Biology subjects

Uetrecht, C.

Publications and source records attributed to Uetrecht, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Variant-specific nucleocapsid mutations shape host innate immune trajectories during SARS-CoV-2 infection

The SARS-CoV-2 nucleocapsid (N) protein is one of the most highly expressed proteins during infection and plays crucial roles in the protection and packaging of viral RNA, replication, suppression of the immune response and virus assembly. The N gene and the overlapping accessory open reading frame ORF9b have continually evolved throughout the circulation of the virus, likely due to ongoing adaptation to the human host. This genetic variability influences the interplay of the N protein and its interactors. Yet, little is known about how specific mutations within the N/ORF9b locus of Variants of Concern (VOCs) shape the progression and outcome of infection. Here, we use a multi-omics approach to decipher how these genetic alterations reprogram the host cell by creating Wuhan-Hu-1-based recombinant viruses carrying an isogenic backbone with respective N mutations from the VOCs (called rNs) and comparing their effects at the transcriptome and proteome levels. We found that the mutations induce distinct transcriptional and translational alterations: rN-Alpha drives a stealth-like infection characterized by sustained translation efficiency and specific evasion of the 2'-5'-oligoadenylate synthetase (OAS) innate immune sensor, whereas rN-Delta and rN-BA.2 trigger a highly elevated inflammatory response. For rN-Delta, hyperphosphorylation of the N-protein drives cellular stress culminating in necroptotic cell death. Because these viruses differ only within the N/ORF9b locus, N sequence variation emerges as a determinant of infection outcome in its own right, warranting increased surveillance attention.

molecular biology↗

bioRxiv2026

CAPRIN1 localizes to CD63-positive MVB-like SARS-CoV-2 egress compartments and limits cytopathic effects

Stress granules (SGs) are cytoplasmic condensates that assemble under cellular stress, including viral infection. The best-characterized evasion strategy in SARS-CoV-2 infection involves the viral nucleocapsid (N) protein hijacking the SG core protein G3BP1, thereby preventing SG assembly. By performing network analysis of N protein interactomes, we identified CAPRIN1 as an underexplored candidate, despite its central role alongside G3BP1 in SG assembly. Here, we provide the first high-resolution spatial and functional data on CAPRIN1 in SARS-CoV-2 infection. Using ultrastructure expansion microscopy (U-ExM), we localized CAPRIN1 and viral egress markers to enlarged, CD63- and LAMP1-positive, multivesicular body (MVB)-like egress compartments, refining the current model of SARS-CoV-2 egress. CAPRIN1 was found on both the limiting membrane of these compartments and within the intraluminal vesicles, a pattern distinct from G3BP1. Knockout of CAPRIN1 increased infection-associated cell death, promoted an aberrant cell-death phenotype, and enhanced syncytia formation. Together, our data reveal distinct activities of CAPRIN1 during infection and identify the egress compartments as MVB-like, raising the possibility that SARS-CoV-2 repurposes more than one cellular pathway for egress.

molecular biology↗

bioRxiv2026

Applying distinct CDMS strategies to observe non-classical virus capsid assembly

In conventional native mass spectrometry (MS), one faces severe limitations when challenged with heterogenous, high mass samples, commonly failing to resolve clear peak distributions and thus mass determination. Charge detection MS (CDMS) has emerged as a premier method to analyze these samples by determining mass-to-charge ratio (m/z) and charge (z) simultaneously. Here, the two currently available commercialized CDMS systems, the Orbitrap-based Direct Mass Technology (DMT) and the electrostatic linear ion trap (ELIT)-based Xevo CDMS are applied to human norovirus capsids from two different strains, GI.1 Norwalk and GII.17 Kawasaki. The norovirus capsid is highly heterogenous due to N-terminal processing on the repeating subunits that it is built from and commonly forms T = 3 and sometimes T = 4 particles. Both CDMS approaches were able to determine similar masses in both strains. GII.17 Kawasaki exhibits both T = 3 and T = 4 particles, though the Xevo CDMS measurements were closer to the theoretical mass than the DMT instrument. Interestingly, GII.17 Kawasaki also displayed non-classical mass distributions with high abundance in-between T = 3 and T = 4 which was then confirmed by cryogenic electron microscopy (cryo-EM), demonstrating an oval capsid shape. GI.1 Norwalk displays a wide mass distribution in both instruments that exceeds the theoretical T = 3 mass by 8-10 %. Proteomics and native MS experiments suggest possible interactions with a protein from the expression system. This study demonstrates the capabilities of two distinct CDMS methodologies on two viral capsids and presents the first non-classical capsid assembly in a GII.17 noroviral capsid.

