Autotaxin (ATX) is a circulating enzyme that plays a major role in the production of the signaling mediator lysophosphatidic acid (LPA). A role for ATX/LPA signaling has been described in multiple disease areas, including fibrosis and cancer. ATX inhibitors are classified in five types (I-V) depending on how they target parts of the tripartite site (active site, pocket and tunnel). We set to explore a "penultimate" type of inhibitors, targeting all these three parts at once. Designing new analogs extending on an ethyl group of the type IV GLPG1690 compound, yielded potent new molecules. Co-crystal structures confirmed compounds that utilize a three-point lock binding mode. The most potent "type VI" inhibitors, 4 and 41, displayed increased inhibitory activity (∼40-fold) compared to the type IV close analog 3. Type VI inhibitors 4 and 41 showed cellular and phenotypic activity similar to type IV inhibitor GLPG1690. Identification of this new binding mode completes this combinatorial puzzle in inhibitor design and calls for further investigation to characterize potential therapeutic benefit.

Zinc finger proteins (ZNFs) are the largest family of transcription factors, yet how they activate gene expression remains unclear. In this study, we identified Zincore, a protein complex consisting of QRICH1 and SEPHS1, as a ZNF-specific coregulator essential for embryonic development in mice and associated with developmental syndromes in humans. We also identified ZFP91 as a representative Zincore client, binding the conserved promoter motif CTTTAAR. Cryo-electron microscopy of a Zincore-ZFP91-DNA complex revealed a SEPHS1 arginine clamp to recognize the DNA-bound zinc finger domains. This mode of binding explains recognition of different ZNFs and stabilizes ZFP91 onto its cognate DNA motif. Thus, our study identified Zincore as a ZNF-specific coregulator essential for development, involving a distinctive mechanism that locks ZNFs onto DNA and regulates transcription.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) regulate numerous biological processes through interaction with signaling effectors at the cell surface. As a unique feature, GPI-APs can be released from their anchors by multi-pass GPI-specific phospholipases (types A2, C, and D) to impact signaling networks, phenotype, and cell fate; however, many questions remain outstanding. Here, we discuss and expand our current understanding of the distinct GPI-specific phospholipases, their substrates, effector pathways, and emerging physiological roles, with a focus on the six-transmembrane ecto-phospholipases GDE2 (GDPD5) and GDE3 (GDPD2). We provide structural insight into their AlphaFold-predicted inner workings, revealing how transmembrane (TM) domain plasticity may enable GPI-anchor binding and hydrolysis. Understanding lipolytic cleavage of GPI-APs adds a new dimension to their signaling capabilities and biological functions.

Despite significant progress made in functional genomics, the roles of a relatively small number of essential genes remain enigmatic. Here, we characterize S1 RNA-binding domain-containing protein 1 (SRBD1), an essential gene with no previously assigned function. Through genetic, proteomic, and functional approaches, we discovered that SRBD1 is a DNA-binding protein and a key component of the mitotic chromatid axis. The loss of SRBD1 results in a pronounced defect in sister chromatid segregation that strikingly resembles the phenotype observed when sister chromatid decatenation is perturbed by topoisomerase IIα (TOP2A) dysfunction. Using genetic screens, we uncovered that the requirement for SRBD1 depends on the presence of condensin II but not condensin I. Moreover, we found that SRBD1 activity is most critical during prophase, when chromosome condensation is established. Taking these results together, we propose that SRBD1 acts during prophase to safeguard the decatenation process to prevent the formation of difficult-to-resolve DNA structures, thereby averting severe chromosome missegregations.

Artificial intelligence (AI)-powered protein structure prediction methods have revolutionised how life scientists explore macromolecular function. Using AI to predict the structure of macromolecular complexes, is gaining attention for modelling known interactions and evaluating the likelihood of proteins forming multimers or interacting with other proteins or nucleic acids. There is a growing need for tools to efficiently evaluate these predicted models. We introduce new tools that use AlphaFold3's "predicted local-distance difference test" (pLDDT), "predicted aligned error" (PAE), and "predicted distance error" (PDE) matrices in a graph-based community clustering approach to label sequence motifs involved in binary interactions. The resulting "interaction image" is processed through a multidimensional image algorithm to cluster interacting sequence motifs in three-dimensional binary interfaces. This method allows us to present the interaction information between multiple proteins and nucleic acids back to two-dimensional space using chord diagrams. These "AlphaBridge" diagrams summarise predicted interfaces and intermolecular interactions, including prediction confidence and sequence conservation scores. They are valuable for efficient screening of predictions, ranking, and scoring the confidence of predicted interactions, prior to more detailed (and resource intensive) analysis. ### Competing Interest Statement The authors have declared no competing interest.

