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.

Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.

Structural insights into the nucleic acid remodeling mechanisms of the yeast THO-Sub2 complex.

The yeast THO complex is recruited to active genes and interacts with the RNA-dependent ATPase Sub2 to facilitate the formation of mature export-competent messenger ribonucleoprotein particles and to prevent the co-transcriptional formation of RNA:DNA-hybrid-containing structures. How THO-containing complexes function at the mechanistic level is unclear. Here, we elucidated a 3.4 Å resolution structure of Saccharomyces cerevisiae THO-Sub2 by cryo-electron microscopy. THO subunits Tho2 and Hpr1 intertwine to form a platform that is bound by Mft1, Thp2, and Tex1. The resulting complex homodimerizes in an asymmetric fashion, with a Sub2 molecule attached to each protomer. The homodimerization interfaces serve as a fulcrum for a seesaw-like movement concomitant with conformational changes of the Sub2 ATPase. The overall structural architecture and topology suggest the molecular mechanisms of nucleic acid remodeling during mRNA biogenesis.

Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling.

The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. We identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. We determined the 2.1 Å resolution structure of MLEcore bound to a U10 RNA and ADP-AlF4. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation.

Structural studies on Mycobacterium tuberculosis RecA: molecular plasticity and interspecies variability.

Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the 'switch' residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. The interspecies variability in the plasticity of the two mycobacterial proteins is particularly surprising as they have similar sequence and 3D structure. Details of the interactions of ligands with the protein, characterized in the structures reported here, could be useful for design of inhibitors against M. tuberculosis RecA.