Udo Heinemann

Processing bodies (P-bodies) are cytoplasmic, non-membrane-bound structures involved in mRNA decay. EDC4 serves as a key scaffold for the decapping complex within P-bodies. Here, we demonstrate that N4BP1 interacts with EDC4, as well as with DCP1A, DCP2, and XRN1 - key components of 5′-cap hydrolysis. Endogenous N4BP1 colocalizes with EDC4 in P-bodies, requiring both of its KH domains. Structural analysis revealed that N4BP1 contains a type-I KH fold but lacks the canonical GXXG motif required for single-stranded RNA binding. Deletion or mutation of KH domains non-canonical GXXG motifs disrupts the N4BP1-EDC4 complex. N4BP1 reduces HIV-1 transcript levels independently of its P-body localization or association with decapping components, implying the involvement of other host factors in regulating viral mRNAs. Similarly, for N4PB1-dependend negative regulation of endogenous transcripts in HaCaT keratinocytes, EDC4 is not essential. For both HIV-1 and endogenous transcripts, the reduction is associated with the activity of the NYN domain.

Wolfram Saenger is remembered.

Transcription factors recognise and bind specific DNA sequence patterns in promoters and enhancers thereby regulating gene expression. Variations in the DNA sequence of transcription factor binding sites (TFBSs) can alter gene regulation and may disrupt development. The transcription factor NKX2.1 is a crucial regulator of thyroid, lung, and neural development. Mutations in its coding gene NKX2-1 may cause choreoathetosis and congenital hypothyroidism with or without pulmonary dysfunction (CAHTP, OMIM #610978). Most genetically solved patients carry mutations in the coding regions of NKX2-1 that affect DNA binding, while the majority of patients with CAHTP-like symptoms do not carry mutations in the NKX2-1 coding sequence. We hypothesise that variations in the DNA-sequence at promoter or enhancer sites to which the transcription factor NKX2.1 binds could cause disease as well. We employed EMSA-seq to quantify the effects of genetic variation on NKX2.1 binding strength and used this data to train neural network models to forecast the influence of DNA variation on NKX2.1 binding. We validated our models using microscale thermophoresis, X-ray crystallography, and publicly available ChIP-seq data sets. The neural networks were able to detect TFBSs in ChIP-seq data and can thus be used to evaluate whole genome sequencing data of CAHTP-patients in order to prioritise potential disease-causing variants in regulatory elements. Graphical

Serotonin (5-HT), a crucial neurotransmitter and peripheral mediator, regulates various physiological processes and is synthesized by tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme responsible for its production. 5-HT overproduction is implicated in multiple diseases, making TPH1 a promising therapeutic target. However, selectivity remains a challenge due to the structural similarity of TPH1 with other members of the aromatic amino acid hydroxylase (AAAH) family, including TPH2, phenylalanine hydroxylase (PAH), and tyrosine hydroxylase (TH). This study aimed to evaluate the selectivity and inhibitory potential of TPT-004, a novel TPH inhibitor, compared with Telotristat (LP778902) and its prodrug (LX1606). We developed high-throughput fluorescence assays to evaluate the inhibitory effects of the test compounds on TPH1, TPH2, PAH, and TH enzymes. TPT-004 demonstrated high selectivity for TPHs compared to LP778902 and LX1606. Structural analysis based on a detailed sequence alignment within the AAAH enzyme family, combined with cocrystal structures of TPH1 and TPH2 bound to different generations of inhibitors, enhances our understanding of the molecular basis of inhibitor binding and provides a framework for explaining TPT-004’s selectivity for TPHs. Selectivity profiling against 97 targets confirmed that TPT-004 showed minimal off-target interactions, underscoring its specificity. A dose–range finding (DRF) study in rats assessed the in vivo safety profile of TPT-004, showing no adverse effects on survival and body weight at doses up to 400 mg/kg/day. Hematology parameters remained normal, with only minor liver changes observed. These results highlight TPT-004’s potential as a selective and safe TPH inhibitor, offering a promising therapeutic option for serotonin-related disorders.

Since 2003, the Macromolecular Crystallography (MX) group at Helmholtz-Zentrum Berlin (HZB) has been operating three MX beamlines at the BESSY II storage ring in Berlin. These beamlines were established to support the emerging structural genomics initiatives founded in Germany, Europe, and overseas around the turn of the century. Over the past two decades, these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory, a spectroscopy laboratory, a Biosafety Level 1 laboratory and all necessary computing resources for the MX and chemical crystallography user community. Currently, more than 100 independent research groups from the greater Berlin area, Germany, and Europe utilize these beamlines. Over time, more than 4500 Protein Data Bank depositions have been accrued based on data collected at the beamlines. This paper presents historical aspects of the beamlines, their current status including their research output, and future directions.

Background: Transcription factors regulate gene expression by binding to transcription factor binding sites (TFBSs). Most models for predicting TFBSs are based on position weight matrices (PWMs), which require a specific motif to be present in the DNA sequence and do not consider interdependencies of nucleotides. Novel approaches such as Transcription Factor Flexible Models or recurrent neural networks consequently provide higher accuracies. However, it is unclear whether such approaches can uncover novel non-canonical, hitherto unexpected TFBSs relevant to human transcriptional regulation. Results: In this study, we trained a convolutional recurrent neural network with HT-SELEX data for GRHL1 binding and applied it to a set of GRHL1 binding sites obtained from ChIP-Seq experiments from human cells. We identified 46 non-canonical GRHL1 binding sites, which were not found by a conventional PWM approach. Unexpectedly, some of the newly predicted binding sequences lacked the CNNG core motif, so far considered obligatory for GRHL1 binding. Using isothermal titration calorimetry, we experimentally confirmed binding between the GRHL1-DNA binding domain and predicted GRHL1 binding sites, including a non-canonical GRHL1 binding site. Mutagenesis of individual nucleotides revealed a correlation between predicted binding strength and experimentally validated binding affinity across representative sequences. This correlation was neither observed with a PWM-based nor another deep learning approach. Conclusions: Our results show that convolutional recurrent neural networks may uncover unanticipated binding sites and facilitate quantitative transcription factor binding predictions.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell–cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.