biochemistry↗

bioRxiv2025

Proteoform-Resolved Phosphorylation Dynamics in Kinase Complexes by Hybrid Precision Mass Spectrometry

Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major challenge. Here, we dissect AMP-activated protein kinase (AMPK) proteoforms during activation through controlled biochemical reactions with a hybrid mass spectrometry (MS) approach integrating bottom-up MS for site-specific kinetics with top-down proteoform characterization. We reveal that AMPK phosphorylation proceeds through hierarchical cascades rather than binary switching, with dual entry points: canonical CaMKK2-mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with 1-S496 showing highest kinetic priority. Proteoform-resolved analysis uncovers channeled {beta}1-S24/25+S108 co-phosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only 1-T183, with all other modifications arising through autophosphorylation. Phosphatase competition reveals asymmetric control where PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected, establishing persistent regulatory states. Resolving AMPKs temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.

biochemistry↗

bioRxiv2025

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

biochemistry↗

bioRxiv2025

Fast-tracking native mass spectrometry: Skipping over buffer exchange

Obtaining sufficient amounts of pure protein for downstream applications such as native mass spectrometry (nMS) is often challenging, especially when expression yields are low or proteins are unstable. In these cases, the commonly required buffer-exchange step is a major bottleneck, as it often leads to substantial protein loss and compromises biophysical characterization. These challenges are exacerbated in insect or eukaryotic expression systems, where protein yields are typically lower than in bacteria, making protein loss during purification particularly detrimental. Standard lysis and purification buffers contain non-volatile components such as Tris, phosphate, HEPES and sodium chloride, which form adducts during electrospray ionization (ESI) interfering with the signal and therefore must be re-moved prior to nMS. To address protein loss associated with this mandatory buffer-exchange, we evaluated an affinity-purification workflow, in which non-volatile salts are excluded throughout purification and proteins are directly eluted into nMS-compatible ammonium acetate-based buffers. This approach eliminates the need for a separate buffer exchange step and enables rapid nMS analysis immediately after affinity purification. We show that common eluents used in His- and Strep- based affinity purification, such as imidazole, biotin, and desthiobiotin, are well tolerated at relevant concentrations, allowing acquisition of high-quality spectra suitable for determining protein stoichiometry and for monitoring enzymatic or assembly processes. Together, this fast-track affinity workflow increases protein recovery, shortens sample preparation and complements online exchange protocols, which are less suited for monitoring processes. It hence expands the applicability of nMS to proteins and protein complexes that are difficult to obtain in sufficient quantity using conventional purification and buffer exchange strategies.

biophysics↗

bioRxiv2025

X-ray Spectroscopy Meets Native Mass Spectrometry: Probing Gas-phase Protein Complexes

Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.

biochemistry↗

bioRxiv2025

Statistical crystallography reveals an allosteric network in SARS-CoV-2 Mpro

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics with atomic resolution remains a key challenge. Here, using thousands of crystals of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the proteins correlated motions. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the correlated motions that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally linked to the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography can determine protein correlated motions and rationalize their biological function. TeaserCrystallography at scale goes beyond a single structure, revealing native-state protein dynamics.

biochemistry↗

bioRxiv2024

Automated high-throughput selection of DNA aptamers using a common optical next-generation sequencer

