CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation.

An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting.

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity.

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.

A novel non-canonical PIP-box mediates PARG interaction with PCNA.

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding.

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD-BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein-protein interactions by intramolecular mimicry.

SIRT2 regulates nuclear envelope reassembly through ANKLE2 deacetylation.

Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.

Noncanonical assembly, neddylation and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex.

Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, 'cullin-RING' and 'RBR', are individually found in several hundred human E3 ligases, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry and cellular assays elucidate a 1.8-MDa hexameric human CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique among RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrates are recruited to various upstream domains, while ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.