Biology subjects

Rozycki, B.

Publications and source records attributed to Rozycki, B..

10 recordsLinked to original sources

bioRxiv2026

Characterization of the N7 RNA cap methyltransferase from Trichomonas vaginalis and inhibitor discovery

RNA cap formation is essential for eukaryotic gene expression, yet the enzymes responsible for cap methylation remain poorly characterized in many eukaryotic parasites, including Trichomonas vaginalis. Here, we functionally and structurally characterized the RNA guanine-N7 methyltransferases (MTases) from T. vaginalis, TvRNMT1 and TvRNMT2. We determined the crystal structure of TvRNMT1 in complex with S-adenosylhomocysteine (SAH) at 1.6 A resolution and additional structures with the inhibitors sinefungin and OBO101, defining the architecture and ligand-recognition properties of its active site. Screening of SAH analogues identified several inhibitors with submicromolar to low-micromolar activity against both homologues. Molecular docking and modelling of capped RNA binding further delineated the inhibitor- and RNA-binding regions of TvRNMT1 and identified residues that may contribute to cap recognition. These results define the molecular basis of ligand recognition by T. vaginalis RNA cap MTases and establish a structural framework for targeting parasite RNA capping.

molecular biology↗

bioRxiv2025

Multi-scale simulations of membrane adhesion mediated by CD47-SIRPα complexes

Adhesion of biological cells is essential for various processes, including tissue formation, immune responses, and signaling. It involves multiple length scales, ranging from nanometers to micrometers, which are characteristic of (a) the intercellular receptor-ligand binding that mediates the cell adhesion, (b) the spatial distribution of the receptor and lignad proteins in the membranes of adhering cells, (c) adhesion-induced deformations and thermal undulations of the membranes, (d) the overall size of the interface between adhering cells. Therefore, computer simulations of cell membrane adhesion require multi-scale modeling and suitable approximations that capture the essential physics of the system under study. Here, we introduce such a multi-scale approach to study membrane adhesion mediated by the CD47-SIRP binding, which is an immunologically relevant process. The synergetic use of coarse-grained molecular dynamics simulations and mesoscale kinetic Monte Carlo simulations allows us to explore both equilibrium properties and dynamical behavior of adhering membranes on the relevant length scales between 1 nm and 1 {micro}m on time scales ranging from 0.1 ns all the way up to about 20 s. The multi-scale simulations not only reproduce available experimental data but also give quantitative predictions on binding-induced conformational changes of SIRP and membrane-mediated cooperativity of the CD47-SIRP binding as well as fluctuation-induced interactions between the CD47-SIRP complexes. Our approach is applicable to various membrane proteins and provides invaluable data for comparison with experimental findings.

biophysics↗

bioRxiv2023

Molecular mechanisms and energetics of lipid droplet formation and directional budding

The formation and budding of lipid droplets (LDs) are known to be governed by the LD size and by membrane tensions in the Endoplasmic Reticulum (ER) bilayer and LD-monolayers. Using coarse-grained simulations of an LD model, we first show that ER-embedded LDs of different sizes can form through a continuous transition from wide LD lenses to spherical LDs at a fixed LD size. The ER tendency to relax its bilayer modulates the transition via a subtle interplay between the ER and LD lipid densities. By calculating the energetic landscape of the LD transition, we demonstrate that this size-independent transition is regulated by the mechanical force balance of ER and LD-tensions, independent from membrane bending and line tension whose energetic contributions are negligible according to our calculations. Our findings explain experimental observation of stable LDs of various shapes. We then propose a novel mechanism for directional LD budding where the required membrane asymmetry is provided by the exchange of lipids between the LD-monolayers. Remarkably, we demonstrate that this budding process is energetically neutral. Consequently, LD budding can proceed by a modest energy input from proteins or other driving agents. We obtain equal lipid densities and membrane tensions in LD-monolayers throughout budding. Our findings indicate that unlike LD formation, LD budding by inter-monolayer lipid exchange is a tension-independent process.

biophysics↗

bioRxiv2023

Protocols for Multi-Scale Molecular Dynamics Simulations in Amber and Gromacs: a Case Study of Intrinsically Disordered Amyloid Beta

