Biology subjects

Urnavicius, L.

Publications and source records attributed to Urnavicius, L..

10 recordsLinked to original sources

bioRxiv2026

RNA 3D Motif Dynamics Guide Assembly of the Replication Initiation Complex in Flaviviruses

Viral RNA genomes contain structured elements that recruit the machinery necessary for their replication. However, these mechanisms remain poorly understood because of RNA structures dynamic nature. Using cryo-EM and single-molecule Forster resonance energy transfer, we show that the flaviviral stem-loop A (SLA)--which recruits the viral polymerase NS5 to initiate negative-strand synthesis--populates a conserved, multi-state conformational ensemble governed by a dynamic three-dimensional (3D) motif. Structures of Zika and dengue SLA-NS5 complexes reveal that NS5 engages a preorganized SLA conformation. Notably, destabilizing this conformation impairs negative-strand synthesis without affecting NS5 binding. Thus, the conserved SLA 3D ensemble guides a post-binding conformational search that converts an initial encounter complex into a productive replication-initiation complex--revealing how RNA conformational ensembles, rather than static structures, dictate function. SUMMARYViral genomes encode dynamic RNA structures that are specifically recognized by proteins to regulate critical steps of the viral life cycle. Although these RNAs exist as multi-state conformational ensembles, structural studies have largely captured single conformations, limiting our mechanistic understanding of RNA-protein recognition and function. Here, using single-particle cryo-electron microscopy (cryo-EM), single-molecule Forster resonance energy transfer, and biochemical assays, we show how flaviviral genomes use structural dynamics encoded in their stem-loop A (SLA) to guide a conserved, multi-step mechanism of negative-strand synthesis initiation by non-structural protein 5 (NS5). Cryo-EM ensembles of SLAs from dengue, Zika, West Nile, and yellow fever viruses reveal a conserved conformational landscape dictated by alternative stacking configurations of a central three-way junction (3WJ). Cryo-EM structures of SLA-NS5 complexes from dengue and Zika show that NS5 engages a specific state within SLA 3D ensemble, one in which the 3WJ adopts a GRR/A tetraloop-like motif that we identify across diverse structural RNAs. Strikingly, mutations that substantially destabilize this state impair negative-strand synthesis without affecting NS5 binding--decoupling binding from function and ruling out a simple conformational-capture mechanism. Instead, the SLAs intrinsic structural dynamics direct a post-binding conformational search that converts an initial, conformationally heterogeneous encounter complex into a productive replication-initiation complex. These results demonstrate a mechanism of RNA-protein recognition in which the 3D conformational ensemble of an RNA guides formation of the functional state downstream of binding, and identify a ubiquitous, intrinsically dynamic 3D motif within the SLAs 3WJ as a critical determinant of flavivirus negative-strand synthesis.

biochemistry↗

bioRxiv2026

SLC33A1 exports oxidized glutathione to maintain endoplasmic reticulum redox homeostasis

The endoplasmic reticulum (ER) requires an oxidative environment to support the efficient maturation of secretory and membrane proteins. This is in part established by glutathione, a redox-active metabolite present in reduced (GSH) and oxidized (GSSG) forms. The ER maintains a higher GSSG:GSH ratio than the cytosol; however, the mechanisms controlling ER redox balance remain poorly understood. To address this, we developed a method for the rapid immunopurification of the ER, enabling comprehensive profiling of its proteome and metabolome. Combining this approach with CRISPR screening, we identified SLC33A1 as the major ER GSSG exporter in mammalian cells. Loss of SLC33A1 leads to GSSG accumulation in the ER and a liposome-based assay demonstrates that SLC33A1 directly transports GSSG. Cryo-EM structures and molecular dynamics simulations reveal how SLC33A1 binds GSSG and identify residues critical for its transport. Finally, an imbalance in GSSG:GSH ratio induces ER stress and dependency on the ER-associated degradation (ERAD) pathway, driven by a shift in protein disulfide isomerases (PDIs) toward their oxidized forms. Altogether, our work establishes SLC33A1-mediated GSSG export as a key mechanism for ER redox homeostasis and protein maturation.

cell biology↗

bioRxiv2025

Antibody-mediated feedback modulates interclonal competition in the germinal center

Serum antibodies from prior immune responses regulate B cell activation and germinal center (GC) access upon recall immunization. However, how antibodies produced by an ongoing immune response influence the outcomes of contemporaneous GCs is less clear. To explore this, we developed mouse models enabling the targeted ablation of plasma cells and antibodies produced by an immune response of interest, without affecting those produced homeostatically or by prior antigen encounters. Our findings show that, whereas antibody-mediated feedback is not required for affinity maturation, it can influence competition between B cells with different epitope specificities, specifically by reducing the abundance of clones that recognize the same epitopes as circulating antibodies. This modality of feedback represents a mechanism by which antibody responses can influence epitope specificity in ongoing GCs. These findings may therefore have implications for vaccination strategies aimed at steering clonal selection towards desired epitopes on complex antigens.

