Structural and biophysical characterisation of ubiquitin variants that inhibit the ubiquitin conjugating enzyme Ube2d2.

The ubiquitin-conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases, and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function relies on interactions with partner proteins, and the disruption of these is an effective way to modulate activity.

Zinc finger 1 of the RING E3 ligase, RNF125, interacts with the E2 to enhance ubiquitylation.

Inflammation is essential for healthy immune function, wound healing, and resolution of infection. RIG-I is a key RNA sensor that initiates an immune response, with activation and termination of RIG-I signaling reliant on its modification with ubiquitin. The RING E3 ubiquitin ligase, RNF125, has a critical role in the attenuation of RIG-I signaling, yet it is not known how RNF125 promotes ubiquitin transfer or how its activity is regulated.

From seeds to trees: how E2 enzymes grow ubiquitin chains.

Modification of proteins by ubiquitin is a highly regulated process that plays a critical role in eukaryotes, from the construction of signalling platforms to the control of cell division. Aberrations in ubiquitin transfer are associated with many diseases, including cancer and neurodegenerative disorders. The ubiquitin machinery generates a rich code on substrate proteins, spanning from single ubiquitin modifications to polyubiquitin chains with diverse linkage types.

Ubiquitin and a charged loop regulate the ubiquitin E3 ligase activity of Ark2C.

A large family of E3 ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. Regulation of RING E3s depends on modulating their ability to stabilise the RING bound E2~ubiquitin conjugate in the activated (or closed) conformation. Here we report the structure of the Ark2C RING bound to both a regulatory ubiquitin molecule and an activated E2~ubiquitin conjugate.

Identification of Ubiquitin Variants That Inhibit the E2 Ubiquitin Conjugating Enzyme, Ube2k.

Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates.

The Structure and Ubiquitin Binding Properties of TRAF RING Heterodimers.

Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases.

Isolation and Characterization of Ice-Binding Proteins from Higher Plants.

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity.

Ubiquitin Variant Inhibitors Meet the Deubiquitinase USP15.

Deubiquitinases (DUBs) are important regulators of cellular function and selective inhibitors are required to reveal their biological role and therapeutic potential. In this issue of Structure, Teyra et al. (2019) report the development of DUB USP15 inhibitors that provide a starting point for the analysis of USP15 function.

A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes.

Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions.

The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains.

The Really Interesting New Gene (RING) Finger protein 11 (RNF11) is a subunit of the A20 ubiquitin-editing complex that ensures the transient nature of inflammatory responses. Although the role of RNF11 as a negative regulator of NF-κB signalling is well-documented, the molecular mechanisms that underpin this function are poorly understood. Here, we show that RNF11 binds both Ubc13 and the Ubc13~ubiquitin conjugate tightly and with similar affinity, but has minimal E3 ligase activity.

Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex.

Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism.

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1.

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains.

Regulation of E2s: A Role for Additional Ubiquitin Binding Sites?

Attachment of ubiquitin to proteins relies on a sophisticated enzyme cascade that is tightly regulated. The machinery of ubiquitylation responds to a range of signals, which remarkably includes ubiquitin itself. Thus, ubiquitin is not only the central player in the ubiquitylation cascade but also a key regulator.

Structure and Function of the RING Domains of RNF20 and RNF40, Dimeric E3 Ligases that Monoubiquitylate Histone H2B.

Monoubiquitylation of histone H2B is a post-translational mark that plays key roles in regulation of transcription and genome stability. In humans, attachment of ubiquitin to lysine 120 of histone H2B depends on the activity of the E2 ubiquitin-conjugating enzyme, Ube2B, and the really interesting new gene (RING) E3 ligases, RING finger protein (RNF) 20 and RNF40. To better understand the molecular basis of this modification, we have solved the crystal structure of the RNF20 RING domain and show that it is a homodimer that specifically interacts with the Ube2B~Ub conjugate.

The molecular basis of lysine 48 ubiquitin chain synthesis by Ube2K.

The post-translational modification of proteins by ubiquitin is central to the regulation of eukaryotic cells. Substrate-bound ubiquitin chains linked by lysine 11 and 48 target proteins to the proteasome for degradation and determine protein abundance in cells, while other ubiquitin chain linkages regulate protein interactions. The specificity of chain-linkage type is usually determined by ubiquitin-conjugating enzymes (E2s).

Use of E2~ubiquitin conjugates for the characterization of ubiquitin transfer by RING E3 ligases such as the inhibitor of apoptosis proteins.

Ubiquitylation of proteins is a versatile posttranslational modification that can serve to promote protein degradation, or it can have nondegradative roles, such as mediating protein-protein interactions. The Inhibitor of APoptosis (IAP) proteins are important regulators of pathways that control cell death, proliferation, and differentiation. A number of IAP family members are RING E3 ubiquitin-protein ligases, which promote direct transfer of ubiquitin from charged E2 enzymes, or E2~ubiquitin (E2~Ub) conjugates, to substrate proteins.

Isolation and characterization of ice-binding proteins from higher plants.

The characterization of ice-binding proteins from plants can involve many techniques, only a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition activity. Two distinct procedures are described; neither is normally used for precise quantitative assays.

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP.

Ice restructuring inhibition activities in antifreeze proteins with distinct differences in thermal hysteresis.

Antifreeze proteins (AFPs) share two related properties: the ability to depress the freezing temperature below the melting point of ice (thermal hysteresis; TH); and the ability to inhibit the restructuring of ice into larger crystals. Since the 'hyperactive' AFPs, which have been more recently discovered, show an order of magnitude more TH than previously characterized AFPs, we have now determined their activities in ice restructuring inhibition (IrI) assays. IrI activities of three TH-hyperactive AFPs and three less TH-active AFPs varied over an 8-fold range.

Compound ice-binding site of an antifreeze protein revealed by mutagenesis and fluorescent tagging.

By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology.

Identification of the ice-binding face of a plant antifreeze protein.

The antifreeze protein of Lolium perenne, a perennial ryegrass, was previously modeled as a beta-roll with two extensive flat beta-sheets on opposite sides of the molecule. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short side-chain residues were replaced by tyrosine. None of these disrupted the fold.