forming the SR module. CUL2 and CUL5 employ a three-protein SR module: a BC-box protein that binds a substrate and an EloBC heterodimer. CUL3 binds the BTB domain of BTB proteins that directly bind substrates. CUL4’s CR1 domain and an N-terminal tail bind DDB1, which in turn binds a substrate-binding DCAF protein. NEDD8, which is ligated to the WHB motif, is not shown for simplicity. SR proteins are shown in grey and labeled by their hallmark domain name. Substrates are shown in purple, and the trigger for substrate–SR interactions such as a PTM as a yellow circle. Abbreviations: CR, cullin repeat; CRL, cullin-RING ubiquitin ligase; CUL, cullin; DCAF, DDB1- and cullin 4–associated factors; Elo, elongin; SR, substrate receptor; Sub, substrate; WHB, winged-helix B.

Figure 3

Myriad ways stimuli control substrate binding. (a) Phosphorylation-dependent regulation. A phosphodegron can directly bind to an SR, bind via an intervening adaptor, or undergo multiple phosphorylation events via a multistep cascade before SR binding. Substrate phosphorylation can also block substrate–SR interactions. (b) Numerous posttranslational modifications can trigger substrate–SR interactions (e.g., prolyl hydroxylation, glycosylation, acetylation). (c) Some SRs are modified to block (as shown here) or activate (not shown) substrate binding. (d) Quality control of complex formation can remove misassembled proteins. In some cases, the SR may recognize defective assemblies, as in noncognate mixed BTB dimers. (e) Small molecules can create binding surfaces between receptors and substrates that promote substrate ubiquitylation. This can occur through molecular glues that seal a gap between the SR and degron—these can be endogenous plant hormones, synthetic molecules such as immunomodulatory imide drugs, or bifunctional molecules (PROTACs) in which binders to a protein of interest and an SR are connected by a linker. (f) The CUL3–SPOP complex, which has two dimerization domains, interacts with a substrate DAXX that harbors many SPOP-binding consensus motifs in phase-separated compartments (gray oval) through multivalent interactions. Abbreviations: CUL, cullin; DAXX, death domain–associated protein; m, modification; P, phosphate; PROTAC, protein-targeting chimeric molecule; SR, substrate receptor; Ub, ubiquitin.

Figure 4

Diverse substrate ubiquitylation effector responses are mediated by mix-and-match ubiquitin-carrying enzymes. Neddylated CRLs can employ E2s or ARIH-family RBR E3s to promote initial ubiquitin transfer to substrates (priming) or chain extension. UBE2R1 and 2 and UBE2G1 are specialized to generate K48-linked chains, while UBE2D can create branched chains. Abbreviations: CUL, cullin; RBR, RING-between-RING; S, substrate; SR substrate receptor; WHB, winged-helix B.

Figure 5

The CRL system regulatory circuit is completed by sensing, transducing, and transmitting feedback through the neddylation-deneddylation-SR exchange cycle. The life history of individual cullin molecules involves cycling through multiple states, shown here in a simplified view of the cycle. Fully assembled and active CRLs that are engaged with substrates are neddylated. When all cognate substrates have been degraded, the CSN complex can assemble with the CRL complex and remove NEDD8 (deneddylation). This CRL complex is then a substrate for CAND-mediated exchange, wherein CAND binding leads to release of the SR from a CUL–RBX complex. The free CUL-RBX complex can then assemble with a different substrate-bound SR from an SR pool (inferred intermediates are shown in brackets). Assembly of a substrate-bound SR blocks CSN binding and therefore favors neddylation to make a fully active CRL engaged in substrate ubiquitylation. An entire cycle is estimated to occur on average every 87 s in HeLa cells. Abbreviations: CAND, cullin-associated NEDD8-dissociated; CRL, cullin-RING ubiquitin ligases; CSN, COP9 Signalosome; CUL, cullin; S, substrate; SR, substrate receptor.