Biology subjects

Holehouse, A. S.

Publications and source records attributed to Holehouse, A. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Generative design of functional disordered regions in a developmental transcription factor

Rapid advances in protein structure design have not translated to intrinsically disordered regions (IDRs). This is largely due to our incomplete understanding of how IDR sequence encodes function. Here, we use stem cell differentiation as a model to map functional sequence features in the IDR of transcription factor RUNX2. We show that RUNX2 requires its C-terminal IDR to bind chromatin and regulate gene expression during osteogenesis. Surprisingly, linear IDR sequence is dispensable, and instead features of the conformational ensemble predict function. We develop an algorithm for rational IDR design based on biophysical principles, and use it to generate functional IDRs de novo. Together, these results illustrate the central role for TF IDRs in chromatin binding and extend generative protein design beyond the folded proteome.

synthetic biology↗

bioRxiv2026

Proteoform plasticity modulates the temperature response in circadian timekeeping

The molecular circadian clock optimally coordinates an organism's physiology with the light/dark cycle. One commonality among circadian molecular clock proteins is that they undergo alternative splicing, yielding multiple proteoforms. The isoform-specific sequences spliced into clock proteins contain intrinsically disordered regions (IDRs), suggesting that these regions might be intricately involved in cellular regulation. However, the functions of these isoform-specific IDRs in the clock remain poorly defined. Here, we use the core clock repressor FREQUENCY (FRQ) from the fungal circadian model system Neurospora crassa to test the hypothesis that alternative splicing of core clock IDRs yields multiple proteoforms that expand the functional regulatory capacity of FRQ. To do so, we biophysically characterized an IDR specific to the long isoform of the core clock repressor FRQ and identified motifs and molecular behaviors that regulate clock robustness in a temperature-dependent manner. We further compared the interactomes of two FRQ isoforms, identifying distinct functions and interacting proteins that could contribute to temperature regulation. Taken together, our data highlight that isoform-specific IDRs in clock-repressor proteins enhance and expand clock function in a context-dependent manner.

biochemistry↗

bioRxiv2026

Lipoproteins modulate the uptake and biological functionof cationic cell-penetrating peptides across the animal lineage.

Cationic cell-penetrating peptides (+CPPs) are pervasive killer peptides produced by all animals and found in innate immune systems, venoms and neurodegenerative diseases. Despite their ubiquity, the mechanisms underlying their uptake remain opaque and intensely debated. Here, we interrogate +CPPs spanning 600 million years of animal evolution and identify endocytosis as a conserved/convergent uptake mechanism across cell types and organisms, at physiological concentrations. By combining multiplex imaging, genetic screening, cryo-electron tomography, and biophysical methods, we uncover that +CPPs seemingly universally enter eukaryote cells via 'hitchhiking' on lipoproteins. We further show that the interaction of +CPPs with lipoproteins modulates their antimicrobial function. Combined, our findings establish a unified molecular framework describing a pan-eukaryote cell penetrance mechanism. As hyperlipidemia is a common comorbidity, our findings have direct implications for human health. Lastly, the insights gleaned from this work highlight design principles that may inform the future engineering of peptide-based therapeutics and delivery vehicles.

cell biology↗

bioRxiv2026

Combinatorial sequence elements fine-tune mitochondrial protein import to facilitate dual localization

The dual targeting of mitochondrial proteins regulates a host of cellular processes, including metabolism, cofactor biosynthesis, mitophagy, and stress responsiveness. Despite this importance, the mechanisms by which proteins dually localize are incompletely defined. Here, we identify multiple sequence elements that compromise the matrix localization of the phosphatase PPTC7 to facilitate its accumulation at the outer mitochondrial membrane (OMM), where it regulates mitophagy. We find that PPTC7 has a moderately weak presequence, but this feature is insufficient to promote dual targeting of a generic cargo protein. Instead, our data suggest that a recently evolved glycine stretch decreases the helical potential of the PPTC7 presequence, weakening its import efficiency in vitro and in cells. Deletion of these glycine residues improves PPTC7 in vitro import and enrichment within the mitochondrial matrix, but only partially suppresses PPTC7-mediated regulation of mitophagy at the OMM. These data suggested additional elements may contribute to PPTC7 dual localization, including its mature phosphatase domain which has robust thermal stability and becomes further stabilized to an import-incompetent state upon binding to its requisite enzymatic co-factor manganese. Simultaneous increases in presequence strength and denaturation of the PPTC7 phosphatase domain are required to promote import in vitro, underscoring the multifactorial challenges associated with its matrix targeting. These data suggest that sequence-specific features can work combinatorially to impart dual-localization capacity to mitochondrial proteins, enabling functions across cellular compartments.