Aptamers are conventionally selected via Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process is, however, laborious, time-consuming, and has a relatively low efficacy. Here, we developed an automated high-throughput screening platform for the selection of DNA aptamers which consists of an optical next-generation sequencer with a modified software and hardware to automatically perform fluorescence-based binding assays on the displayed DNA sequences subsequent to sequencing. Using this platform, after only three to five SELEX rounds we selected highly affine DNA aptamers for the lectins LecA and LecB of Pseudomonas aeruginosa as well as for the Pseudomonas Exotoxin A. In comparison, twelve rounds of conventional SELEX resulted in three-fold less affine aptamers for LecA and PEA and none for LecB. Our high throughput-approach bears great potential to augment SELEX as it significantly increases time efficiency, enabling the selection of aptamers within only one week.

biochemistry↗

bioRxiv2024

Subtleties in Clathrin Heavy Chain Binding Boxes provide selectivity among Adaptor Proteins of Budding Yeast

Clathrin, forming the triskelion network, orchestrates highly regulated cellular processes facilitating cargo internalization and trafficking in eukaryotes, with its N-terminal domain (NTD) pivotal for adaptor protein (AP) interactions. The NTD contains up to four AP-binding sites, and their roles in preferential occupancy by APs have not been addressed. Here, employing a combination of integrative biophysical and structural approaches together with in vivo functional experiments, we investigated the binding hierarchy and selectivity of adaptors for clathrin, aiming to understand the evolutionary conservation of redundant APs and their specialized roles in endocytosis and cellular trafficking mechanisms. We found that yeast epsin Ent5 displayed the highest affinity for clathrin, indicating its significant role in cellular trafficking processes. Epsins Ent1 and Ent2, which are crucial for endocytosis but described to have redundant functions, revealed distinct binding patterns; Ent1 demonstrated stronger interactions with clathrin than Ent2, explaining its functional divergence towards actin binding. Despite both having actin anchoring domains, since Ent1 is actually more stably recruited by clathrin, it would provide a better actin anchoring function. These results offer molecular insights into AP selectivity, suggesting they competitively bind clathrin while also targeting different clathrin sites.

molecular biology↗

bioRxiv2024

Glycan-induced structural activation softens the human papillomavirus capsid for entry through reduction of intercapsomere flexibility

High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigated the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data will help further devising prophylactic strategies against HPV infections.

microbiology↗

bioRxiv2024

The kinetics of SARS-CoV-2 nsp7-11 polyprotein processing and impact on complexation with nsp16

In severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex formation is critical and offers potential therapeutic targets. However, the interplay of polyprotein processing and RTC-assembly are poorly understood. Here, we studied two key aspects: The influence of the pp1a terminal nsp11 on the order of polyprotein processing by viral main protease Mpro and the influence of polyprotein processing on core enzyme complex formation. We established a method based on native MS to determine rate constants k considering the structural environment. This enabled us to quantify the multi-reaction kinetics of coronavirus polyprotein processing for the first time. Our results serve as a blueprint for other multi-cleavage reactions. Further, it offers a detailed and quantifiable perspective to the dynamic reactions of SARS-CoV-2 polyprotein processing, which is required for development of novel antivirals.

biophysics↗

bioRxiv2023

Structural Insights into the Interaction Between Adenovirus C5 Hexon and Human Lactoferrin

Adenovirus (AdV) infection of the respiratory epithelium is common, but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexons hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.

biochemistry↗

bioRxiv2023

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming the ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs have to be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly and budding, and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment and release in Lassa virus.

biophysics↗

bioRxiv2021

Mapping the peptide binding groove of MHC class I

An essential element of adaptive immunity is the selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes on the cell surface. Using native mass spectrometry, we here analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule. This novel approach allows us to examine the binding properties of diverse peptides. The unique stability of our MHC class I even enables us to determine the binding affinity of complexes, which are suboptimally loaded with truncated or charge-reduced peptides. Notably, a unique erucamide adduct decouples affinity analysis from peptide identity alleviating issues usually attributed to clustering during electrospray ionization. We discovered that two anchor positions at the binding surface between MHC and peptide can be stabilized independently and further identify the contribution of other peptidic amino acids on the binding. We propose this as an alternative, likely universally applicable method to artificial prediction tools to estimate the binding strength of peptides to MHC class I complexes quickly and efficiently. This newly described MHC class I-peptide binding affinity quantitation represents a much needed orthogonal, independent approach to existing computational affinity predictions and has the potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

