Biology subjects

Scheltema, R. A.

Publications and source records attributed to Scheltema, R. A..

17 recordsLinked to original sources

bioRxiv2025

Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 [A] structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

molecular biology↗

bioRxiv2025

An optimized SP3 sample processing workflow for in-depth and re-producible phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf. 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

biochemistry↗

bioRxiv2023

Fast and accurate disulfide bridge detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multi-billion-dollar industry. Essential here is quality control assessment of critical attributes such as sequence fidelity, proper folding, and post-translational modifications (PTMs). Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two Cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis (MAAH), the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer (XlinkX/PD) to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within one hour, allowing for high-throughput, high-accuracy disulfide mapping.

biochemistry↗

bioRxiv2023

Type III-B CRISPR-Cas signaling-based cascade of proteolytic cleavages

Type III CRISPR-Cas systems provide a sequence-specific adaptive immune response that protects prokaryotic hosts against viruses and other foreign genetic invaders. These crRNA-guided Cas effector complexes bind and cleave complementary RNA targets. Specific target binding stimulates the Cas10 subunit to generate cyclic oligoadenylate (cOA) signaling molecules, that in turn allosterically activate proteins carrying cognate sensory domains: CARF or SAVED. Here, we characterize an elaborate set of genes associated with the type III-B CRISPR-Cas system from Haliangium ochraceum, which includes a signal transduction module of a CBASS defense system with two caspase-like proteases, SAVED-CHAT and PCaspase (Prokaryotic Caspase). We show that binding of a 3-nucleotide cOA (cA3) to the SAVED domain of SAVED-CHAT induces its oligomerization into long filaments that activate the proteolytic activity of the CHAT domain. Surprisingly, we find that activated SAVED-CHAT specifically cleaves and activates the second protease, PCaspase. In turn, activated PCaspase cleaves a multitude of other proteins, including a putative sigma factor and a PCaspase-inhibitor. We expressed the type III-B system and its associated genes in E. coli and observed a strong abortive phenotype when offering a complementary target RNA, but only in the presence of both SAVED-CHAT and PCaspase. Together, our findings show an intriguing cascade of proteolytic activities (conceptually similar to eukaryotic caspases) in this bacterial immune system that reveals yet another strategy to effectively defend against mobile genetic elements.

biochemistry↗

bioRxiv2023

Thrombin activation of the factor XI dimer is a multi-staged process for each subunit

Factor XI (FXI), a protein in the intrinsic coagulation pathway, can be activated by two enzymes. In hemostasis, FXI is activated by thrombin, while FXIIa-mediated activation is prothrombotic. The interactions between FXI and its activating enzymes are poorly understood due to their transient nature. Here, we applied structural proteomics, molecular dynamics simulations and binding assays to investigate the interface between thrombin and FXI including the dynamics underlying FXI activation. We demonstrate that activation of FXI is a multi-staged process, where thrombin first binds to Pro520 on FXI, after which it migrates towards the activation site by engaging the apple 1 domain and finally Arg378. We validated with known mutation sites and additionally found that Pro520 is conserved in prekallikrein (PK). This enables binding of thrombin even though it cannot activate PK. Understanding the exact binding of thrombin to FXI points a way for future interventions for bleeding or thrombosis.

biochemistry↗

bioRxiv2022

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 cooperates with the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pol{varepsilon}) for PCNA binding, but not with the lagging strand DNA polymerase Delta (Pol{delta}). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand both processes are coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

biochemistry↗

bioRxiv2022

MRPS36 provides a missing link in the eukaryotic 2-oxoglutarate dehydrogenase complex for recruitment of E3 to the E2 core

The tricarboxylic acid (TCA) cycle, or Krebs cycle, is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. The enzymes involved in this cycle generate the reducing equivalents NADH and FADH2 by a series of enzymatic reactions, which are utilized by the electron transport chain to produce ATP. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled just from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive XL-MS data obtained from measurements in mice and bovine heart mitochondria, supported by phylogenetic analyses, we provide evidence that MRPS36 is an essential member of OGDHC, albeit only in eukaryotes. Comparative sequence analysis and computational structure predictions reveal that in eukaryotic OGDHC, E2o does not contain the peripheral subunit-binding domain (PSBD), present in bacterial and archaeal E2os. We propose that in eukaryotes MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC containing 16 E1o, 12 E3, and 6 subunits of MRPS36 accommodated around the OGDHC core composed of 24 E2o subunits (3.45 MDa). The model provides new insights into the OGDH complex topology and stipulates putative mechanistic implications.

biochemistry↗

bioRxiv2022

Thriving in the heat - Lysine acetylation stabilizes the quaternary structure of a Mega-Dalton hyperthermoactive PEP-synthase

