NanT and NanX are bacterial transporters that import the sialic acids, N-acetylneuraminate and 2,7-anhydro-n-acetylneuraminate, respectively. Here, we used complementary biophysical and computational approaches to structurally characterise Escherichia coli NanX. Size exclusion chromatography, analytical ultracentrifugation and low-resolution cryo-electron microscopy reveal that NanX exists in both monomeric and dimeric states following purification. Molecular modelling and substrate docking identify key residues likely involved in 2,7-anhydro-n-acetylneuraminate recognition. Using this information, we engineered a mutant NanX transporter that can import the NanT-specific substrate, N-acetylneuraminate, which we verified using a bacterial growth assay. These data identify amino acids involved in major facilitator superfamily mediated sialic acid transport and offer a new research perspective of its metabolism.

Pseudokinases are the catalytically-dead counterparts of protein kinases and, over the past 20 years, have increasingly garnered attention as crucial signaling entities-comprehensively dispelling the possibility that they are merely evolutionary remnants or cellular passengers. The field has been framed by a sequence-based definition of a pseudokinase, where the absence of one or more of the three critical catalytic residues required for phosphoryl transfer in conventional protein kinases has allowed their classification. As a result, pseudokinases have been defined by their dissimilarity to active kinases, meaning they are the outcasts or black sheep of the kinome. Pseudokinases are prevalent in nature, accounting for 10% or more of the kinase complement throughout phyla, and have been reported to mediate diverse functions in controlling the activities of other enzymes allosterically, mediating signaling complex assembly, serving as conformational switches and as negative regulators of signaling flux. Here, we review our current understanding of the varied pseudokinase functions as a window toward understanding non-catalytic functions of conventional protein kinases, the challenges associated with defining pseudokinases-especially in cases where cryptic catalytic activities have been reported-and the emergence of pseudokinases as pharmacological targets.

To define and systematically characterize the human E3 ubiquitin ligase (E3) landscape, we generated the E3-ome, a compendium of E3s encoded by the human genome. The E3-ome integrates experimental data, bioinformatics, and published research, revealing 672 high-confidence E3s. We standardized E3 classifications to create a unified framework for annotation and comparative analysis. The E3-ome identified several previously unrecognized domains, motifs, E3 candidates, and relationships, expanding the diversity of E3s. Furthermore, the E3-ome mapped the spatial and physiological organization of E3s across human tissues and cell types, revealing context-dependent E3s. Genetic analyses identified disease-associated variants across the E3-ome, linking E3s to diverse human pathologies. Together, these analyses define the human E3 landscape at high resolution and deliver a foundational resource to drive mechanistic and therapeutic discovery.

Transcription factor ubiquitination is a decisive regulator of growth and development. The DET1-DDB1-DDA1 (DDD) complex associates with the Cullin-4 ubiquitin ligase (CRL4) and a second ubiquitin ligase, COP1, to control ubiquitination of transcription factors involved in neurological, metabolic, and immune cell development. Here, we report the structure of the human DDD complex, revealing a specific segment of DET1 that can recruit ubiquitin-conjugating (E2) enzymes. Structural variability analysis, mass spectrometry, and mutagenesis based on AlphaFold predictions suggest that dynamic closure of DET1, stabilized by DDA1, underlies coordinated recruitment of E2 enzymes and COP1. Biochemical assays suggest that the E2 acts as a recruitment factor to bring COP1 to DET1 for more effective substrate ubiquitination, which parallels a catalytically inactive E2 enzyme (COP10) in plant DDD complexes. This work provides a clear architecture for regulation and cooperative CRL4DET1-COP1 complex assembly, which can affect degradation of diverse targets by COP1 complexes.

Sulfate-reducing bacteria import organosulfur compounds from the environment for anaerobic respiration. They contribute to human disease and are problematic in industrial settings because they produce hydrogen sulfide. Here, we demonstrate how the sulfate-reducing bacterium Oleidesulfovibrio alaskensis imports isethionate, a common organosulfonate, using a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM). The cryo-EM structure of isethionate-bound OaIseQM to 2.98 Å resolution defines the substrate-binding site, two Na+-binding sites, and a distinct fusion helix. Key residues within the OaIseQM substrate-binding site are identified using substitution and proteoliposome assays. Functional studies demonstrate that OaIseQM requires the substrate-binding protein (OaIseP) and a Na+ gradient to drive transport. Modeling of the OaIsePQM complex supports that elevator-type conformational changes are involved in this unique coupled transport process. This work expands our knowledge of the transport of organosulfur compounds in bacteria and establishes OaIsePQM as a new model system for exploring the mechanism of TRAP transporters.

