Biology subjects

Onck, P. R.

Publications and source records attributed to Onck, P. R..

13 recordsLinked to original sources

bioRxiv2026

A two-bead-per-aminoacid coarse-grained MD model with hydrogen bonding (2BPA-HB) to probe DNAJB6b-mediated suppression of polyglutamine aggregation in Huntingtons disease

Polyglutamine (polyQ) aggregation plays a central role in several neurodegenerative diseases, including Huntington's disease. DNAJB6b, a molecular chaperone involved in protein quality control, is known to efficiently suppress polyQ aggregation, but its anti-aggregation mechanism remains unclear. In this work we investigate the interaction between DNAJB6b and the polyQ region (Q48) of mutant Huntingtin Exon 1 (mHttEx1) using a custom-built coarse-grained molecular dynamics model. The model incorporates a two-bead-per-amino-acid representation with hydrogen bonding (termed 2BPA-HB), and is calibrated against all-atom molecular dynamics data in terms of geometry, hydrophobicity, and hydrogen bonding. The model reproduces the tertiary structure of DNAJB6b and its interactions with Q48, and reveals an inverse correlation between DNAJB6b concentration and Q48 aggregation propensity. Our simulations show that DNAJB6b co-condensates with polyQ molecules, thereby shielding the polyQ from forming the intermolecular hydrogen bonds necessary for amyloid formation. The 2BPA-HB CGMD model en- ables efficient exploration of DNAJB6b conformations, supporting future studies of chaperone-mediated aggregation suppression and therapeutic development.

biophysics↗

bioRxiv2026

Modeling disorder, secondary structure formation, and amyloid growth in FG-nucleoporins

Nuclear transport relies on intrinsically disordered FG-nucleoporins (FG-Nups) that form a dynamic selective barrier, yet experiments show that these proteins can also adopt highly ordered fibrillar structures. Capturing this duality within a single computational framework remains a major challenge. Here, we introduce 2BPA-HB, a sequence-resolved coarse-grained model that combines explicit, directional backbone hydrogen bonding with residue-specific side chain interaction centers derived from all-atom simulations. This design enables microsecond-scale simulations of the transitions between disordered, condensed, and amyloid-like states while maintaining computational efficiency. We first validate the model by simulating several experimentally resolved Nup98 fibril polymorphs, which retain their characteristic {beta}-sheet registry and protofibril architecture. Seeded growth simulations further show that disordered FG fragments align with and extend these fibrillar templates in a sequence-specific manner, and can initiate secondary nucleation events on fibril surfaces. Applying the model to yeast FG-Nups, we find that cohesive FG domains form stable condensates with interaction networks dominated by FG motif contacts, consistent with experimental observations and prior simulations. The increased structural resolution of 2BPA-HB additionally reveals transient - and {beta}-structures within these condensates. Together, these results demonstrate that 2BPA-HB captures both liquid-like and fibrillar behavior of intrinsically disordered proteins within a single coarse-grained framework. The model provides a platform to study how disorder, secondary structure formation, and aggregation coexist in nuclear pore biology, and represents a step forward to a transferable coarse-grained approach for low-complexity proteins that undergo both phase separation and amyloid formation, as implicated in neurodegenerative diseases.

biophysics↗

bioRxiv2025

Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

Nucleocytoplasmic transport occurs via nuclear pore complexes (NPCs), [~]40-60 nm wide pores lined with intrinsically disordered proteins that are rich in Phe-Gly motifs (FG-Nups) that form a selective barrier. Molecules larger than [~]50 kDa are increasingly blocked for transport unless they are bound to a nuclear transport receptor (NTR). How the amino acid sequence of FG-Nups contribute to this is not fully understood. Here, we present de novo designed artificial FG-Nups with a systematically varied FG-repeat spacing and charge-to-hydrophobicity ratio (C/H). Starting from a reference sequence termed NupY (with the average properties of natural yeast GLFG- Nups), we designed, synthesized, and experimentally tested a library of NupY variants using QCM-D experiments and phase separation assays. We find that the spacing between FG-motifs governs Kap95 absorption into the FG- Nup phase, while increasing C/H results in higher avidity for Kap95 due to an increased accessibility of FG-motifs. Molecular dynamics simulations of transport through NupY-coated pores show a reduced barrier function for noncohesive high-C/H-ratio variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between entropic repulsion and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.