Over time structural adaptations enabled proteins and enzymes to have sufficient stability and flexibility to perform the basic functions of life under various environmental conditions. The catalytic cores of key metabolic enzymes of hyperthermophilic archaea work at a temperature range of 80-120 {degrees}C, similar to the conditions wher the earliest life forms may have thrived. Here we characterize a key enzyme of the central carbon metabolism of Pyrococcus furious, through an integrative approach combining structural mass spectrometry, cryo-electron microscopy, mass photometry and molecular modelling with molecular dynamics simulations. From our investigation, we unveil the structural organization of phosphoenolpyruvate synthase (PPSA). Its 24-meric assembly - weighing over 2 MDa - harbors flexible distal domains, whose proper functioning and coordination depends on widespread chemical acetylation of lysine residues. This non-enzymatic post-translational modification, along with other types of lysine modifications, also occurs on most other major protein complexes of P. furiosus. These modifications likely originated in the chemically favorable primordial conditions and gradually became highly specialized and enzyme-driven in more distantly related mesophiles and Eukaryotes.

biochemistry↗

bioRxiv2022

Oxonium Ion-Guided Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

biochemistry↗

bioRxiv2022

Xlink Mapping and AnalySis (XMAS) - Smooth Integrative Modeling in ChimeraX

Crosslinking mass spectrometry (XL-MS) adds tremendous value to structural biology investigations. Its main strength lies in uncovering structural information in the form of distance constraints between neighboring amino acids of proteins, protein regions and complex samples, which are difficult to assess by other analytical techniques. However, although several approaches have been proposed, interpreting XL-MS data in a structural context has been cumbersome. ChimeraX has gained momentum as a flexible and widely used software package for the visualization of structural data, but is currently lacking functionalities for integration of experimental XL-MS data. Here, we introduce XMAS, a bundle that allows users to load results from several XL-MS search engines directly into ChimeraX and map the information onto protein structures. Besides automatically locating distance constraints on protein structures, XMAS offers the possibility to work with replicate experiments and/or different crosslinkers, and filter this data based on the number of replicates for which a given distance constraint was detected, thereby increasing the data quality. Additionally, we introduce the concept of self-links, which allows easy modeling of homo-dimeric interactions. Its core functionality is extended by the implementation of seamless connections to the HADDOCK suite to streamline otherwise time-consuming tasks in structural modeling pipelines. We demonstrate these key elements of the XMAS bundle by modeling crosslinking data obtained from human fibrin clots. The software is freely available from the ChimeraX toolshed, with an extensive user manual and example datasets.

bioinformatics↗

bioRxiv2021

Towards a structurally resolved human protein interaction network

All cellular functions are governed by complex molecular machines that assemble through protein-protein interactions. Their atomic details are critical to the study of their molecular mechanisms but fewer than 5% of hundreds of thousands of human interactions have been structurally characterized. Here, we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human interactions. We show that higher confidence models are enriched in interactions supported by affinity or structure-based methods and can be orthogonally confirmed by spatial constraints defined by cross-link data. We identify 3,137 high confidence models, of which 1,371 have no homology to a known structure, from which we identify interface residues harbouring disease mutations, suggesting potential mechanisms for pathogenic variants. We find groups of interface phosphorylation sites that show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple interactions as signalling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies. Accurate prediction of protein complexes promises to greatly expand our understanding of the atomic details of human cell biology in health and disease.

bioinformatics↗

bioRxiv2021

Notch-Jagged signaling complex defined by an interaction mosaic

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand Jagged1 to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical and structural techniques on the full extracellular complex and targeted sites,we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-13 and Jagged1 C2-EGF3 activation sites, as well as to two additional Jagged1 regions, EGF 8-11 and cysteine-rich domain (CRD). XL-MS and quantitative interaction experiments show that the three Notch1 binding sites on Jagged1 also engage intramolecularly.These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility determined by small-angle X-ray scattering (SAXS), support the formation of backfolded architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal, but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating new routes for therapeutic applications.

molecular biology↗

bioRxiv2021

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

biochemistry↗

bioRxiv2020

Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments.

biochemistry↗

bioRxiv2020

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 [A]2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

biochemistry↗

bioRxiv2019

Flexible regions in the molecular architecture of Human fibrin clots structurally resolved by XL-MS and integrative structural modeling

Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots that assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and insoluble character of fibrin clots, have however restricted our ability to develop novel treatments for clotting diseases. The so far resolved disparate structural details did provide insights into linear elongation; however, molecular details like the C-terminal domain of the -chain, the heparin-binding domain on the {beta}-chain, and others involved in lateral aggregation are lacking. To illuminate these dark areas, we applied crosslinking mass spectrometry (XL-MS) to obtain biochemical evidence in the form of over 300 distance constraints and combined this with structural modeling. These restraints additionally define the interaction network of the clots and e.g. provide molecular details for the interaction with Human Serum Albumin (HSA). We were able to construct the models of fibrinogen (excluding two highly flexible regions) and {beta}, confirm these models with known structural arrangements and map how the structure laterally aggregates to form intricate lattices together with fibrinogen{gamma} . We validate the final model by mapping mutations leading to impaired clot formation. From a list of 22 mutations, we uncovered structural features for all, including a crucial role for {beta} Arg196 in lateral aggregation. The resulting model will be invaluable for research on dysfibrinogenemia and amyloidosis, as it provides insights into the molecular mechanisms of thrombosis and bleeding disorders related to fibrinogen variants. The structure is provided in the PDB-DEV repository.

biochemistry↗

bioRxiv2019

PhoX - an IMAC-enrichable Crosslinking Reagent

Chemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

biochemistry↗