Biology subjects

Dym, O.

Publications and source records attributed to Dym, O..

14 recordsLinked to original sources

bioRxiv2025

Systematic DNA nicking reveals the structural logic of protein recognition

Transcription factors (TFs) bind to specific genomic sites to regulate gene expression1,2. These interactions almost universally require DNA deformation and the accumulation of local mechanical strain within the double helix. As a result, TF-DNA recognition is determined not only by the linear base sequence but also by the spatial alignment of bases and phosphates, as well as their ability to adopt and retain structural deformations3. However, the sequence-centric focus of existing studies makes it challenging to directly probe DNA structural determinants and to decouple their impact from alterations in base sequences, limiting our ability to unravel the key factors influencing binding beyond the sequence identity and leaving significant gaps in our understanding of the principles governing TF-DNA recognition. Here, we introduce a high-throughput strategy to perturb TF binding sites without altering their base sequence, enabling systematic investigation of the structural features of DNA that govern TF binding. Our method, PIC-NIC, introduces single-strand breaks (SSBs) at every position within the binding site, selectively disrupting backbone continuity while preserving nucleotide identity, with the resulting effects on TF binding measured quantitatively. Applied to 15 human TFs spanning eight structural classes, and supported by seven high-resolution TF-DNA crystal structures and molecular dynamics simulations, PIC-NIC uncovers discrete backbone positions serving as structural anchor points where nicks can abolish binding, rewire sequence preferences, or even enhance affinity. By decoupling structural and chemical contributions, we demonstrate that DNA mechanics--encoded in backbone geometry and continuity--can independently shape binding specificity beyond the linear code of base identity. These findings shift the paradigm of TF-DNA recognition, establishing the backbone not as a passive scaffold, but as a functional determinant capable of directing regulatory mechanisms through its physical architecture.

biophysics↗

bioRxiv2025

High-efficiency Kemp eliminases by complete computational design

We present a fully computational workflow for de novo design of efficient enzymes using backbone fragments from natural proteins and without recourse to iterative experimental optimization. The best designed Kemp eliminase exhibits >140 mutations from any natural protein, high stability (>85 {degrees}C) and unprecedented catalytic efficiency (12,700 M-1s-1), surpassing previous computational designs by two orders of magnitude. We find that mutations both inside and outside the active site contribute synergistically to the high observed activity and stability. Mutation of an aromatic residue used in all prior Kemp eliminase designs increases efficiency to >105 M-1s-1. Our approach addresses critical limitations in design methodology, generating stable, high-efficiency, new-to-nature enzymes in complex folds and enables testing hypotheses on the fundamentals of biocatalysis through a limited experimental effort.

biochemistry↗

bioRxiv2024

An unconventional interaction interface between the peroxisomal targeting factor Pex5 and Eci1 enables PTS1 independent import

Accurate and regulated protein targeting to organelles is crucial for eukaryotic cellular function and homeostasis. This has driven the evolution of targeting signals on proteins and the targeting factors that recognize them. One example for this is peroxisomal matrix proteins, the majority of which rely on the targeting factor Pex5 to correctly localize and function. While most Pex5 cargos contain a Peroxisomal Targeting Signal type 1 (PTS1), in recent years it has become clear that more binding interfaces exist, and that targeting by Pex5 is more complex than previously thought. Here, we uncover that the matrix protein Eci1 can reach peroxisomes in the absence of its PTS1. By solving the structure of a complex between full length yeast Pex5 and Eci1 using Cryo-Electron Microscopy, we could identify their binding interfaces. This allowed us to map an additional binding interface that is independent of the canonical PTS1-mediated binding site. Our work brings forward a solution to a long-standing mystery regarding Eci1 targeting to peroxisomes. More globally, it demonstrates the intricate and complex nature of organelle targeting and how it has evolved to serve the complex eukaryotic environment.

cell biology↗

bioRxiv2024

Cathepsin X is a conserved cell death protein involved in algal response to environmental stress

