Peter D. Mace

Screening of COP1 E3 ligase with pooled, peptide-based combinatorial libraries and affinity selection mass spectrometry revealed new peptide binder sequences for further development as cellular probes for studying COP1 biology.

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.

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.

Protein–protein interactions (PPIs) are critical to all cellular activities. Despite having a large number of proteins, cells have spatial and temporal control over PPIs to avoid dysregulation in cellular pathways. Considerable research efforts have aimed to find new PPIs, curate PPIs from the literature and build searchable PPI databases. These databases have been widely used by experimental and computational scientists. Here we find that the PPIs captured by these databases are highly heterogeneous and concentrated on a small number of species. These issues hamper researchers from capturing the full landscape of reliable PPIs, affecting the accuracy of machine-learning models and the effectiveness of experimental designs. However, there are opportunities to fill gaps computationally and experimentally. We suggest developing a phylogenetically informed approach to test PPIs experimentally and computationally.

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.

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 CRL4 ^DET1-COP1 complex assembly, which can affect degradation of diverse targets by COP1 complexes.

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-4 ^DET1 and offers multiple opportunities for physiological regulation.

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 (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM 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 HiSiaQM 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 HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. 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.