biochemistry↗

bioRxiv2026

Subnuclear cofactor partitioning underlies auxin-dependent transcriptional regulation

Cellular and organismal function relies on the precise activation and repression of gene expression by DNA-binding transcription factors (TFs). Many TFs occur in large gene families, and a key question in biology is how divergent functions emerge in TF families during evolution. Here we discover the biochemical mechanism for transcriptional activation by the Marchantia polymorpha AUXIN RESPONSE FACTOR1 (MpARF1) TF, which relies on direct recruitment of the Mediator complex into subnuclear MpARF1 clusters. We find that the Mediator recruitment region was the evolutionary innovation that converted ARF repressors into activators, switching binding specificity from co-repressor to co-activator. We demonstrate that this evolutionary innovation can be recreated, thereby revealing a deeply conserved mechanism based on competition between ARF clusters at the heart of the transcriptional auxin response.

plant biology↗

bioRxiv2026

C9ORF72-derived polyGR polypeptides disrupt passive nucleocytoplasmic transport by tuning protein affinity for the nuclear pore barrier

Nucleocytoplasmic partitioning is an essential determinant of eukaryotic cellular function, governed by the nuclear pore complex, a molecular portal filled by a disordered phenylalanine-glycine (FG)-rich phase that governs selective entry and exit. Disruption of nucleocytoplasmic partitioning is seen across many different diseases, including viral infection, cancer, and neurodegeneration. However, what determines whether a given protein mislocalises when nucleocytoplasmic transport is disrupted remains unknown. This question is central to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), where cytosolic mislocalisation of the nuclear RNA-binding protein TDP-43 is a defining pathological feature. The most common genetic cause of these diseases is a G4C2 repeat expansion in the gene C9ORF72, which produces aberrant neurotoxic polypeptides that induce nucleocytoplasmic transport defects. Here, we show how the highly toxic poly(glycine-arginine/GR) polypeptide engages the nuclear pore FG-rich selectivity barrier and retunes passive nucleocytoplasmic transport according to client surface chemistry. Using coarse-grained simulations, in vitro FG-phase reconstitution and human cell lines and neurons, we find that polyGR produces a non-linear, biphasic modulation of nuclear pore passage. Proteins with low affinity for the FG phase are unaffected, whereas proteins with higher affinity due to solvent-exposed hydrophobic residues exhibit enhanced transport up to a critical threshold, beyond which highly hydrophobic proteins experience transport suppression, cytoplasmic accumulation and aggregation. Together, these findings establish how disease-associated polypeptides retune the physicochemical rules governing passive nuclear pore transport, leading to biphasic outcomes determined by protein surface chemistry that alter protein compartmentalisation and aggregation. This provides a biophysical mechanism by which polyGR drives selective protein vulnerability to nuclear pore dysfunction in C9ORF72-associated ALS/FTD.

cell biology↗

bioRxiv2025

Direct Optical Quantification of Chain Collapse, Reduced Dielectric, and Water Release Driving Protein Phase Separation

Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. Our experimental and computational results reveal that the intrinsically disordered N-terminal domain of the RNA Deadbox helicase 4 (DDX4) deviates from random coil behavior and undergoes chain collapse that correlates with phase separation, which leads to lower dielectric and reduced water content inside condensates. Our data support a model of DDX4 phase separation whereby chain collapse, reduced dielectric, and water release enhance the strength of multivalent protein-protein interactions within condensates, driving condensate growth and phase separation through positive feedback. Our study addresses the critical driving forces of biomolecular phase separation across a range of length scales, providing quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors.