Phytoplankton play a crucial role in global primary production and can form vast blooms in aquatic ecosystems. Bloom demise and the rapid turnover of phytoplankton are suggested to involve programmed cell death (PCD) induced by diverse environmental stressors. However, fundamental knowledge of the PCD molecular components in algae and protists in general remains elusive. Previously, we revealed that early oxidation in the chloroplast predicted subsequent cell death or survival in isogenic subpopulations that emerged following H2O2 treatment in the diatom Phaeodactylum tricornutum. Here, we performed transcriptome analysis of sorted sensitive oxidized cells and resilient reduced cells, to discover genes linked to their contrasting fates. By cross-comparison with a large-scale mutant screen in the green alga Chlamydomonas reinhardtii, we identified functionally relevant conserved PCD gene candidates, including the cysteine protease cathepsin X/Z (CPX). CPX mutants in P. tricornutum CPX1 and C. reinhardtii CEP12 both exhibited profound resilience to oxidative stress, supporting a conserved function in algal PCD. P. tricornutum cpx1 mutants, generated using CRISPR-Cas9, also exhibited resilience to the toxic diatom-derived infochemical cyanogen bromide. Phylogenetic and predictive structural analyses show that CPX is highly conserved in eukaryotes, and algae of the green and red lineages exhibit strong structural similarity to human cathepsin CTSZ. CPX is expressed by diverse algae across the oceans and during toxic Pseudo-nitzschia blooms, supporting its ecological importance. Elucidating PCD components in algae sheds light on the evolutionary origin of PCD in unicellular organisms, and on the cellular strategies employed by the population to cope with stressful conditions.

microbiology↗

bioRxiv2023

The impact of molecular variants, crystallization conditions and space group on structure-ligand complexes: A case study on Bacterial Phosphotriesterase Variants and complexes

While attempting to study the 3D structure of proteins with bound ligands, one often encounters considerable difficulties. We illustrate, as an example, the bacterial enzyme phosphotriesterase and specifically examine the effects of multiple factors such as the molecular constructs, ligands used during protein expression and purification, crystallization precipitance, and space group on the visualization of molecular complexes of organophosphate ligands bound to the enzyme. We analyzed twelve crystal structures of the different phosphotriesterase constructs derived by directed evolution in both apo and holo forms (in complex with organophosphate analogs), with resolutions up to 1.38 [A]. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing molecular complexes of ligands bound to the enzyme. The study revealed that residual tags used for protein expression can lodge in the active site and hinder ligand binding. Additionally, the space groups in which the proteins are crystallized can significantly impact the visualization of the organophosphate ligands bound to the phosphotriesterase. The study also reveals that the crystallization precipitants can compete with and even preclude ligand binding, leading to false positives or the incorrect identification of lead drug candidates, which is particularly crucial for ligands with pharmacological and toxicological contexts. Overall, this study provides valuable insights into the challenges and considerations involved in studying the 3D structure of proteins with bound ligands, highlighting the importance of careful experimental design and rigorous data analysis to ensure the accuracy and reliability of the resulting protein-ligand structures.

biochemistry↗

bioRxiv2023

Stable mammalian serum albumins designed for bacterial expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40{degrees}C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/534334v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@71ef93org.highwire.dtl.DTLVardef@27a975org.highwire.dtl.DTLVardef@1e657fforg.highwire.dtl.DTLVardef@19b3de3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Computational design stabilized human and bovine serum albumins - Designs express solubly in E. coli and exhibit up to 40 {degrees}C increased thermostability - Some designs exhibit identical ligand binding properties - Crystal structure confirms design accuracy - Designs can be used in cell culture and in vitro applications

biochemistry↗

bioRxiv2022

Phylogeny and biogeography of the algal DMS-releasing enzyme

Phytoplankton produce the volatile dimethyl sulfide (DMS), an important infochemical, which is emitted to the atmosphere and affecting the global climate. Albeit the enzymatic source for DMS in eukaryotes was elucidated, namely a DMSP lyase (DL) called Alma1, we still lack basic knowledge regarding its taxonomy and biogeographic distribution. We defined unique sequence motifs which enable the identification of DL homologs (DLHs) in model systems and environmental populations. We used these motifs to predict DLHs in diverse algae by analyzing hundreds of genomic and transcriptomic sequences from model systems under stress conditions and from environmental samples. Our findings show that the DL enzyme is more taxonomically widespread than previously thought, as it is encoded by known algal taxa as haptophytes and dinoflagellates, but also by chlorophytes, pelagophytes and diatoms, which were conventionally considered to lack the DL enzyme. By exploring the Tara Oceans database, we showed that DLHs are widespread across the oceans and are predominantly expressed by dinoflagellates. Certain dinoflagellate DLHs were differentially expressed between the euphotic and mesopelagic zones, suggesting a functional specialization and an involvement in the metabolic plasticity of mixotrophic dinoflagellates. In specific regions as the Southern Ocean, DLH expression by haptophytes and diatoms was correlated with environmental drivers such as nutrient availability. The expanded repertoire of putative DL enzymes from diverse microbial origins and geographic niches suggests new potential players in the marine sulfur cycle and provides a foundation to study the cellular function in marine microbes.

