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Post-translational protein modifications by ubiquitin and ubiquitin-like modifiers regulate many major pathways in the cell. These modifications can be reversed by de-ubiquitinating enzymes such as ubiquitin-specific proteases (USPs). Proteolytic activity towards ubiquitin-modified substrates is common to all USP family members except for USPL1, which shows a unique preference for the ubiquitin-like modifier SUMO. Here, we present the crystal structure of USPL1 bound to SUMO2, defining the key structural elements for the unusual deSUMOylase activity of USPL1. We identify specific contacts between SUMO2 and the USPL1 subdomains, including a unique hydrogen bond network of the SUMO2 C-terminal tail. In addition, we find that USPL1 lacks major structural elements present in all canonical USPs members such as the so-called blocking loops, which facilitates SUMO binding. Our data give insight into how a structural protein scaffold designed to bind ubiquitin has evolved to bind SUMO, providing an example of divergent evolution in the USP family. USPL1 is a non-canonical member of the ubiquitin-specific protease (USP) family with activity toward SUMO instead of ubiquitin. Here, the authors present a crystal structure of USPL1 bound to SUMO2, revealing how this enzyme has evolved to bind SUMO as an example of divergent evolution in the USP family.

Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat TTR amyloidoses. Here we repurpose tolcapone, an FDA-approved molecule for Parkinson’s disease, as a potent TTR aggregation inhibitor. Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. Crystal structures of tolcapone bound to wild-type TTR and to the V122I cardiomyopathy-associated variant show that it docks better into the TTR T 4 pocket than tafamidis, so far the only drug on the market to treat TTR amyloidoses. These data indicate that tolcapone, already in clinical trials for familial amyloid polyneuropathy, is a strong candidate for therapeutic intervention in these diseases, including those affecting the central nervous system, for which no small-molecule therapy exists. Misfolding of transthyretin can cause amyloid aggregation disorders that can be treated by stabilizing the tetrameric form with tafamidis. Here the authors show that tolcapone, a drug already FDA-approved for Parkinson disease, has strong transthyretin stabilizing function and might be a superior therapeutic option for CNS amyloidosis as it can cross the blood brain barrier.

USP25 deubiquitinating enzyme is a key member of the ubiquitin system, which acts as a positive regulator of the Wnt/β-catenin signaling by promoting the deubiquitination and stabilization of tankyrases. USP25 is characterized by the presence of a long insertion in the middle of the conserved catalytic domain. The crystal structure of USP25 displays an unexpected homotetrameric quaternary assembly that is directly involved in the inhibition of its enzymatic activity. The tetramer is assembled by the association of two dimers and includes contacts between the coiled-coil insertion domain and the ubiquitin-binding pocket at the catalytic domain, revealing a distinctive autoinhibitory mechanism. Biochemical and kinetic assays with dimer, tetramer and truncation constructs of USP25 support this mechanism, displaying higher catalytic activity in the dimer assembly. Moreover, the high stabilization of tankyrases in cultured cells by ectopic expression of a constitutive dimer of USP25 supports a biological relevance of this tetramerization/inhibition mechanism. USP25 is a deubiquitinating enzyme and a positive regulator of Wnt/β-catenin signaling. Here the authors present the crystal structure of USP25 in a tetrameric inactive state and their biochemical and kinetic assays support an USP25 autoinhibitory mechanism that is mediated through a dimer to tetramerization transition.

Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage. Nse2 is a SUMO E3 ligase component of the Smc5/6 multisubunit complex involved in the DNA repair and chromosome integrity. Here, the structure of the Nse2 in complex with an E2-SUMO thioester mimetic reveals the combined action of two SIM motifs during the E3- dependent conjugation reaction.