biophysics↗

bioRxiv2025

A coarse-grained MD model for disorder-to-order transitions in polyQ aggregation

Polyglutamine (polyQ) aggregation plays a central role in several neurodegenerative diseases, including Huntingtons disease. To investigate the underlying mechanisms of polyQ aggregation, we developed a coarse-grained molecular dynamics model calibrated using atomistic simulations and experimental data. To assess the models predictive power beyond the calibrated parameter set, we systematically varied side chain hydrophobicity and hydrogen bonding strength to explore a broader range of aggregation pathways. These pathways ranged from nucleated growth to liquid-to-solid phase transitions. Through seeded aggregation simulations, we observed that amyloid growth occurs primarily in the {beta}-sheet elongation direction, although growth through steric zippering was also observed. Longer polyQ sequences (Q48) exhibited significantly faster growth compared to shorter sequences (Q23), underscoring the role of chain length in aggregation kinetics. Our model provides a versatile framework for studying polyQ aggregation and offers a foundation for investigating broader aggregation mechanisms and sequence variations.

biophysics↗

bioRxiv2024

Nuclear transport at full amino-acid resolution

Recent studies of nuclear pore complexes (NPCs) have provided detailed descriptions of the core scaffold structures, yet fall short in resolving the dynamic FG-meshwork with similar precision. Here, we present a novel modeling framework that enables the simulation of nuclear transport at full amino-acid resolution. We describe the distributions of the different FG-Nups in the central transporter and highlight the dynamic nature of the FG-meshwork, with FG-FG interaction lifetimes on the order of nanoseconds. Our findings reveal that Nsp1, the most abundant FG-Nup in the NPC, creates a central meshwork due to its unique bimodal structure, that is essential for controlling both passive and active transport. By adding nuclear transport receptors (NTRs)--specifically Kap95--to the pore, we demonstrate that NTRs play a key role in increasing the energy barrier for translocation of inert particles. The NTRs are subject to a dynamic interplay between binding to FG motifs and the temporal fluctuations of the FG-meshwork, leading to transient voids through which they move. Overall, our simulations identify a dense GLFG-ring coated by lower-mobility Kaps and a central dynamic FG-FG meshwork to create a reduced-dimensional transport surface of optimal binding avidity that drives Kap translocation.

biophysics↗

bioRxiv2024

The effect of dipeptide repeat proteins on FUS/TDP43-RNA condensation in C9orf72 ALS/FTD

Condensation of RNA binding proteins (RBPs) with RNA is essential for cellular function. The most common familial cause of the diseases ALS and FTD are C9orf72 repeat expansion disorders that produce dipeptide repeat proteins (DPRs). We explore the hypothesis that DPRs disrupt the native condensation behaviour of RBPs and RNA through molecular interactions resulting in toxicity. FUS and TDP43 are two RBPs known to be affected in ALS/FTD. We use our previously developed 1-bead-per-amino acid and a newly developed 3-bead-per-nucleic acid molecular dynamics model to explore ternary phase diagrams of FUS/TDP43-RNA-DPR systems. We show that the most toxic arginine containing DPRs (R-DPRs), can disrupt the RBP condensates through cation-{pi} interactions, and can strongly sequester RNA through electrostatic interactions. The native droplet morphologies are already modified at small additions of R-DPRs leading to non-native FUS/TDP43-encapsulated condensates with a marbled RNA/DPR core.

biophysics↗

bioRxiv2024

Selective phase separation of transcription factors is driven by orthogonal molecular grammar