The polycomb repressive-deubiquitinase (PR-DUB) complex removes ubiquitin from lysine residue 119 on histone H2A (H2AK119Ub) in humans. The PR-DUB is composed of two central protein factors, the catalytic breast cancer type 1 susceptibility protein (BRCA1)-activating protein 1 (BAP1) and one of three additional sex combs-like 1-3 (ASXL1-3) proteins. A plant homeodomain (PHD) at the C terminus of ASXL proteins is recurrently truncated in cancer, was previously proposed to recognise epigenetic modifications on the N-terminal tail of histone H3 and was recently shown to bind an auxiliary set of PR-DUB interactors, named methyl CpG-binding domain proteins 5 (MBD5) and 6 (MBD6). Here, we demonstrate that the ASXL PHD domain lacks features required for histone tail recognition and is unable to bind histone H3 epigenetic marks. Modelling the structure of the ASXL PHD using AlphaFold3 suggests that the domain has an atypical fold and that the isolated ASXL PHD can chelate a single zinc ion in vitro, compared with the two ions conventionally bound by PHD domains. Alternatively, we show that the ASXL PHD-MBD5 and PHD-MBD6 complexes are stable in vitro. A composite zinc-binding site was shown to form at the interface between the ASXL2 PHD and MBD6 MBD domains, and is required for stable complex formation. Overall, these data suggest an unconventional pairing of domains coordinate key functions of the PR-DUB-a noncanonical PHD domain from ASXL proteins partners with MBD5 or 6, which were themselves misannotated because they cannot bind to methylated DNA.

Ubiquitin ligases regulate core cellular processes through diverse mechanisms. The ubiquitin ligase COP1 is conserved from plants to humans and is particularly important for targeting developmental transcription factors for ubiquitination. COP1 can function independently, but can also be recruited to Cullin-4 ubiquitin ligase complexes via the DET1 adaptor protein. However, the mechanism of action of complexes containing COP1 and DET1 is not well understood. Here we report the cryo-electron microscopy structure of human DET1, bound to proteins that enable Cullin-4 recruitment (DDB1-DDA1) and an additional ubiquitin ligase enzyme (Ube2e2). We observe that DDA1 stabilises a closed conformation of DET1, binding adjacent to a unique Ube2e2 binding-insert in DET1. Moreover, we demonstrate that closure of DET1 underlies COP1 recruitment, which binds in an antiparallel dimeric state. Disrupting either the Ube2e2-binding insertion of DET1, or distinct recruitment sites on COP1, abolish DET1–COP1 binding and DET1-mediated modulation of COP1 levels. The multifaceted architecture provides an efficient platform for ubiquitination of substrates, or COP1 itself, by Cullin-4DET1 and offers multiple opportunities for physiological regulation. ### Competing Interest Statement The authors have declared no competing interest.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N -acetylneuraminate TRAP transporter ( Hi SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous Hi SiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na + sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the Hi SiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity ( K D ) for the complex between the soluble Hi SiaP protein and Hi SiaQM is in the micromolar range and that a related SiaP can bind Hi SiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

Bacteria evolve mechanisms to compete for limited resources and survive in new niches. Here we study the mechanism of isethionate import from the sulfate-reducing bacterium Oleidesulfovibrio alaskensis. The catabolism of isethionate by Desulfovibrio species has been implicated in human disease, due to hydrogen sulfide production, and has potential for industrial applications. O. alaskensis employs a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM) to import isethionate, which relies on the substrate-binding protein (OaIseP) to scavenge isethionate and deliver it to the membrane transporter component (OaIseQM) for import into the cell. We determined the binding affinity of isethionate to OaIseP by isothermal titration calorimetry, KD = 0.95 µM (68% CI = 0.6-1.4 µM), which is weaker compared with other TRAP substrate-binding proteins. The X-ray crystal structures of OaIseP in the ligand-free and isethionate-bound forms were obtained and showed that in the presence of isethionate, OaIseP adopts a closed conformation whereby two domains of the protein fold over the substrate. We serendipitously discovered two crystal forms with sulfonate-containing buffers (HEPES and MES) bound in the isethionate-binding site. However, these do not evoke domain closure, presumably because of the larger ligand size. Together, our data elucidate the molecular details of how a TRAP substrate-binding protein binds a sulfonate-containing substrate, rather than a typical carboxylate-containing substrate. These results may inform future antibiotic development to target TRAP transporters and provide insights into protein engineering of TRAP transporter substrate-binding proteins.