Cell division is a central process in all living organisms and requires the coordinated action of many proteins and regulatory elements. In most bacteria, the division and cell wall (dcw) gene cluster is regulated by the first gene of the dcw operon, mraZ, a highly conserved DNA-binding transcriptional regulator. Here we report the structural basis of MraZ transcriptional regulation by the resolution of three different cryo-EM structures of MraZ in complex with the upstream promoter region of the dcw cluster from Mycoplasma genitalium at 3.36, 3.57 and 3.87 Å resolution. The structures reveal the specific interactions between MraZ DNA-binding motif and nucleobases of the binding boxes, which induces distortion in the MraZ octamer to enable the interaction with the four repetitive binding boxes of the promoter DNA. The “cradle-like” DNA-binding motif of MraZ exposes three highly conserved basic residues, Lys13, Arg15 and Arg86, which are essential for binding to the consensus sequence of its cognate promoter. Ultimately, the mechanism behind MraZ’s DNA binding and regulation of the dcw operon could be translated to other species, working as a general mechanism for the regulation of dcw gene cluster in bacteria. MraZ, a conserved transcription factor, controls bacterial cell division by binding the dcw operon promoter. Here, the authors present cryo-EM structures of M.genitalium MraZ with its 4-box promoter, revealing its DNA-binding mechanism

The large family of deubiquitinating enzymes (DUBs) are involved in the regulation of a plethora of processes carried out inside the cell by protein ubiquitination. Ubiquitination is a basic pathway responsible for the correct protein homeostasis in the cell, which could regulate the fate of proteins through the ubiquitin–proteasome system (UPS). In this review we will focus on recent advances on the molecular mechanisms and specificities found for some types of DUBs enzymes, highlighting illustrative examples in which the regulatory mechanism for DUBs has been understood in depth at the molecular level by structural biology. DUB proteases are responsible for cleavage and regulation of the multiple types of ubiquitin linkages that can be synthesized inside the cell, known as the ubiquitin-code, which are tightly connected to specific substrate functions. We will display some strategies carried out by members of different DUB families to provide specificity on the cleavage of particular ubiquitin linkages. Finally, we will also discuss recent progress made for the development of drug compounds targeting DUB proteases, which are usually correlated to the progress of many pathologies such as cancer and neurodegenerative diseases.

Post-translational SUMO modification is a widespread mechanism for regulating protein function within cells. In humans, SUMO-conjugated proteins are partially regulated by the deconjugating activity of six SENP family members. The proteolytic activity of these enzymes resides within a conserved catalytic domain that exhibits specificity for the two primary SUMO isoforms: SUMO1 and SUMO2/3. SENP5, along with SENP3, are nucleolar proteins involved in ribosome biogenesis and preferentially target SUMO2/3 isoforms. Here, we present the crystal structures of human SENP5 in complex with both SUMO1 and SUMO2 isoforms. These structures reveal a minimal complex interface and elucidate the molecular basis for SENP5’s preference for the SUMO2 isoform. This preference can be attributed to a basic patch surrounding SENP5 Arg624 at the interface. Swapping mutagenesis and structural analysis demonstrate that Arg624 is favorably oriented to interact with Asp63 in SUMO2/3, while its interaction with the equivalent Glu67 in SUMO1 is less favorable. These results suggest that subtle structural differences within SUMO isoforms can significantly influence their deconjugation by SENP enzymes, opening new avenues for exploring the regulation of SUMOylation in various cellular processes and for developing therapeutic agents targeting SUMOylation pathways. SUMO modification regulates protein function, with SENP enzymes controlling SUMO removal. Here, the authors present crystal structures of SENP5 bound to SUMO1 and SUMO2, revealing how structural features drive its preference for SUMO2 and offering insights into SUMOylation regulation.

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810–828. A crystal structure of Nek7 Y97F bound to Nek9 810–828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7 Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families. NEK7, a kinase involved in mitosis, is regulated by the kinase NEK9. Here the authors identify the region in NEK9 that binds NEK7 and find that the mechanism of regulation involves dimerization coupled to structural changes in the active site.

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection. The unique structural features of the NopD catalytic domain, an effector protease from symbiotic bacteria rhizobia, grant a surprising versatility to remove various regulatory tags (SUMO, ubiquitin, Nedd8) from host plant proteins.