Biology subjects

Efremov, R. G.

Publications and source records attributed to Efremov, R. G..

16 recordsLinked to original sources

bioRxiv2026

An ancient receptor family illuminates the evolution of animal sensation

Animals use nervous systems to sense and respond to their environment. Yet single-celled organisms can also detect cues to execute diverse behaviors, suggesting that core components for animal sensation predate multicellularity and nervous systems. Here, we report that choanoflagellates, the closest living animal relatives, use an ancient sensory receptor family to detect bacterial prey. These receptors are related to transient receptor potential ion channels but are distinguished by WD40 domains, defining TRPW. TRPW1 detects specific bacterial lipids to modulate flagellar beating, providing a mechanism for attraction towards prey. TRPW emerged in early eukaryotes and reveals ancestral architectural and ligand-binding features that predate animal somatosensory receptors. In multicellular choanoflagellates, TRPW1 elicits collective responses, linking bacterial ecology to the evolution of receptors, sensory organelles, and multicellular life.

evolutionary biology↗

bioRxiv2025

Immune Antibodies Recognizing the Stem Region of SARS-CoV-2 Spike Protein: Molecular Modelling and In Vitro Study of Synthetic Peptides Presentation to the Antibodies

Antibodies to peptide 1147 (amino acids 1147-61) of the SARS-CoV-2 protein S are highly diagnostic. Peptide 1147, although located in a region that is partly spatially hidden in the intact protein, is not subject to mutations, suggesting therapeutic potential. The aim of this study was to elucidate the architecture of this region and the way in which it is presented to antibodies. As a model system, this peptide carrying a single lipophilic tail and the same peptide carrying a lipophilic tail at both ends (pseudocyclic) were incorporated into lipid membrane. Isolated anti-1147 antibodies interacted with it regardless of how the peptide was presented, be that freely exposed via the N-terminus, organized as a pseudocycle, or adsorbed on the surface. MDS showed that peptide 1147 is capable of closely approaching the membrane. Analysis of the surface properties of peptide 1147 in membrane-bound states and in available conformations in the full-sized S protein reveals interface for interaction with antibodies. Interestingly, the latter bears similarities to one published peptide-antibody complex. However, these antibodies, in spite of their high diagnostic significance, show no virus-neutralizing activity, indicating that peptide 1147 has no therapeutic value as a synthetic vaccine.

immunology↗

bioRxiv2025

MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

Single particle cryo-EM enables reconstructing near-atomic or even atomic resolution 3D maps of proteins by visualizing thousands to a few million purified protein particles embedded in nanometer- thick vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of one of the most sensitive analytical methods that deliver a high-resolution protein structure as a readout. In practice, more than a million times more starting biological material is required to prepare cryo-EM grids. To close the gap, we developed a micro isolation (MISO) method that combines microfluidics-based protein purification with cryo-EM grid preparation. We validated the method on soluble bacterial and eukaryotic membrane proteins. We showed that MISO enables protein structure determination starting from below one microgram of a target protein and going from cells to cryo-EM grids within a few hours. This scales down the purification by a factor of a few hundred to a few thousand and opens possibilities for the structural characterization of hitherto inaccessible proteins.

biophysics↗

bioRxiv2024

Lipids modulate open probability of RyR1 under cryo-EM conditions

Ryanodine receptors (RyRs) are intracellular tetrameric ion channels responsible for Ca2+ release from the sarcoplasmic and endoplasmic reticulum. Among the three known mammalian RyR isoforms, RyR1 is critical for muscle contraction and has been studied most extensively. The cytoplasm-exposed multidomain fragment of RyRs integrates multiple cellular signals that regulate channel gating and small deviations from the physiological open probability of RyRs leads to life-threatening diseases. While cryo-EM has been instrumental in revealing near-atomic details of RyR gating mechanisms, the open probability of RyR1 under cryo-EM conditions is notably lower than that observed in electrophysiological studies, complicating structural investigations of RyR1 gating modulation. Here, we present a cryo-EM study examining the open probability of RyR1 solubilized in CHAPS with varying lipid concentrations. We found that increasing lipid concentration from 0.001% to 0.05% raised the RyR1 open probability from 16 to 84%; however, RyR1 reconstituted into lipid nanodiscs remained closed. We modelled 72 lipid molecules in the map reconstructed at the highest lipid concentration. These findings demonstrate critical role of lipids in modulating RyR1 gating under cryo-EM conditions and suggest optimal lipid-mimetics for structural studies of RyR1 gating modulation.

biochemistry↗

bioRxiv2024

Lipid specificity of action of SARS-CoV-2 fusion peptide fragments on model membranes