immunology↗

bioRxiv2023

Structure of the γ-tubulin ring complex-capped microtubule

Microtubules are composed of /{beta}-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale /{beta}-tubulin tracks for intracellular transport that align with, rather than spiral along, the filaments long-axis. We report that the human [~]2.3MDa {gamma}-tubulin ring complex ({gamma}-TuRC), an essential regulator of microtubule formation that contains 14 {gamma}-tubulins, selectively nucleates 13-protofilament microtubules. Cryo-EM reconstructions of {gamma}-TuRC-capped microtubule minus-ends reveal the extensive intra- and inter-domain motions of {gamma}-TuRC subunits that accommodate its actin-containing luminal bridge and establish lateral and longitudinal interactions between {gamma}- and -tubulins. Our structures reveal how free {gamma}-TuRC, an inefficient nucleation template due to its splayed conformation, transforms into a stable cap that blocks addition or loss of /{beta}-tubulins from minus-ends and sets the lattice architecture of cellular microtubules. One Sentence SummaryStructural insights into how the {gamma}-tubulin ring complex nucleates and caps a 13-protofilament microtubule.

biochemistry↗

bioRxiv2023

Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to [~]3-fold. Our cryo-EM studies resulted in structures ([~]2.9-3.5 [A]-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry. SignificanceThe loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCPs ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.

biochemistry↗

bioRxiv2020

Targeting Allostery in the Dynein Motor Domain with Small Molecule Inhibitors

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dyneins unusually large size, four distinct nucleotide-binding sites, and the existence of closely-related isoforms with different functions, pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with single molecule experiments and mutagenesis studies, indicate that the compounds likely inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dyneins main catalytic site in the AAA1 domain. A double Walker B mutant in AAA3 and AAA4 is an inactive enzyme, suggesting that inhibiting these regulatory sites can have a similar effect to inhibiting AAA1. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dyneins AAA domains.

biochemistry↗

bioRxiv2020

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The formation of cellular microtubule networks is regulated by the{gamma} -tubulin ring complex ({gamma}-TuRC). This [~]2.3 MDa assembly of >31 proteins includes{gamma} -tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge". The challenge of reconstituting the{gamma} -TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human{gamma} -TuRC ({gamma}-TuRC-GFP), a [~]35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex,{gamma} -TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that{gamma} -TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native{gamma} -TuRCs. Electron microscopy reveals that{gamma} -TuRC-GFP resembles the native{gamma} -TuRC architecture, while{gamma} -TuRCmini-GFP adopts a partial cone shape presenting only 8-10{gamma} -tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the{gamma} -TuRC.

biochemistry↗

bioRxiv2019

Asymmetric molecular architecture of the human γ-tubulin ring complex

The{gamma} -tubulin ring complex ({gamma}-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation 1-4. Metazoan{gamma} -TuRCs isolate as [~]2 MDa complexes containing the conserved proteins{gamma} -tubulin, GCP2 and GCP3, as well as the expanded subunits GCP4, GCP5, and GCP6 3,5,6. However, in current structural models,{gamma} -TuRCs assemble solely from subcomplexes of{gamma} -tubulin, GCP2 and GCP3 7. The role of the metazoan-specific subunits in{gamma} -TuRC assembly and architecture are not currently known, due to a lack of high resolution structural data for the native complex. Here, we present a cryo-EM structure of the native human{gamma} -TuRC at 3.8[A] resolution. Our reconstruction reveals an asymmetric, single helical-turn and cone-shaped structure built from at least 34 polypeptides. Pseudo-atomic models indicate that GCP4, GCP5 and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes and are distal to the{gamma} -TuRC \"seam\". Evolutionary expansion in metazoan-specific subunits diversifies the{gamma} -TuRC by introducing large (>100,000 [A]2) surfaces that could interact with different regulatory factors. We also identify an unanticipated structural bridge that includes an actin-like protein and spans the{gamma} -TuRC lumen. Despite its asymmetric composition and architecture, the human{gamma} -TuRC arranges{gamma} -tubulins into a helical geometry poised to nucleate microtubules. The observed compositional complexity of the{gamma} -TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g. within biological condensates 8 or alongside existing filaments 9.

biochemistry↗

bioRxiv2018

The CryoEM Structure of the Ribosome Maturation Factor Rea1

The biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an -helical bundle of AAA2 as a major ATPase activity regulator. The -helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

molecular biology↗

bioRxiv2017

Cryo-EM shows how dynactin recruits two dyneins for faster movement

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased toward recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double dynein complex increases both force and speed. A 3.5 [A] cryo-EM reconstruction of a dynein tail/dynactin/BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side by side. Our work provides a structural basis for how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein/dynactin transport machine.

molecular biology↗