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Abstract: henylketonuria (PKU) is an inherited metabolic disorder caused by pathogenic variants in phenylalanine hydroxylase (PAH), leading to toxic phenylalanine accumulation and severe neurological complications if untreated. Current pharmacological treatment relies on tetrahydrobiopterin (BH4), which benefits only a subset of patients, highlighting a major unmet need for alternative therapies. Here, we combined high-throughput screening, computational modelling, and drug repurposing to identify pharmacological chaperones capable of rescuing PAH function. We evaluated 26 structurally diverse small molecules in HEK293T cells expressing wild-type PAH or one of eight PKU-associated variants spanning phenotypes from mild to classical disease. Chaperoning efficacy was strongly variant-dependent, and for every variant tested at least one compound produced a greater activity increase than BH4 under identical assay conditions. Notably, belinostat, a clinically approved histone deacetylase inhibitor, emerged as the most effective compound for several clinically severe variants. Mechanistically, functional rescue consistently correlated with an increased population of tetrameric, catalytically competent PAH, as quantified by mass photometry. The crystal structure of the PAH–belinostat complex (PDB ID: 9T1O), together with structural models for all compounds, provide a framework for rational optimization. These results establish a preclinical proof-of-concept for genotype-guided pharmacological chaperone therapy in PKU and support the feasibility of personalized, variant-specific treatment strategies.

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Abstract: The bacteriophages with single-stranded RNA (ssRNA) genomes (class Leviviricetes) are among the simplest known viruses that encode only three core proteins: a receptor-binding protein, a capsid protein, and an RNA-dependent RNA polymerase. The number of isolated ssRNA phages has remained very low, but the accumulating RNA metagenome data have uncovered a large variety of these viruses in many environments. Besides the core proteins, many of these genomes putatively encode additional proteins, which up to now have remained uncharacterized. We looked for non-conserved open reading frames (ORFs) in Leviviricetes sequences from the IMG/VR virus metagenome database and used sequence- and structure-based clustering to organize them into similarity groups. Potential ORFs were found throughout the ssRNA phage genomes but almost exclusively on the positive-sense RNA strand, suggestive of their protein-coding potential. The prevalence of the non-conserved ORFs varied in various phage lineages, and their distribution among different genome positions was markedly uneven. Most of the identified ORFs encode all-α proteins, a portion of which contain transmembrane segments that resemble a group of known ssRNA phage lysis proteins, while many others represent previously uncharacterized families of globular or semi-globular α-helical proteins. We additionally uncovered a major class of globular α/β proteins and experimentally determined the structure of a representative protein of this group. These results pave the way for further functional studies of novel ssRNA phage proteins for a better understanding of this diverse virus group.

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Abstract: Nicotinamidases (PncA) catalyze the hydrolysis of nicotinamide to nicotinic acid, a key step in NAD+ salvage pathways. In the Lyme disease spirochete Borrelia burgdorferi, the plasmid-encoded gene bbe22 encodes a PncA enzyme that is essential for survival in both mammalian and tick hosts. Previous genetic and biochemical studies demonstrated that translation of B. burgdorferi PncA initiates from a rare non-canonical AUU start codon, resulting in a protein that is 24 residues longer than the sequence currently annotated in major databases. Despite these findings, public resources such as UniProt and KEGG still list a truncated protein beginning at residue 36, which lacks part of the N-terminal region required for enzymatic activity. Here we report the crystal structure of full-length B. burgdorferi PncA determined at 3.2 Å resolution. The structure reveals the canonical fold of bacterial nicotinamidases and clear electron density for a ligand in the active site consistent with nicotinic acid, the product of the enzymatic reaction. Structural comparison with homologous PncA enzymes demonstrates conservation of the catalytic architecture, including residues involved in substrate binding and catalysis. Importantly, the experimentally determined structure confirms that the longer N-terminal sequence described previously is required for formation of the correct fold and active-site geometry, whereas the truncated annotation is structurally inconsistent with the observed fold and with AlphaFold predictions. Our results provide the first structural characterization of B. burgdorferi PncA and resolve the long-standing annotation discrepancy for bbe22, validating the correct protein sequence and providing the structural basis for nicotinamidase activity in this essential metabolic enzyme.

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Abstract: The most recently acquired and transcriptionally active family of human endogenous retroviruses (HERVs) is HERV-K. Of the approximately 100 copies of HERV-K in our genome, many retain the potential to proliferate by retrotransposition, express viral proteins, and form functional virus particles. Aberrant expression of the HERV-K envelope glycoprotein (Env) has been associated with cancer and neurodegeneration. Autoantibodies against HERV-K Env have been found in patients with various autoimmune diseases. Here, we report the crystal structure of the Env surface subunit (SU) from HERV-K HML-2, determined at 2.25-Å resolution. The overall fold is somewhat similar to Syncytin-2 SU and distantly related to HIV-1 gp120. The structure contains five disulfides, four N-linked glycans, and two sulfate ions bound to a basic surface groove. Two extended loops form a surface for potential interactions with cell-surface receptors or other cellular factors. The structure also contains three steroid molecules bound to hydrophobic surface patches. This crystal structure provides a platform for future studies to map autoantigenic epitopes, identify small molecules that interfere with HERV-K activity, and extend our mechanistic understanding of retroviruses.

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Abstract: We present the structural and functional characterization of a single-stranded DNA-binding protein (SSB-M5) identified from a hot spring metagenome in Vatnajökull National Park, Iceland. This small protein (136 aa; 15,695 Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic Fervidobacterium species. Functional complementation assay demonstrated that SSB-M5 can substitute for Escherichia coli SSB in an ssb− mutant strain, confirming its biological activity. A recombinant C-terminally His-tagged SSB-M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB-M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel β-sheet. The flat surfaces of the β-sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB-M5. Electrophoretic mobility shift assays showed that SSB-M5, after heat treatment up to 100°C, forms stable DNA-protein complexes with the (dT)40 oligo. Quantitative analyses revealed that SSB-M5 binds (dT)70 oligonucleotide with very high affinity (KD = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC50 of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB-M5 in PCR reactions significantly enhanced amplification by eliminating non-specific products. Together, these findings identify SSB-M5 as a hyperthermostable, high-affinity single-stranded DNA-binding protein with potential applications in molecular biology and biotechnology.