Abstract Calmodulin (CaM) regulates diverse signaling proteins through recognition of short linear motifs, including isoleucine‐glutamine (IQ) motifs that commonly bind Ca 2+ ‐free CaM (apo‐CaM). The homologous to E6AP carboxyl terminus (HECT) E3 ubiquitin ligases ubiquitin protein ligase E3B (UBE3B) and ubiquitin protein ligase E3C (UBE3C) are closely related enzymes that both contain N ‐terminal IQ motifs, yet whether these motifs encode conserved modes of apo‐CaM recognition has remained unclear. Here, we combined isothermal titration calorimetry, nuclear magnetic resonance (NMR) spectroscopy, and computational modeling to compare apo‐CaM binding by the UBE3B and UBE3C IQ motifs. The UBE3B IQ motif bound apo‐CaM with high affinity and preferentially engaged the CaM C ‐lobe, whereas the UBE3C IQ motif interacted more weakly under the same conditions. NMR titrations revealed distinct molecular recognition modes—UBE3B IQ binding occurred in slow exchange and produced a localized C ‐lobe interaction surface, while UBE3C IQ binding occurred in fast exchange and generated smaller, more distributed chemical shift perturbations. Structural models and molecular dynamics simulations provide a framework for interpreting these divergent apo‐CaM engagement modes despite conservation of canonical IQ motif features. Phylogenetic analysis further suggests that the UBE3B and UBE3C IQ motifs have followed divergent evolutionary trajectories within the broader IQ motif family. Together, these findings show that conserved IQ motifs in related HECT ligases can encode different apo‐CaM binding modes, providing a molecular basis for divergent CaM‐mediated regulation of UBE3B and UBE3C.
Abstract RepDBmaker is a reproducible Snakemake pipeline designed for the automated construction of large‐scale protein sequence databases. It enables the seamless downloading of sequences from different public databases and the integration of user‐specified proteomes. The pipeline also performs standardized taxonomic annotations to all compiled sequences and contamination screening in eukaryotic proteomes. In addition, RepDBmaker generates queryable database files for Basic Local Alignment Search Tool Program (BLASTP), DIAMOND, and MMseqs2, with the option of taxonomy‐aware sequence clustering to produce less redundant datasets. To showcase its utility, we used RepDBmaker to build a Tree‐of‐Life‐scale database comprising 482 million protein sequences from 160,045 publicly available genomes and transcriptomes. RepDBmaker offers an off‐the‐shelf but customizable solution for researchers undertaking large‐scale evolutionary projects that require extensive, well‐annotated protein databases. The pipeline is available at https://github.com/Gabaldonlab/RepDBmaker .
Abstract Protein‐nucleic acid (protein‐Nu) interactions are crucial for many cellular processes and biological functions. Crosslinking mass spectrometry (XL‐MS) has emerged as a powerful method for identifying these interactions. However, the analysis of protein‐Nu XL‐MS data requires an integrated workflow from raw data processing to results inspection and validation. Existing workflows for protein‐Nu XL‐MS often require switching between tools and considerable manual processing. Here, we introduce NuXLApp, an interactive web‐based application for data processing and results visualization in protein‐Nu XL‐MS studies. NuXLApp integrates the protein‐Nu crosslink search engine “NuXL,” machine‐learning models for peptide property prediction, and interactive result visualization within a reproducible workflow. It enables rapid validation of false discovery rate‐controlled crosslink spectrum match‐level identifications and facilitates collaboration and intuitive exploration of experimental results. In addition, NuXLApp generates a spectral library from identified protein‐Nu crosslinks to enable quantitative analysis of crosslinks using data‐independent acquisition (DIA). NuXLApp supports discovery‐driven identification of protein‐nucleic acid crosslinks and enables DIA‐based follow‐up experiments for their quantitative analysis. NuXLApp is open‐source and available via the OpenMS WebApps platform at https://nuxl.webapps.openms.de/ .
Abstract Membrane‐embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand‐discovery studies. Pseudomonas aeruginosa relies on the TonB‐dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterization of the two soluble HasR domains directly involved in heme uptake: the N‐terminal plug and the Secretin/TonB short N‐terminal domain. Each domain was expressed individually in Escherichia coli and purified to homogeneity, adopting well‐folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilization tags. A GST‐His dual‐affinity strategy enabled efficient purification of the construct, whereas His6‐tag alone resulted in insoluble protein and HLT‐tag fusions suffered from non‐specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N‐terminal domain remains stably folded within the fusion construct, while the N‐terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand‐screening applications, including aptamer and nanobody discovery.
Abstract Histone deacetylases (HDACs) are critical epigenetic regulators of fungal growth and pathogenicity and constitute promising targets for antifungal drug development. Hos3 defines a fungal‐specific HDAC subclass that lacks a clear mammalian ortholog; however, the structural determinants underlying its selective inhibition remain poorly characterized. Here, we integrated x‐ray crystallography with biochemical and biophysical analyses to comprehensively characterize the catalytic properties of Hos3 from the pathogenic fungus Aspergillus fumigatus ( Af Hos3) and its inhibition by the hydroxamate‐based compound T2383. Despite high conservation of catalytic residues, Af Hos3 displayed significantly higher intrinsic catalytic activity and markedly enhanced sensitivity to T2383 compared with its yeast homolog Sc Hos3. The crystal structure of the Af Hos3–T2383 complex revealed a canonical zinc‐dependent inhibitor‐binding mode, coupled with a distinct, fungal‐specific architecture of the substrate and inhibitor binding pocket. Structural and functional studies identified an aromatic clamp, comprising residues F371 and F443, that is indispensable for both catalytic activity and ligand stabilization. Furthermore, fungal‐specific residues M538 and V205, together with structural elements loopA and α 12, shape the catalytic‐pocket environment and differentially influence enzymatic activity and T2383 responsiveness. Notably, the dimeric assembly of Af Hos3 was associated with optimal enzymatic activity but did not influence inhibitor recognition. Collectively, these findings elucidate how fungal‐specific structural elements synergize with the evolutionarily conserved catalytic core to govern selective inhibitor recognition, providing a robust structural and mechanistic framework for the rational design of fungal‐selective HDAC inhibitors.
