Juri Rappsilber

Regulated helicase activation by DDK kinase is central for genome stability. However, how DDK phosphorylation primes the MCM2-7 double hexamer (DH) for Sld3-Sld7 binding and Cdc45 loading remained unclear. We define this mechanism through cryo-EM structures of MCM2-7 DH-Sld3-Sld7 (MS) and MCM2-7 DH-Sld3-Sld7-Cdc45 (MSC). We reveal that the autoinhibitory Mcm4 tail engages not only Mcm4 but also Mcm6. Upon DDK-dependent phosphorylation, both of these sites become accessible. In the context of the MS structure, we identify that two short Sld3 motifs that contact Mcm4 and Mcm6 read out the DH phosphorylation state, while the Sld3 Treslin domain (STD) binds to Mcm2. In the MSC structure, Cdc45 dislodges the Sld3 STD from Mcm2, allowing Sld3 to position Cdc45 at the Mcm2/Mcm5 interface. Mutagenesis of the Sld3 STD-Cdc45 interface disrupts Cdc45 loading, validating this interaction. Together, our data reveal a phosphorylation-encoded mechanism coupling DDK-activated Mcm4/Mcm6 surfaces to distal Cdc45 placement, explaining how firing factors choreograph the DH-to-CMG transition.

Protein–protein interaction (PPI) information is distributed across resources that differ in organism coverage, identifier systems, evidence models, confidence scores and access mechanisms, so assembling and comparing evidence for a protein requires source-specific queries, identifier conversion and extensive post-processing. We present KlinkPPI, a web server that retrieves, compares and exports PPI evidence from STRING, BioGRID, IntAct, CORUM, HuRI and Predictomes from a single query. KlinkPPI accepts UniProtKB accessions, NCBI Gene and Ensembl identifiers and gene names, and performs taxonomy-aware mapping to a common identifier space. Users can query individual proteins across all resources available for an organism, or retrieve organism-wide interaction sets. Results are presented per source so that database-specific evidence, annotations and confidence values are retained, while an integrated view exposes coverage and agreement between resources. KlinkPPI deliberately does not merge heterogeneous confidence scores, nor collapse functional associations, complex co-membership, binary interactions and structural predictions into a single consensus network. Results can be exported in PSI-MI TAB 2.8-compatible or Apache Parquet format with user-selected evidence fields. KlinkPPI is freely available at https://rappsilberlab.org/KlinkPPI/ and the source code at https://github.com/Rappsilber-Laboratory/KlinkPPI . Graphical

Mass spectrometry-based proteomics enables in-depth investigation of protein phosphorylation, quantifying tens of thousands of phosphosites per sample following phosphopeptide enrichment. However, critical information on phosphosite occupancy (stoichiometry) is typically lost during the phospho-enrichment process. Here, we introduce SILAC-Site, a SILAC-based fractionation-free and chemical labelling-free workflow for direct phosphosite stoichiometry evaluation using stable isotope labeling and phosphatase treatment. By acquiring treated or untreated peptides together with their heavy-labelled dephosphorylated counterparts within the same LC-MS runs, this approach provides internally controlled stoichiometry estimates compatible with high-throughput data-independent acquisition proteomics. Applying SILAC-Site to S. cerevisiae , we show that the majority of phosphopeptides identified only after enrichment possess low stoichiometries, and that inferred stoichiometry strongly correlates with the direct detection of phosphopeptides in samples without enrichment. Based on these findings, we propose the analysis of samples without enrichment as a simple complementary addition to a typical phosphoproteomics workflow, facilitating recovery of phosphorylation stoichiometry information.

In mass-spectrometry-based proteomics it remains challenging to ensure the accuracy of protein quantities. Here we introduce QuantUMS (quantification using an uncertainty-minimizing solution), a machine learning-based method that dynamically tunes the quantification algorithm to minimize quantitative errors. When applied to data-independent acquisition proteomics, QuantUMS increases accuracy and precision, ameliorates ratio compression bias and enhances differential expression analysis. It further reports an uncertainty measure enabling quality control of individual quantities.

Orthogonal translation systems (OTSs) enable site‐specific incorporation of non‐canonical amino acids (ncAAs) and are central to genetic code expansion. Current engineering strategies typically rely on hyperstable aminoacyl tRNA synthetase (aaRS) scaffolds to tolerate destabilizing mutations required for substrate diversification. Here, we introduce an alternative design principle: exploiting the intrinsic conformational flexibility of a psychrophilic pyrrolysyl‐tRNA synthetase (PylRS) enzyme to enhance mutational tolerance and in vivo performance. We identified a cold‐adapted PylRS from Methanococcoides burtonii and established a psychrophilic OTS (“Cold‐OTS”) compatible with Escherichia coli . This system consistently outperformed established mesophilic and thermophilic PylRS variants in single‐ and multi‐site ncAA incorporation. Notably, Cold‐OTS maintained high suppression efficiency at low ncAA concentrations and exhibited enhanced absolute performance at reduced cultivation temperatures, even under globally diminished protein synthesis rates. In addition, engineered variants accommodated a broader set of substrates, consistent with elevated substrate promiscuity. These findings establish psychrophilic aaRS scaffolds as potentially powerful resources for genetic code expansion. Given the broad host compatibility of the PylRS platform, Cold‐OTS provides a scalable strategy for efficient production of ncAA‐modified proteins across diverse biological systems.