Intrinsically disordered proteins (IDPs) present challenges to conventional experimental techniques due to their large-scale conformational fluctuations and the transient occurrence of structural elements. This work illustrates computational methods for studying IDPs at various levels of resolution. The included simulation protocol offers a step-by-step guide on how to conduct molecular dynamics (MD) simulations and analyze the results using the Amber and Gromacs packages, employing both all-atom and coarse-grained approaches. This protocol can be easily adapted to study other biomacromolecules, including folded and disordered proteins and peptides. Furthermore, it is discussed in this work how to perform standard molecular modeling operations, such as amino-acid substitutions (mutagenesis) and insertions of residues missing in a protein structure, as well as how to incorporate post-translational modifications into the simulations, such as disulfide bonds, which are often crucial for proteins to attain their physiologically functional structure. In conventional MD studies, disulfide bonds are typically fixed at the preparation step and remain unchanged throughout the simulations, unable to break or reform. Here, in contrast, a dynamic approach is presented. It involves adequate distance restraints applied to the sulfur atoms of selected cysteine residues, allowing disulfide bonds to break and reform during the simulation. The effectiveness of these methodologies is demonstrated by examining a model IDP, the monomeric form of 1-42 amyloid-{beta} (A{beta}42), both with and without disulfide bonds, at different levels of resolution. This study not only contributes to our understanding of the role of disulfide bonds but also provides detailed simulation protocols that can serve as a foundation for future investigations. SUMMARYGiven the challenges of experimental studies on intrinsically disordered proteins, this manuscript demonstrates step-by-step protocols for conducting all-atom and coarse-grained molecular dynamics simulations using two widespread packages, Amber and Gromacs. The monomeric form of 1-42 amyloid-{beta} (A{beta}42) is used as an example, from which insights into the structure, dynamics and physicochemical properties of this protein can be obtained.

molecular biology↗

bioRxiv2023

Coordination of transporter, cargo, and membrane properties during non-vesicular lipid transport.

Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol-4 phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to their charge and fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.

biophysics↗

bioRxiv2023

Membrane curvature sensing by model biomolecular condensates

Biomolecular condensates (BCs) are fluid droplets that form in biological cells by liquid-liquid phase separation. Their major components are intrinsically disordered proteins. Vast attention has been given in recent years to BCs inside the cytosol and nucleus. BCs at the cell membrane have not been studied to the same extent so far. However, recent studies provide increasingly more examples of interfaces between BCs and membranes which function as platforms for diverse biomolecular processes. Galectin-3, for example, is known to mediate clathrin-independent endocytosis and has been recently shown to undergo liquid-liquid phase separation, but the function of BCs of galectin-3 in endocytic pit formation is unknown. Here, we use dissipative particle dynamics simulations to study a generic coarse-grained model for BCs interacting with lipid membranes. In analogy to galectin-3, we consider polymers comprising two segments - one of them mediates multivalent attractive interactions between the polymers, and the other one has affinity for association with specific lipid head groups. When these polymers are brought into contact with a multi-component membrane, they spontaneously assemble into droplets and, simultaneously, induce lateral separation of lipids within the membrane. Interestingly, we find that if the membrane is bent, the polymer droplets localize at membrane regions curved inward. Although the polymers have no particular shape or intrinsic curvature, they appear to sense membrane curvature when clustered at the membrane. Our results indicate toward a generic mechanism of membrane curvature sensing by BCs involved in such processes as endocytosis.

biophysics↗

bioRxiv2022

Mechanical forces impair antigen discrimination by reducing differences in T cell receptor off-rates

T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic simulations. Consistent with these findings, we show that the best characterized catch-bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination and we suggest this new force-shielding role for the adhesion receptors CD2 and LFA-1. One sentence summaryMechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination

immunology↗

bioRxiv2021

Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to 1 to 3 foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here is relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.

immunology↗

bioRxiv2021

Structural variability and concerted motions of the T cell receptor - CD3 complex

We investigate the structural and orientational variability of the membrane-embedded T cell receptor (TCR) - CD3 complex in extensive atomistic molecular dynamics simulations based on the recent cryo-EM structure determined by Dong et al. (2019). We find that the TCR extracellular (EC) domain is highly variable in its orientation by attaining tilt angles relative to the membrane normal that range from 15{degrees} to 55{degrees}. The tilt angle of the TCR EC domain is both coupled to a rotation of the domain and to characteristic changes throughout the TCR - CD3 complex, in particular in the EC interactions of the C{beta} FG loop of the TCR, as well as in the orientation of transmembrane helices. The concerted motions of the membrane-embedded TCR - CD3 complex revealed in our simulations provide atomistic insights for force-based models of TCR activation, which involve such structural changes in response to tilt-inducing forces on antigen-bound TCRs.

immunology↗

bioRxiv2019

smFRET Detects Dual Binding Modes Modulated by Proline Isomerization in a Mega-Dalton Multi-Enzyme Complex

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