Protein production is critically dependent on gene transcription rates, which are regulated by RNA polymerase and a large collection of transcription factors (TFs). Previous studies identified the formation of super enhancer regions where increased transcriptional activity is observed. This has been linked to phase separation, in which the differential condensation behaviour of separate TF families has been hypothesised to cause the selectivity in gene expression. The underlying molecular forces that are responsible for this selectivity, however, are unknown. Here, we conduct phase separation studies on six TFs (FUS, EWS, TAF15, SP1, SP2, and HNF1A) from three different TF families by carrying out residue-scale coarse-grained molecular dynamics simulations. Our exploration of ternary TF phase diagrams revealed four dominant sticker motifs and two orthogonal driving forces, consisting of hydrophobic (aromatic, aliphatic) interactions and electrostatic/cation-{pi} interactions. The contribution of these driving forces to the homotypic and heterotypic intermolecular strengths dictate the resultant condensate morphology. These results point to sequence-dependent orthogonal grammar as a generic mechanism responsible for selective transcriptional condensation in gene expression. Interestingly, our results also show how RNA polymerase is able to overcome this orthogonality to coalesce with TF condensate providing a framework in which co-condensation is the primary nuclear organisational principle that controls selective partitioning of RNAP to targeted genes for transcription.

biophysics↗

bioRxiv2023

Structural properties of the HNF-1A transactivation domain

Hepatocyte nuclear factor 1 (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic {beta}-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic {beta}-cells.

biochemistry↗

bioRxiv2023

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Despite its importance in maintaining cellular homeostasis, the structure-functional relationship of DNAJB6b is not yet known. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a novel, less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

biophysics↗

bioRxiv2023

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis. HighlightsO_LINups form native cytosolic condensates C_LIO_LINsp1 reduction mimics NPC aging phenotypes C_LIO_LINsp1 acts as phase state modulator of FG-Nups C_LIO_LINsp1 shares surveillance function with classical chaperones C_LI

cell biology↗

bioRxiv2022

Molecular basis of C9orf72 poly-PR interference with the β-karyopherin family of nuclear transport receptors

Nucleocytoplasmic transport (NCT) is affected in several neurodegenerative diseases including C9orf72-ALS. It has recently been found that arginine-containing dipeptide repeat proteins (R-DPRs), translated from C9orf72 repeat expansions, directly bind to several importins. To gain insight into how this can affect nucleocytoplasmic transport, we use coarse-grained molecular dynamics simulations to study the molecular interaction of poly-PR, the most toxic DPR, with several Kap{beta}s (importins and exportins). We show that poly-PR-Kap{beta} binding depends on the net charge per residue (NCPR) of the Kap{beta}, salt concentration of the solvent, and poly-PR length. Poly-PR makes contact with the inner surface of most importins, which strongly interferes with Kap{beta} binding to cargo-NLS, IBB, and RanGTP in a poly-PR length-dependent manner. Longer poly-PRs at higher concentrations are also able to make contact with the outer surface of importins that contain several binding sites to FG-Nups. We also show that poly-PR binds to exportins, especially at lower salt concentrations, interacting with several RanGTP and FG-Nup binding sites. Overall, our results suggest that poly-PR might cause length-dependent defects in cargo loading, cargo release, Kap{beta} transport and Ran gradient across the nuclear envelope.

biophysics↗

bioRxiv2022

Nuclear transport under stress phenocopies transport defects in models of C9Orf72 ALS

The nucleus is the hallmark of eukaryotic life and transport to and from the nucleus occurs through the nuclear pore complex (NPC). There is a multitude of data connecting the nuclear transport machinery - i.e. the NPCs and associated nuclear transport factors - to neurodegenerative diseases, but the mechanisms are not well understood. Using Saccharomyces cerevisiae, we systematically studied how the expression of polyPR and polyGA related to C9Orf72 amyotrophic lateral sclerosis impacts the nuclear transport machinery. We measured the abundance and localization of NPC components and transport factors, and assessed the kinetics of import and export by four transport receptors. PolyPR and polyGA cause distinct, and transport receptor dependent effects. We compared the specific changes in transport to those obtained when cells were exposed to different stress situations or mutations. This comparison showed similar patterns of transport defects in cells lacking specific NTRs and cells expressing polyPR. In contrast, polyGA expressing cells bear resemblance to stress conditions where energy maintenance is decreased. The similarity of the patterns of transport deficiencies suggests that polyPR has a direct effect on nuclear transport via NTRs, while polyGA impacts the energy state of the cell and subsequently changes transport.

cell biology↗

bioRxiv2020

A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs.

biophysics↗