The study focuses on investigating the interaction of SARS-CoV-2 fusion peptide fragment with model membranes of various lipid composition to elucidate the molecular mechanisms of peptide-derived membrane fusion. The work utilized the short fragment of SARS-CoV-2 fusion peptide which is homologous to 816-827 region of the native SARS-CoV-2 FP (FP816-827) and contains the highly conserved LLF motif responsible for membrane fusion, and its ineffective analogue (mFP816-827), where LLF motif was replaced for AAA. Using fluorescence fusion assay, it was demonstrated that the LLF motif plays a key role in inducing liposome fusion, whereas its replacement completely abolishes this capability. The fusogenic activity of the peptide strictly depended on the vesicle lipid composition. It was potentiated by phosphatidylethanolamine and inhibited by phosphatidylserine. Molecular dynamics revealed that both peptides predominantly adopt an -helical conformation; however, the native peptide interacts more strongly with the hydrophobic core of the membrane by increasing peptide-lipid hydrophobic contacts, while the mutant version exhibits a more superficial localization. Differential scanning microcalorimetry data indicated that the ability of FP816- 827 to disturb lipid packing increased with decreasing membrane lipid tail length. The molecular mechanisms underlying the fusogenic activity of the SARS-CoV-2 fusion peptide were identified, specifically its ability to cluster phospholipid head groups in its own vicinity. As a result, local regions with positive spontaneous curvature are formed in the outer monolayer, facilitating membrane fusion. The findings highlight the role of membrane composition and lipid architecture in the mechanism of viral fusion with host cells.

biophysics↗

bioRxiv2024

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs were identified in mammals, disfunction of which was associated with a series of life-threatening diseases. Advances in structural studies of RyRs are limited by the need for large amounts of native tissue or eukaryotic cell cultures. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification starting from isolated membranes to cryo-EM grade protein is completed within four hours on the bench and produces protein usable for cryo-EM as we show by solving the structures of rabbit RyR1 and bovine and mouse RyR2 solubilized in detergent, reconstituted into lipid nanodiscs or liposomes. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in the cases where the amount of starting material is limited.

biochemistry↗

bioRxiv2023

Structure of γ-secretase (PSEN1/APH-1B) in complex with Aβ46 provides insights into amyloid-β processing and modulation by the APH-1B isoform

Deposition of amyloid-{beta} (A{beta}) peptides in the brain is a hallmark of Alzheimers disease. A{beta}s are generated through sequential proteolysis of the amyloid precursor protein by the {gamma}-secretase complexes (GSECs). A{beta} peptide length, which is modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimers pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of A{beta}, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate A{beta}46 substrate. We found a divergent APH-1 loop to be involved with PSEN1 in substrate-binding-induced concerted rearrangements. Upstream the catalytic site, A{beta}46 structure is similar to the endopeptidase substrates and is stabilised by polar interactions including a previously unseen interaction with PSEN1 loop1. The hybrid {beta}-sheet was not observed downstream the catalytic site.

molecular biology↗

bioRxiv2023

Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the first cryo-EM structure of a lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a clockwise quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.

biochemistry↗

bioRxiv2022

Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting

Using single particle cryogenic electron microscopy (cryo-EM) high-resolution structures of proteins in different conformations can be reconstructed. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid initiation of reaction and rapid plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a spatial resolution of 2.1 [A]. We performed trEM experiments on GroEL:GroES chaperonin complex, these resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.

biophysics↗

bioRxiv2022

On a mechanistic impact of transmembrane tetramerization in pathological activation of RTKs

Constitutive activation of receptor tyrosine kinases (RTKs) via different mutation has a strong impact into development of severe human disorders, including cancer. While pathological effect of such mutations can be common on the phenotypical level, mechanistic understanding of their contribution into the receptor activation depends on the exact molecular context. Mutations in transmembrane (TM) domains represent an interesting class of pathological modifications since they can directly affect a signal transduction pathway from the receptor to the kinase domains of RTKs. Here we propose a putative activation scenario of RTK, whereby TM mutations can also promote higher order oligomerization of the receptors that leads to the subsequent ligand-free activation. To illustrate this model with all-atom resolution for a previously characterized oncogenic TM mutation V536E in platelet-derived growth factor receptor alpha (PDGFRA), we use a computational modeling framework including sequence-based structure prediction and all-atom 1 {micro}s molecular dynamics (MD) simulations in a model membrane for the predicted configuration of the PDGFRA TM tetramers. We show that in the course of MD simulations the mutant tetramer retains stable and compact configuration, which is strengthened by tight protein-protein interactions. In contrast, the wild type tetramer demonstrates looser packing and tendency to dissociate. Such a structural organization shapes the dynamics of TM helices in the oligomeric state. Specifically, the mutation affects the characteristic motions of mutated TM helical segments by introducing additional non-covalent crosslinks in the middle of the TM tetramer, which work as mechanical hinges. This leads to dynamic decoupling of the C-termini from the rigidified N-terminal parts and facilitates higher possible displacement between the C-termini of the mutant TM helical regions. This, in turn, can provide more freedom to downstream kinase domains in their mutual rearrangement. The observed structural and dynamic effects of the V536E mutations in the context of PDGFRA TM tetramer provide an interesting possibility that an effect of oncogenic TM mutations can go beyond alternating structure and dynamics of TM dimeric states and also promote formation of higher-order oligomers that may directly contribute into the ligand independent signaling of PDGFRA and other RTKs.

