Structure of a Glomulin-RBX1-CUL1 Complex: Inhibition of a RING E3 Ligase through Masking of Its E2-Binding Surface

Abstract

Summary The ~300 human Cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1’s RING domain, regulates the RBX1-CUL1-containing SCFFBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN’s selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.

Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

“…GLMN originally was identified as a gene responsible for familial glomuvenous malformations, a disorder that results in localized cutaneous vascular lesions because of loss-of-function mutations in the Glmn gene (45)(46)(47)(48). More recently, GLMN was characterized as a Cullin ring ligase inhibitor that blocks interaction with its ubiquitin-conjugating enzyme (E2) (49,50).…”

“…The same group recently reported that GLMN binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity (49,50). Rbx1 regulates the cullin RING ligase-mediated turnover of Fbw7, a substrate receptor for Cul1-RING-CRL, which facilitates the ubiquitination and degradation of cyclin E and c-Myc (49,50). If this activity also is involved in the GLMN-inflammasome interplay, GLMN may suppress NLR inflammasome activation, e.g., by interacting with and inhibiting a hypothetical E3 ubiquitin ligase that activates the inflammasome.…”

“…Cullins are subunits of the cullin RING ligase (CRL) family. The same group recently reported that GLMN binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity (49,50). Rbx1 regulates the cullin RING ligase-mediated turnover of Fbw7, a substrate receptor for Cul1-RING-CRL, which facilitates the ubiquitination and degradation of cyclin E and c-Myc (49,50).…”

Suramin inhibits cullin-RING E3 ubiquitin ligases

“…Glomulin, a HEAT-repeat-containing protein, was shown to bind the E2-interacting surface of the ROC1/RBX1 RING domain and to inhibit CRL-mediated chain formation by Cdc34 (40,41). Likewise, the small-molecule inhibitor of Cdc34a, CC0651, stabilizes a noncovalent complex between the E2 and the donor Ub, thereby blocking Ub discharge (18).…”

Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation

“…For most RING-E3s, the cycles of E2 engagement and dissociation are thought to occur constitutively (32), and only a few examples of controlled E2 activation are known. Access of Cdc34 to its specific RING-E3, the Skp1-Cul1-F box (SCF) complex, can be regulated by phosphorylation or competition with the inhibitory protein glomulin (33,34). Reminiscent of this situation, Ube2S interacts with the APC/C in a cell cycle-dependent manner, and depletion of Cdc20 prevents Ube2S from stably binding to the APC/C in cells (12,15).…”