Biology subjects

Schuck, P.

Publications and source records attributed to Schuck, P..

10 recordsLinked to original sources

bioRxiv2026

The dynamic and heterogeneous structure of the non-canonical inflammasome

Inflammasomes are high molecular weight complexes that play an integral role in the innate immune system, triggering an inflammatory cascade to protect against cellular stresses such as pathogenic bacteria. Both canonical and non-canonical inflammasomes have been described in the literature and detailed structural studies of many components of the more complex and larger canonical versions have been reported. In contrast, corresponding structures of the non-canonical inflammasome have not emerged even though it consists of only two components: lipopolysaccharide (LPS) from gram-negative bacteria, and one of caspase-4 or caspase-5 in humans or caspase-11 in mice. Here we determine the stoichiometry of the non-canonical inflammasome using size-exclusion chromatography coupled with UV, refractive index, and light-scattering measurements, showing that the non-canonical inflammasome is heterogeneous, comprised of three major complexes with different numbers of LPS and caspase molecules. Solution Nuclear Magnetic Resonance (NMR) spectroscopy studies of the N-terminal Caspase Activation and Recruitment Domain (CARD) of caspase-11, that binds LPS, show that it is largely unstructured in the absence of lipid, with pervasive dynamics on the s-ms timescale. Formation of this complex increases the alpha-helical content of the CARD but the dynamics persist, multiple conformers are formed, and tertiary contacts are transient, consistent with formation of a molten globule. Our NMR results establish that the protease domain of caspase-11 is monomeric in isolation. As proteolysis is linked with dimerization, the protease domains are inactive in this state, but upon formation of the non-canonical inflammasome dimerization occurs, priming the complex for rapid processing of substrates. Significance StatementAnimals respond to injury, infection, or toxic materials via a process called inflammation. At the molecular level inflammation involves formation of large machines - inflammasomes - that are instrumental in triggering cascades that lead to an immune response. Although structural studies of canonical inflammasomes have emerged, much less is known about the structures of non-canonical inflammasomes. Using solution Nuclear Magnetic Resonance (NMR) spectroscopy in concert with other biophysical approaches we show that non-canonical inflammasomes are highly dynamic and structurally heterogeneous, and we characterize the different sized inflammasome particles that are formed in terms of their composition. We also show that once formed, non-canonical inflammasomes are primed to rapidly cleave substrate molecules, necessary for propagating the immune response.

biophysics↗

bioRxiv2026

Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.

biophysics↗

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗

bioRxiv2023

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

biophysics↗

bioRxiv2023

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

biophysics↗

bioRxiv2023

Diversity of Short Linear Interaction Motifs in SARS-CoV-2 Nucleocapsid Protein

Molecular mimicry of short linear interaction motifs has emerged as a key mechanism for viral proteins binding host domains and hijacking host cell processes. Here, we examine the role of RNA-virus sequence diversity in the dynamics of the virus-host interface, by analyzing the uniquely vast sequence record of viable SARS-CoV-2 species with focus on the multi-functional nucleocapsid protein. We observe the abundant presentation of motifs encoding several essential host protein interactions, alongside a majority of possibly non-functional and randomly occurring motif sequences absent in subsets of viable virus species. A large number of motifs emerge ex nihilo through transient mutations relative to the ancestral consensus sequence. The observed mutational landscape implies an accessible motif space that spans at least 25% of known eukaryotic motifs. This reveals motif mimicry as a highly dynamic process with the capacity to broadly explore host motifs, allowing the virus to rapidly evolve the virus-host interface.

biophysics↗

bioRxiv2023

An automated interface for sedimentation velocity analysis in SEDFIT

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an indispensable tool for the study of particle size distributions in biopharmaceutical industry, for example, to characterize protein therapeutics and vaccine products. In particular, the diffusion-deconvoluted sedimentation coefficient distribution analysis, in the software SEDFIT, has found widespread applications due to its relatively high resolution and sensitivity. However, a lack of available software compatible with Good Manufacturing Practices (GMP) has hampered the use of SV-AUC in this regulatory environment. To address this, we have created an interface for SEDFIT so that it can serve as an automatically spawned module with controlled data input through command line parameters and output of key results in files. The interface can be integrated in custom GMP compatible software, and in scripts that provide documentation and meta-analyses for replicate or related samples, for example, to streamline analysis of large families of experimental data, such as binding isotherm analyses in the study of protein interactions. To test and demonstrate this approach we provide a MATLAB script mlSEDFIT.

biophysics↗

bioRxiv2022

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. Tyrosine phosphorylation of C3G and interaction with Crk adaptor proteins, whose expression is increased in multiple human cancers, participate in C3G activation. However, the molecular details of C3G activation and the interplay between C3G phosphorylation and Crk interaction are poorly understood. Here, we combine biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. CrkL interacts through its SH3N domain with the proline-rich motifs P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Phosphorylation and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. The simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases.

molecular biology↗

bioRxiv2022

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

biophysics↗

bioRxiv2021

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.

biophysics↗