biophysics↗

bioRxiv2021

Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin ({beta}2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the 3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the 3 domain of one FHC binds to another FHC in a manner similar to the {beta}2m light chain. Significance StatementMHC class I molecules are cell surface transmembrane proteins with key functions in adaptive immunity against viral infections. The spatiotemporal organization of fully assembled MHC I at the cell surface and its function with respect to trans-interactions with T and NK cells has been studied in detail. By contrast, the consequences of peptide and {beta}2m dissociation yielding to formation of free heavy chains (FHC) have remained unclear. We have discovered that class I free heavy chains form distinct non-covalent dimers at the cell surface rather than non-specific clustering, and we have identified a dimerization interface mediated by the 3 domain. We propose that these non-covalent dimers are the basis of distinct signaling and endocytic sorting of MHC I FHC. This is to be explored in further work.

cell biology↗

bioRxiv2021

Protein secondary structure affects glycan clustering in native mass spectrometry

Infection with human noroviruses (hNoV) for the vast majority of strains requires attachment of the viral capsid to histo blood group antigens (HBGA). The HBGA binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for binding of HBGAs to P dimers and, in some cases, disagreed whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of the reference protein is crucial. We analyzed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of {beta}-sheet has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

biophysics↗

bioRxiv2021

Murine norovirus capsid plasticity - Glycochenodeoxycholic acid stabilizes P domain dimers and triggers escape from antibody recognition

The murine norovirus (MNV) capsid protein is the target for various neutralizing antibodies binding to distal tips of its protruding (P)-domain. The bile acid glycochenodeoxycholic acid (GCDCA), an important co-factor for murine norovirus (MNV) infection, has recently been shown to induce conformational changes in surface-loops and a contraction of the virion. Here, we employ protein NMR experiments using stable isotope labeled MNV P-domains to shed light on underlying molecular mechanisms. We observe two separate sets of NMR resonance signals for P-domain monomers and dimers, permitting analysis of the corresponding exchange kinetics. Unlike human norovirus GII.4 P-dimers, which exhibit a half-life in the range of several days, MNV P-dimers are very short lived with a half-life of about 17 s. Addition of GCDCA shifts the equilibrium towards the dimeric form by tightly binding to the P-dimers. In MNV virions GCDCA-mediated stabilization of the dimeric arrangement of P-domains generates a more ordered state, which in turn may entropically assist capsid contraction. Numerous long-range chemical shift perturbations (CSPs) upon addition of GCDCA reflect allosteric conformational changes as a feature accompanying dimer stabilization. In particular, CSPs indicate rearrangement of the EF loop, a target for various neutralizing antibodies. Indeed, treating MNV virions with GCDCA prior to neutralizing antibody exposure abolishes neutralization. These findings advance our understanding of GCDCA-induced structural changes of MNV capsids and experimentally support an intriguing viral immune escape mechanism relying on GCDCA-triggered conformational changes of the P-dimer. Significance StatementThis study sheds light on the role of glycochenodeoxycholic acid (GCDCA) in promoting murine norovirus (MNV) infection and immune escape. Binding of GCDCA to the dimeric P-domain has been well characterized by crystallography and cryo EM studies, showing that upon GCDCA binding, a 90{degrees} rotation of the P-domain occurs, which results in its collapse onto the underlying shell of the virus. Our NMR experiments now reveal P-dimer stability as a new dimension of plasticity of MNV capsids and suggest that capsid contraction is entropically assisted. Conformational changes as a feature of P-dimer stabilization eliminate recognition by neutralizing antibodies, no longer being able to prevent infection. These findings highlight key differences between human and MNV capsid structures, promote our understanding of MNV infection on a molecular level, and reveal a novel immune escape mechanism.

biochemistry↗