microbiology↗

bioRxiv2022

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. Mutation of multiple conserved residues leads to increased substrate inhibition, suggesting a positive selection during evolution. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity. Significance StatementHow conformational dynamics affect the catalytic activity of enzymes remains a topic of active debate. We focus here on the domain closure dynamics of adenylate kinase (AK) and how they are affected by substrate inhibition. By screening an extensive mutant library, we show that this feature of the enzyme is well conserved in evolution. Importantly, domain closure is required in order to bring AKs substrates close together for their chemical reaction; single-molecule FRET studies directly measure the populations of the open and closed states. We find that overpopulation of the closed state can be detrimental to activity. The results allow us to develop a kinetic model that properly accounts for AK kinetics by combining conformational dynamics and biochemical steps.

biophysics↗

bioRxiv2022

gUMI-BEAR, a modular, unsupervised population barcoding method to track variants and evolution at high resolution

Cellular lineage tracking provides a means to observe population makeup at the clonal level, allowing exploration of heterogeneity, evolutionary and developmental processes and individual clones relative fitness. It has thus contributed significantly to understanding microbial evolution, organ differentiation and cancer heterogeneity, among others. Its use, however, is limited because existing methods are highly specific, expensive, labour-intensive, and, critically, do not allow the repetition of experiments. To address these issues, we developed gUMI-BEAR (genomic Unique Molecular Identifier Barcoded Enriched Associated Regions), a modular, cost-effective method for tracking populations at high resolution. We first demonstrate the systems application and resolution by applying it to track tens of thousands of Saccharomyces cerevisiae lineages growing together under varying environmental conditions applied across multiple generations, revealing fitness differences and lineage-specific adaptations. Then, we demonstrate how gUMI-BEAR can be used to perform parallel screening of a huge number of randomly generated variants of the Hsp82 gene. We further show how our method allows isolation of variants, even if their frequency in the population is low, thus enabling unsupervised identification of modifications that lead to a behaviour of interest.

evolutionary biology↗

bioRxiv2022

PGRL1A redox states alleviate photoinhibition in Arabidopsis during step changes in light intensity

Non-motile plants have evolved regulatory mechanisms to maintain homeostasis for optimal growth. Responses to environmental changes in light are particularly important not only during the diurnal transition from night to day but also to react to light changes caused by passing clouds or by wind. Thioredoxins rapidly orchestrate redox control during environmental change by modifying cysteine residues. Here, we assign a function to regulatory cysteines of PGRL1A, a constituent of the ferredoxin-dependent cyclic electron flow (Fd-CEF) pathway and show their role in the regulation of proton motive force (PMF) and nonphotochemical quenching (NPQ). During step increase of low light intensity (10-60 E*m-2*s-1), the intermolecular disulfide of the PGRL1A 59-kDa complex is reduced transiently within seconds to the 28 kDa form. In contrast, step increases to higher light intensity (60-600 E*m-2*s-1) stimulated a stable partially reduced redox state in PGRL1A. Measurements of NPQ, PMF and resultant photosynthetic controls Y(ND) and Y(NA) were found to correlate with the redox state of PGRL1A during step increases in light intensity but not in PGRL1mutant plants pgrl1ab or PGRL1A cysteine mutant (PGRL1AC1,2A). Continuous light regimes did not affect mutant growth; however, fluctuating regimes of light intensity showed significant growth reduction in the mutants. Inhibitors of photosynthesis placed control of the PGRL1A redox state as dependent on the penultimate ferredoxin redox state that fuels reducing equivalents to the large set of chloroplasts thioredoxins. Our results showed that redox state changes in PGRL1A are crucial to the optimization of photosynthesis and are regulated by the photosynthetic electron flux.

plant biology↗

bioRxiv2021

SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor

SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the "European" (20E-EU1), "British" (501.V1),"South African" (501.V2), and Brazilian" variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9[A] Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

biochemistry↗

bioRxiv2020

Bringing BOS to light: Uncovering the key enzyme in the biosynthesis of the neurotoxin β-ODAP in Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, {beta}-N-oxalyl-L-,{beta}-diaminopropionic acid ({beta}-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of {beta}-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme {beta}-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

plant biology↗

bioRxiv2020

A molecular lid mechanism for K+ channel blockers revealed by a cone peptide

Many venomous organisms carry in their arsenal short polypeptides that block K+ channels in a highly selective manner. These toxins may compete with the permeating ions directly via a “plug” mechanism or indirectly via a “pore-collapse” mechanism. An alternative “lid” mechanism was proposed but remained poorly defined. Here we study the block of the Drosophila Shaker channel by Conknunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and in biophysical assays, implying discrete modes of action. We show that while Conknunitzin-S1 binds tightly to the channel turret and acts via a “pore-collapse” mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a “molecular-lid”. Our study provides an atomic description of the “lid” K+ blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Pore-modulating toxins exploit inherent slow inactivation to block K+ channels

Voltage dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified to \"pore-blockers\" that physically plug the ion conduction pathway and \"gating modifiers\" that disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here we show that Cs1, a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

pharmacology and toxicology↗