Biology subjects

Hernychova, L.

Publications and source records attributed to Hernychova, L..

8 recordsLinked to original sources

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Open or closed? Exploring the conformational heterogeneity of human plasminogen across multiple resolution scales

Plasminogen (Plg), the zymogen of the fibrinolytic protease plasmin, is a multidomain, conformationally-rich protein that plays a crucial role in targeted thrombolysis for stroke and heart attack treatments. Yet, the conformational state that affects binding to Plg activators and fibrinolytic activity is rarely validated, contributing to poor reproducibility and failures in drug development. Here, we establish a structural biology workflow to characterize human Plg suspended in an aqueous solution, preserving it in a near-native state and eliminating the need for crystal growth. Two variants, cleavage-resistant Plg-RV (R561A) and catalytically inactive Plg-CAT (S741A), were expressed in mammalian cells. Both variants were correctly folded and stable (Tm {approx} 60.6{degrees}C) and, unlike commercial plasma-derived Plg, were resistant to staphylokinase-mediated activation. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry revealed substantial conformational heterogeneity. The recombinantly-produced Plg variants adopted a closed conformation, exhibiting a good fit to the closed structure determined by X-ray diffraction. Conversely, the plasma-derived Plg populated an extended, open-like state. Cryo-EM analysis of the Plg-RV variant yielded a 4.4 [A] resolution map, and a rigid-body-fitted model revealed the closed-state architecture. Our findings demonstrate that rigorous structural validation of Plg is essential for future functional studies and rational development of next-generation thrombolytic agents.

biochemistry↗

bioRxiv2026

Uncovering Functional Distant Mutations by Ultra-High-Throughput Screening of Dehalogenases

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Investigating the Conformational Flexibility of Staphylokinase Across Multiple Time Scales

Cardiovascular diseases, including ischemic stroke, necessitate improved thrombolytic agents. A microbe-encoded plasminogen activator staphylokinase (SAK) is a promising alternative to the widely used tissue plasminogen activator (tPA) due to its high fibrin specificity and low production cost. To overcome potential immunogenicity hampering its use in clinical settings, the low-immunogenic variants SAK SY155 and SAK THR174 were previously engineered. However, the molecular basis underlying their reduced immunogenicity is not understood and requires detailed elucidation. Here, we determine molecular structures and compare flexibility between low-immunogenic and immunogenic SAK variants, using a combination of experimental and computational structural techniques. Our analyses show that all variants share the canonical SAK fold and retain similar plasminogen activation kinetics, despite the number of introduced substitutions. Crucially, the low-immunogenic variants exhibit distinct flexibility profiles, with SAK THR174 showing substantially increased flexibility in the H1 helix and B3 region. SAK SY155 exhibits an increased flexibility in the H1-B3 loop and propensity to homodimerize. These flexibility changes are found in the known immunogenic epitopes. Our multi-scale flexibility analysis provides the molecular explanation for the reduced immunogenicity, altered thermostability, and retained fibrinolytic function of the engineered variants. This information is critical for the design of next-generation thrombolytics.

biochemistry↗

bioRxiv2025

Taurine Inhibits Apolipoprotein E4 Aggregation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor in many neurodegenerative diseases, yet effective therapeutic strategies targeting its associated pathologies remain unresolved. The aggregation of ApoE4, a key pathological feature, can be attenuated by tramiprosate and its metabolite 3-sulfopropanoic acid. In this study, we investigated the potential of taurine, a close chemical analogue of tramiprosate, to modulate ApoE4-mediated pathological processes. Using an integrated approach--including molecular dynamics simulations, static light scattering, mass spectrometry, and cerebral organoid models--we investigated taurines effects on ApoE4 aggregation. We found that taurine effectively inhibits ApoE4 aggregation. Notably, taurine significantly ameliorates the pathophysiological characteristics of ApoE4, bringing its phenotype closer to the more benign ApoE3 variant. By leveraging its neuroprotective properties, taurine may offer effects comparable to tramiprosate and 3-sulfopropanoic acid, positioning it as an accessible and promising candidate for mitigating neurodegeneration, particularly in individuals with the high-risk ApoE4/E4 genotype.

biochemistry↗

bioRxiv2023

Structural insights into inherited anemia CDA-I: disease-associated mutations disrupt CDIN1-Codanin1 complex

Congenital dyserythropoietic anemia type I (CDA-I) is a rare hereditary disease marked by ineffective erythropoiesis, a characteristic spongy heterochromatin structure in erythroblasts, and mutations in the genes CDAN1 and CDIN1, which encode the proteins Codanin1 and CDIN1. Codanin1 regulates histone shuttling via the chaperone ASF1, yet the role of CDIN1 in CDA-I pathology remains unclear. Notably, CDIN1 is known to interact directly with the C-terminus of Codanin1. Although mutations in both genes are critical to the disease phenotype, their molecular-level effects have not been fully elucidated. Here, we present a comprehensive structural and functional analysis of the CDIN1-Codanin1 C-terminus complex. Using complementary biophysical techniques, we show that CDIN1 and Codanin1 C-terminus form a high-affinity heterodimeric complex with equimolar stoichiometry. We further delineate the essential interacting regions of CDIN1 and Codanin1. We demonstrate that CDA-I-associated mutations in either protein disrupt CDIN1-Codanin1 interaction, suggesting a potential molecular mechanism underlying the disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/542057v3_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ca4364org.highwire.dtl.DTLVardef@e74ccorg.highwire.dtl.DTLVardef@1160f92org.highwire.dtl.DTLVardef@158536b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Domino-like Effect of C112R Mutation on APOE4 Aggregation and Its Suppression by Alzheimer's Disease Drug Candidate

BackgroundApolipoprotein E (ApoE) {varepsilon}4 genotype is the most prevalent risk factor for late-onset Alzheimers Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. MethodsHere, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE {varepsilon}3/{varepsilon}3 and {varepsilon}4/{varepsilon}4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. ResultsWe found that C112R substitution in ApoE4 induces long-distance (>15 [A]) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE {varepsilon}4/{varepsilon}4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. ConclusionsOur results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

neuroscience↗

bioRxiv2022

SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression

IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFN{gamma} stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFN{gamma}. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins. SignificanceIFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.

cancer biology↗