biophysics↗

bioRxiv2025

Accurate predictions of conformational ensembles of disordered proteins with STARLING

Intrinsically disordered proteins and regions (collectively IDRs) are found across all kingdoms of life and play critical roles in virtually every eukaryotic cellular process. In contrast to folded proteins, IDRs lack a stable 3D structure and are instead described in terms of a conformational ensemble, a collection of energetically accessible interconverting structures. This unique structural plasticity facilitates diverse molecular recognition and function; thus, a convenient way to view IDRs is through their ensembles. Here, we combine advances in physics-based force fields for IDPs with the power of modern multi-scale generative modeling to develop STARLING, an approach for the rapid and accurate prediction of IDR ensembles directly from sequence. STARLING enables ensembles of hundreds of conformers to be generated in seconds and works on GPUs and CPUs. This, in turn, dramatically lowers the barrier to the computational interrogation of IDR function through the lens of emergent biophysical properties complementing bioinformatic protein sequence analysis. We evaluate STARLINGs accuracy against extant experimental data and offer a series of vignettes illustrating how STARLING can enable rapid hypothesis generation for IDR function and aid the interpretation of experimental data.

biophysics↗

bioRxiv2024

Cellular Function of a Biomolecular Condensate Is Determined by Its Ultrastructure

Biomolecular condensates play key roles in the spatiotemporal regulation of cellular processes. Yet, the relationship between atomic features and condensate function remains poorly understood. We studied this relationship using the polar organizing protein Z (PopZ) as a model system, revealing how its material properties and cellular function depend on its ultrastructure. We revealed PopZs hierarchical assembly into a filamentous condensate by integrating cryo-electron tomography, biochemistry, single-molecule techniques, and molecular dynamics simulations. The helical domain drives filamentation and condensation, while the disordered domain inhibits them. Phase-dependent conformational changes prevent interfilament contacts in the dilute phase and expose client binding sites in the dense phase. These findings establish a multiscale framework that links molecular interactions and condensate ultrastructure to macroscopic material properties that drive cellular function.

cell biology↗

bioRxiv2024

Disentangling folding from energetic traps in simulations of disordered proteins

Protein conformational heterogeneity plays an essential role in a myriad of different biological processes. Extensive conformational heterogeneity is especially characteristic of intrinsically disordered proteins and protein regions (collectively IDRs), which lack a well-defined three-dimensional structure and instead rapidly exchange between a diverse ensemble of configurations. An emerging paradigm recognizes that the conformational biases encoded in IDR ensembles can play a central role in their biological function, necessitating understanding these sequence-ensemble relations. All-atom simulations have provided critical insight into our modern understanding of the solution behavior of IDRs. However, effectively exploring the accessible conformational space associated with large, heterogeneous ensembles is challenging. In particular, identifying poorly sampled or energetically trapped regions of disordered proteins in simulations often relies on qualitative assessment based on visual inspection of simulations and/or analysis data. These approaches, while convenient, run the risk of masking poorly-sampled simulations. In this work, we present an algorithm for quantifying per-residue local conformational heterogeneity in protein simulations. Our work builds on prior work and compares the similarity between backbone dihedral angle distributions generated from molecular simulations in a limiting polymer model and across independent all-atom simulations. In this regime, the polymer model serves as a statistical reference model for extensive conformational heterogeneity in a real chain. Quantitative comparisons of probability vectors generated from these simulations reveal the extent of conformational sampling in a simulation, enabling us to distinguish between situations in which protein regions are well-sampled, poorly-sampled, or folded. To demonstrate the effectiveness of this approach, we apply our algorithm to several toy, synthetic, and biological systems. Accurately assessing local conformational sampling in simulations of IDRs will help better quantify new enhanced sampling methods, ensure force field comparisons are equivalent, and provide confidence that conclusions drawn from simulations are robust.