Most proteins consist of both folded domains and Intrinsically Disordered Regions (IDRs). However, the widespread occurrence of intrinsic disorder in human proteins, along with its characteristics, is often overlooked by the broader communities of structural and molecular biologists. Building on the MobiDB database of intrinsically disorder in proteins, here we develop a comprehensive dataset (Comprehensive analysis of Human proteins And their disOrdered Segments (CHAOS)). We implement empirical internally consistent definitions of what constitutes a disordered region, annotate general characteristics such as cellular location, essentiality, and post-translational modifications, and cross-reference to structure predictions from AlphaFold. Most proteins contain at least one disordered region, predominantly located at the protein termini. IDRs are less hydrophobic and are enriched in post-translational modifications compared to non-IDRs. Additionally, we discovered that proteins residing in different cellular locations possess distinct disorder profiles. Finally, the predicted AlphaFold models of proteins in CHAOS suggest that while protein disorder may be intrinsic, it does not have to be extrinsic. Hereby we enhance the visibility and understanding of intrinsic disorder in human proteins. ### Competing Interest Statement The authors have declared no competing interest.

Ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family members (ENPP1-7) have been implicated in key biological and pathophysiological processes, including nucleotide and phospholipid signaling, bone mineralization, fibrotic diseases, and tumor-associated immune cell infiltration. ENPPs are single-pass transmembrane ecto-enzymes, with notable exceptions of ENPP2 (Autotaxin) and ENNP6, which are secreted and glycosylphosphatidylinositol (GPI)-anchored, respectively. ENNP1 and ENNP2 are the best characterized and functionally the most interesting members. Here, we review the structural features of ENPP1-7 to understand how they evolved to accommodate specific substrates and mediate different biological activities. ENPPs are defined by a conserved phosphodiesterase (PDE) domain. In ENPP1-3, the PDE domain is flanked by two N-terminal somatomedin B-like domains and a C-terminal inactive nuclease domain that confers structural stability, whereas ENPP4-7 only possess the PDE domain. Structural differences in the substrate-binding site endow each protein with unique characteristics. Thus, ENPP1, ENPP3, ENPP4, and ENPP5 hydrolyze nucleotides, whereas ENPP2, ENPP6, and ENNP7 evolved as phospholipases through adaptions in the catalytic domain. These adaptations explain the different biological and pathophysiological functions of individual members. Understanding the ENPP members as a whole advances our insights into common mechanisms, highlights their functional diversity, and helps to explore new biological roles.

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

Autotaxin (ATX/ENPP2) has attracted widespread attention as a target for various pathologies, notably idiopathic pulmonary fibrosis (IPF). ATX is predominantly (~90%) responsible for the extracellular production of the signaling molecule lysophosphatidic acid (LPA), which acts through two specific families of G protein-coupled receptors. We show that ATX, even when catalytically inactive, is more potent than LPA alone in inducing downstream signaling events in cells. While catalytic activity is dispensable for ATX-mediated signaling, the allosteric tunnel of ATX is necessary to mediate this effect. Notably, this signal is transmitted through Edg family (LPA1-3) but not non-Edg family (LPA4-6) LPA receptors, and is affected by the presence of α5β1 integrin (Fig). Crucially, while orthosteric type I ATX inhibitors do not abrogate ATX-mediated signaling, hybrid (orthosteric-allosteric) type IV ATX inhibitors that occupy the ATX tunnel do. Finally, this correlates with previous results (Heckmann B, et al. ATS 2019; poster A108), where ziritaxestat, but not Cpd A, inhibited bleomycin-induced lung fibrosis in mice. In conclusion, we describe a new, unexpected and unique direct ATX signaling mechanism. Both catalytic production and presentation of bioactive LPA are necessary for biological activity, explaining the predicted clinical benefit of ziritaxestat in treating IPF. Funding: Galapagos

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and the regulation of macrophage biology through unknown mechanisms. The 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. Here we identify specific interactions of MACIR using a fragment complementation-based affinity pull down of cellular proteins prepared with a membrane solubilization buffer. Quantitative mass spectrometry showed enrichment of nuclear and mitochondrial proteins and of 63 significant interacting proteins, binding to the nuclear transport receptor TNPO1 and trafficking proteins UNC119 homolog A and B were validated by immunoprecipitation. Analysis of mutations in two candidate recognition motifs in the MACIR amino acid sequence confirmed TNPO1 binds via a PY-NLS motif (aa98-117). Characterizing nuclear MACIR activity in macrophage and fibroblasts is a priority with respect to developing strategies for treatment of autoimmune disease.