The pseudokinase Tribbles Homolog 1 ( TRIB1 ) is a known driver of tumorigenesis in acute myeloid leukemia and is encoded upstream of the oncogene MYC at the 8q24 locus. We observed that TRIB1/MYC co-amplification is associated with decreased relapse-free and overall survival in breast cancer patients, but the role of TRIB1 in this disease has not been well characterized. TRIB1 knockdown in multiple breast cancer cell lines inhibited cell proliferation and suppressed MYC expression, implicating TRIB1 in breast cancer cell proliferation. Transcriptomic and cell cycle analysis revealed cell cycle regulation as the likely mechanism through which TRIB1 influences breast cancer cell proliferation. TRIB1 knockdown also resulted in significant changes in both estrogen receptor (ER) and β-catenin associated transcription. Interrogating the TRIB1 interactome in breast cancer cells by qPLEX-RIME reinforced the known association between TRIB1 and ubiquitination, while revealing a range of previously undescribed TRIB1 associated factors. Further analysis of the association between TRIB1, β-catenin and FERMT2 suggests TRIB1 may regulate β-catenin activity by controlling the levels of both β-catenin, and its co-factor FERMT2. Together, these results suggest that coregulation of β-catenin and ER-driven transcription by TRIB1, facilitates regulation of MYC expression and breast cancer cell proliferation. Significance The pseudokinase TRIB1 is frequently co-amplified in breast cancers with the potent oncogene MYC , although the functional consequences of this event are not well understood. This study demonstrates TRIB1 is a regulator of cell cycle progression and MYC expression in breast cancer cells. It also profiles TRIB1 -associated proteins in breast cancer cells, demonstrating conservation of TRIB1’s canonical interaction with COP1 and reveals associations with members of the wider ubiquitination machinery, a range of transcriptional regulators and chromatin remodelers. The data presented provide insight into the function of TRIB1 in breast cancer and the role of TRIB1 in transcriptional regulation.

AbstractTripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of theHaemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previousHiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, theHiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the solubleHiSiaP protein andHiSiaQM is in the micromolar range and that a related SiaP can bindHiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential nutrients. TRAP transporters receive their substrates via a secreted soluble substrate-binding protein. How a sodium ion-driven secondary active transporter is strictly coupled to a substrate-binding protein is poorly understood. Here we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 Å resolution. SiaM comprises a “transport” domain and a “scaffold” domain, with the transport domain consisting of helical hairpins as seen in the sodium ion-coupled elevator transporter VcINDY. The SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, suggesting that TRAP transporters may operate as monomers, rather than the typically observed oligomers for elevator-type transporters. We identify the Na + and sialic acid binding sites in SiaM and demonstrate a strict dependence on the substrate-binding protein SiaP for uptake. We report the SiaP crystal structure that, together with docking studies, suggest the molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We thus propose a model for substrate transport by TRAP proteins, which we describe herein as an ‘elevator-with-an-operator’ mechanism.

Structural analysis reveals how the decision to induce apoptotic cell death is regulated.

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function.

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. The structure shows that the RING domain and non-covalently bound ubiquitin molecule together make contacts that stabilise the activated conformation of the conjugate, revealing why ubiquitin is a key regulator of Ark2C activity. We also identify a charged loop N-terminal to the RING domain that enhances activity by interacting with both the regulatory ubiquitin and ubiquitin conjugated to the E2. In addition, the structure suggests how Lys48-linked ubiquitin chains might be assembled by Ark2C and UbcH5b. Together this study identifies features common to RING E3s, as well elements that are unique to Ark2C and related E3s, which enhance assembly of ubiquitin chains.

Tribbles proteins are pervasive pseudokinases in cellular signaling. They play a major role in the differentiation of myeloid cells, hepatocytes and adipocytes, and more widely in immune function, metabolism and cancer. Like many other pseudokinases, an inherent lack of catalytic activity has meant that a specialized cadre of techniques has been required to investigate Tribbles function. A prerequisite to most in vitro biochemistry has been robust methods for purifying useful quantities of Tribbles protein, which can sometimes exhibit non-optimal behavior upon recombinant expression. For instance, structural studies of the Tribbles family have largely focused on TRIB1, in part because of more readily available protein. Here we describe methods we have developed to routinely produce milligram quantities of TRIB1, and specific considerations when employing TRIB1 protein for various downstream analyses. Namely, we describe preparation and crystallization of TRIB1 for structural studies, and using fluorescence polarization and isothermal titration calorimetry to analyze interactions with TRIB1. We hope that applying these considerations can facilitate further understanding of TRIB1 function, specifically, and can be selectively applied to improve studies of other Tribbles proteins and pseudokinases more generally.

The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09–1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.

Self-incompatibility (SI) is a feature of many flowering plants, whereby self-pollen is recognized and rejected by the stigma. In grasses (Poaceae), the genes controlling this phenomenon have not been fully elucidated. Grasses have a unique two-locus system, in which two independent genetic loci (S and Z) control self-recognition. S and Z are thought to have arisen from an ancient duplication, common to all grasses. With new chromosome-scale genome data, we examined the genes present at S- and Z-loci, firstly in ryegrass (Lolium perenne), and subsequently in ~20 other grass species. We found that two DUF247 genes and a short unstructured protein (SP/ZP) were present at both S- and Z- in all SI species, while in self-compatible species these genes were often lost or mutated. Expression data suggested that DUF247 genes acted as the male components and SP/ZP were the female components. Consistent with their role in distinguishing self- from non-self, all genes were hypervariable, although key secondary structure features were conserved, including the predicted N-terminal cleavage site of SP/ZP. The evolutionary history of these genes was probed, revealing that specificity groups at the Z-locus arose before the advent of various grass subfamilies/species, while specificity groups at the S-locus arose after the split of Panicoideae, Chloridoideae, Oryzoideae and Pooideae. Finally, we propose a model explaining how the proteins encoded at the S and Z loci might function to specify self-incompatibility.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.