Clustering is ubiquitous across science, yet a text-mining audit of 736,399 open-access articles identified as using clustering (2000-2025) reveals common practice leaves key parameters undocumented or untuned, contributing to the reproducibility crisis in science. We developed an interactive web platform featuring 11 widely adopted clustering algorithms to enable transparent clustering analysis and reporting, aligning practical use with best practices in computational research. Code availability: The browser-based clustering analysis platform RapCluster is available for download at https://github.com/lutfia95/RapCluster under MIT License and accessible at https://rappsilberlab.org/rapcluster/ The web version is capacity-limited to 8 GB of memory (ca. 12,000 candidates with 130 features). All source codes used for the analysis are included with the publication.

The activation of chemical reactions in living cells using ultraviolet (UV) light enables the interrogation of biomolecules in their native environment with photoreactive probes or crosslinking reagents. Although numerous photo-crosslinking approaches have been successfully employed, they often suffer from common limitations, including low reaction yields, the need for long exposure times, and irradiation-induced cellular damage from heat, desiccation, or side reactions. We recently showed that 365 nm light-emitting diodes enable rapid, biorthogonal protein–DNA crosslinking in living cells, incurring minimal photodamage. Here, we generalize this approach and demonstrate that high-intensity, longwave UV light reduces the irradiation time for in-cell photo-crosslinking reactions by up to 1000-fold, allowing protein–drug, protein–protein, protein–DNA, and protein–RNA interactions to be fixed within seconds. Benchmarking this rapid photo-activation for the analysis of RNA-interacting proteomes responding to RNA-binding drugs or UV-induced RNA damage, we demonstrate both qualitative and quantitative advantages of controlled, high-intensity UV irradiation, uncovering emergent experimental opportunities that were previously inaccessible to light-activated chemistry in intact cells and tissues.

The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na ⁺ /H ⁺ antiporters and an Na ⁺ -translocating NADH:quinone oxidoreductase (Na ⁺ -NQR). The exact contribution of the various Na ⁺ /H ⁺ antiporters to maintain Na ⁺ and H ⁺ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na ⁺ /H ⁺ antiporter NhaA and the Na ⁺ -NQR are required by the bacteria for Na ⁺ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na ⁺ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na ⁺ /H ⁺ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the P _nhaB and P _pepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na ⁺ homeostasis in V. cholerae . IMPORTANCE: The emergence and spread of multi-resistant bacteria are a major problem for humans and animals. Therefore, the identification of novel targets for the development of novel antibiotics to combat pathogenic bacteria is extremely important. Since sodium ion homeostasis is an essential process in many pathogenic bacteria, especially those living in marine environments, it represents an interesting target for antibiotics. The perturbation of sodium ion homeostasis does indeed impair the fitness of the human pathogenic bacterium Vibrio cholerae but can be restored by the rapid evolution of the bacteria. This suggests that inhibiting multiple targets with different antibiotics might be more effective in preventing the development of resistant bacteria.

Kinetoplastids, such as Trypanosoma brucei , are eukaryotes that likely separated from the main lineage at an exceptionally early point in evolution. Consequently, many aspects of kinetoplastid biology differ significantly from other eukaryotic model systems, including yeasts, plants, worms, flies, and mammals. As in many eukaryotes, the T. brucei genome contains repetitive elements at various chromosomal locations, including centromere- and telomere-associated repeats and interspersed retrotransposon elements. T. brucei also contains intermediate-sized and mini-chromosomes that harbour abundant 177 bp repeat arrays and 70 bp repeat elements implicated in Variable Surface Glycoprotein (VSG) gene switching. In many eukaryotes, repetitive elements are assembled in specialised chromatin such as heterochromatin; however, apart from centromere- and telomere-associated repeats, little is known about chromatin-associated proteins that decorate these and other repetitive elements in kinetoplastids. Here, we utilise affinity selection of synthetic TALE DNA binding proteins designed to target specific repeat elements to identify enriched proteins by proteomics. Validating the approach, a telomere repeat binding TelR-TALE identifies many proteins previously implicated in telomere function. Furthermore, the 70R-TALE designed to bind 70 bp repeats indicates that proteins involved in DNA repair are enriched on these elements that reside adjacent to VSG genes. Interestingly, the 177 bp repeat binding 177R-TALE enriches for many kinetochore proteins, suggesting that intermediate-sized and mini-chromosomes assemble kinetochores related in composition to those located on the main megabase chromosomes. This provides a first insight into the chromatin landscape of repetitive regions of the trypanosome genome with relevance for their mechanisms of chromosome integrity, immune evasion, and cell replication.