biophysics↗

bioRxiv2024

Metapredict enables accurate disorder prediction across the Tree of Life

Intrinsically disordered proteins and protein regions (collectively IDRs) are critical in numerous cellular processes. To understand how IDRs facilitate function, we need tools to accurately and rapidly identify them from sequence. While many methods for disorder prediction exist, we are currently limited by throughput and accuracy for evolutionary scale analyses. To bridge this gap, we developed metapredict V3, an updated version of our disorder predictor that enables evolutionary-scale disorder prediction. Metapredict V3 enables proteome-scale prediction with state-of-the-art accuracy in seconds and was developed with a focus on usability. It is distributed as a web server, Python software package, command-line interface, and Google Colab notebook. Here, we leverage the accuracy and throughput of metapredict V3 to predict disorder for over 20,000 proteomes to evaluate the prevalence of disorder across the kingdoms of life.

bioinformatics↗

bioRxiv2024

The dimerization domain of SARS CoV 2 Nucleocapsid protein is partially disordered as a monomer and forms a high affinity dynamic complex.

The SARS-CoV-2 Nucleocapsid (N) is a 419 amino acids protein that drives the compaction and packaging of the viral genome. This compaction is aided not only by protein-RNA interactions, but also by protein-protein interactions that contribute to increasing the valence of the nucleocapsid protein. Here, we focused on quantifying the mechanisms that control dimer formation. Single-molecule Forster Resonance Energy Transfer enabled us to investigate the conformations of the dimerization domain in the context of the full-length protein as well as the energetics associated with dimerization. Under monomeric conditions, we observed significantly expanded configurations of the dimerization domain (compared to the folded dimer structure), which are consistent with a dynamic conformational ensemble. The addition of unlabeled protein stabilizes a folded dimer configuration with a high mean transfer efficiency, in agreement with predictions based on known structures. Dimerization is characterized by a dissociation constant of [~] 12 nM at 23 OC and is driven by strong enthalpic interactions between the two protein subunits, which originate from the coupled folding and binding. Interestingly, the dimer structure retains some of the conformational heterogeneity of the monomeric units, and the addition of denaturant reveals that the dimer domain can significantly expand before being completely destabilized. Our findings suggest that the inherent flexibility of the monomer form is required to adopt the specific fold of the dimer domain, where the two subunits interlock with one another. We proposed that the retained flexibility of the dimer form may favor the capture and interactions with RNA, and that the temperature dependence of dimerization may explain some of the previous observations regarding the phase separation propensity of the N protein.

biophysics↗

bioRxiv2024

LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation

Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.

biochemistry↗

bioRxiv2024

Protein surface chemistry encodes an adaptive resistance to desiccation

Cellular desiccation - the loss of nearly all water from the cell - is a recurring stress in an increasing number of ecosystems that can drive protein unfolding and aggregation. For cells to survive, at least some of the proteome must resume function upon rehydration. Which proteins tolerate desiccation, and the molecular determinants that underlie this tolerance, are largely unknown. Here, we apply quantitative and structural proteomic mass spectrometry to show that certain proteins possess an innate capacity to tolerate rehydration following extreme water loss. Structural analysis points to protein surface chemistry as a key determinant for desiccation tolerance, which we test by showing that rational surface mutants can convert a desiccation sensitive protein into a tolerant one. Desiccation tolerance also has strong overlap with cellular function, with highly tolerant proteins responsible for production of small molecule building blocks, and intolerant proteins involved in energy-consuming processes such as ribosome biogenesis. As a result, the rehydrated proteome is preferentially enriched with metabolite and small molecule producers and depleted of some of the cells heaviest consumers. We propose this functional bias enables cells to kickstart their metabolism and promote cell survival following desiccation and rehydration. TeaserProteins can resist extreme dryness by tuning the amino acids on their surfaces.

biophysics↗

bioRxiv2024

Phosphorylation of disordered proteins tunes local and global intramolecular interactions

Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically disordered protein regions (IDRs) are subject to phosphorylation as a means to modulate their interactions and functions. Toward understanding the relationship between phosphorylation in IDRs and specific functional outcomes, we must consider how phosphorylation affects the IDR conformational ensemble. Various experimental techniques are suited to interrogate the features of IDR ensembles; molecular simulations can provide complementary insights and even illuminate ensemble features that may be experimentally inaccessible. Therefore, we sought to expand the tools available to study phosphorylated IDRs by all-atom Monte Carlo simulations. To this end, we implemented parameters for phosphoserine (pSer) and phosphothreonine (pThr) into the OPLS version of the continuum solvent model, ABSINTH, and assessed their performance in all-atom simulations compared to published findings. We simulated short (< 20 residues) and long (> 80 residues) phospho-IDRs that, collectively, survey both local and global phosphorylation-induced changes to the ensemble. Our simulations of four well-studied phospho-IDRs show near-quantitative agreement with published findings for these systems via metrics including changes to radius of gyration, transient helicity, and persistence length. We also leveraged the inherent advantage of sequence control in molecular simulations to explore the conformational effects of diverse combinations of phospho-sites in two multi-phosphorylated IDRs. Our results support and expand on prior observations that connect phosphorylation to changes in the IDR conformational ensemble. Herein, we describe phosphorylation as a means to alter sequence chemistry, net charge and charge patterning, and intramolecular interactions, which can collectively modulate the local and global IDR ensemble features. SIGNIFICANCESpatially and temporally controlled phosphorylation in disordered protein regions is critical to many facets of protein function and broader cellular health. Intrinsically disordered protein regions (IDRs) are overrepresented as targets of phosphorylation, but the structural and functional consequences of such modifications remain elusive for many systems. Toward rigorous modeling of phosphorylated IDRs using all-atom simulations, we present new parameters for phosphoserine and phosphothreonine for the ABSINTH implicit solvent paradigm. Through the study of four example phospho-IDRs, we demonstrate excellent agreement between our phospho-IDR simulations and published datasets.

biophysics↗

bioRxiv2024

Molecular insights into the interaction between a disordered protein and a folded RNA

Intrinsically disordered protein regions (IDRs) are well-established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, SERF. At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 TAR RNA (TAR) with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation. SIGNIFICANCESubcellular organization through the formation of biomolecular condensates has emerged as an important contributor to myriad cellular functions, with implications in homeostasis, stress response, and disease. To understand the general and specific principles that support condensate formation, we must interrogate the interactions and assembly of their constituent biomolecules. To this end, this study introduces a simple model system comprised of a small, disordered protein and small RNA that undergo charge-driven, associative phase separation. In addition to extensive biophysical characterization of these molecules and their complex, we also generate new insights into mode of interaction and assembly between an unstructured protein and a structured RNA.

biochemistry↗

bioRxiv2024

Direct prediction of intermolecular interactions driven by disordered regions

Intrinsically disordered regions (IDRs) are critical for a wide variety of cellular functions, many of which involve interactions with partner proteins. Molecular recognition is typically considered through the lens of sequence-specific binding events. However, a growing body of work has shown that IDRs often interact with partners in a manner that does not depend on the precise order of the amino acid order, instead driven by complementary chemical interactions leading to disordered bound-state complexes. Despite this emerging paradigm, we lack tools to describe, quantify, predict, and interpret these types of structurally heterogeneous interactions from the underlying amino acid sequences. Here, we repurpose the chemical physics developed originally for molecular simulations to develop an approach for predicting intermolecular interactions between IDRs and partner proteins. Our approach enables the direct prediction of phase diagrams, the identification of chemically-specific interaction hotspots on IDRs, and a route to develop and test mechanistic hypotheses regarding IDR function in the context of molecular recognition. We use our approach to examine a range of systems and questions to highlight its versatility and applicability.

biophysics↗

bioRxiv2024

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponins function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Forster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the {Delta}E160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies. SIGNIFICANCE STATEMENTTroponin plays a central role in regulating heart contraction, and mutations in troponin can cause human cardiomyopathies. There are several functionally-significant regions of troponin that have not been structurally resolved, including the troponin T linker region that contains multiple cardiomyopathy mutations. In these unresolved regions, it is not possible to understand how changes in sequence affect function. We used computational and experimental techniques to demonstrate that this linker is dynamic and intrinsically disordered both in isolation and in the fully regulated thin filament. Moreover, we show how a cardiomyopathy mutation in this region affects function via allosteric disruption of intermolecular interactions. Our results highlight the need to consider how key mutations affect troponin disorder rather than the structure-function relationship.

biophysics↗