biophysics↗

bioRxiv2022

AFM-based force spectroscopy unravels the stepwise-formation of a DNA transposition complex driving multi-drug resistance dissemination

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. So far, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds to a single transposon end and then undergoes a structural transition that enables it to bind the second end co-operatively and to become activated for transposition catalysis. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.

biophysics↗

bioRxiv2022

A uniquely stable trimeric model of SARS-CoV-2 spike transmembrane domain

The spike (S) protein of SARS-CoV-2 effectuates membrane fusion and virus entry into target cells. Its transmembrane domain (TMD) represents a homotrimer of -helices anchoring the spike in the viral envelope. Although S-protein models available to date include the TMD, its precise configuration was given brief consideration. Understanding viral fusion entails realistic TMD models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD (S-TMD) based solely on its primary structure. First, we performed amino acid sequence pattern matching and compared molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and thus selected the TMD of the tumour necrosis factor receptor 1 (TNFR-1) for subsequent template-based modelling. We then iteratively built an all-atom homotrimer model of S-TMD based on "dynamic MHP portraits" and residue variability motifs. In this model each helix possessed two overlapping interfaces interacting with either of the remaining helices, which include conservative residues I1216, F1220, I1227, M1229, and M1233. Finally, the stability of this and several alternative models (including a recent NMR structure) and a set of mutant forms was tested in all-atom molecular dynamics (MD) simulations in a POPC bilayer mimicking the viral envelope membrane. Unlike other configurations, our model trimer remained extraordinarily tightly packed over a microsecond-range MD and retained its stability when palmitoylated in accordance with experimental data. Palmitoylation had no significant impact on the TMD conformation nor the way in which the lipid bilayer was perturbed in the presence of the trimer. Overall, the resulting model of S-TMD conforms to known basic principles of TM helix packing and will be further used to explore the complex machinery of membrane fusion from a broader perspective beyond the TMD.

bioinformatics↗

bioRxiv2022

Metamorphism of catalytic domain controls transposition in Tn3 family transposases

Transposons account for a remarkable diversity of mobile genetic elements that play the role of genome architects in all domains of life. Tn3 is a family of widespread and among first identified bacterial transposons notorious for their contribution to dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase facilitating both transposition and target immunity. The structural framework for understanding the mechanism of the TnpA transposition is however absent. Here, we describe the cryo-EM structures of TnpA from Tn4430 in apo form and paired with transposon ends. We show that TnpA has a unique architecture and exhibits a family-specific regulation mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a unique topology that suggests a specific role for target DNA in transpososome assembly, activation, and target immunity.

molecular biology↗

bioRxiv2022

Predicting TCR-peptide recognition based on residue-level pairwise statistical potential

Prediction of TCR-peptide interactions has great importance for therapy of cancer, infectious and autoimmune diseases, but remains a major challenge, particularly for unseen epitopes. We present a structure-based method that enables scoring of TCR-peptide interactions using an energy potential (TCRen) derived from statistics of TCR-peptide contacts in existing crystal structures. We show that TCRen has high performance in discriminating cognate/unrelated peptides and can facilitate the identification of cancer neoepitopes recognized by tumor-infiltrating lymphocytes.

immunology↗

bioRxiv2022

Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales

Non-membrane-bound biomolecular condensates have been proposed to represent an important mode of subcellular organization in diverse biological settings. However, the fundamental principles governing the spatial organization and dynamics of condensates at the atomistic level remain unclear. The S. cerevisiae Lge1 protein is required for histone H2B ubiquitination and its N-terminal intrinsically disordered fragment (Lge11-80) undergoes robust phase separation. This study connects single- and multi-chain all-atom molecular dynamics simulations of Lge11-80 with the in vitro behavior of Lge11-80 condensates. Analysis of modelled protein-protein interactions elucidates the key determinants of Lge11-80 condensate formation and links configurational entropy, valency and compactness of proteins inside the condensates. A newly derived analytical formalism, related to colloid fractal cluster formation, describes condensate architecture across length scales as a function of protein valency and compactness. In particular, the formalism provides an atomistically resolved model of Lge11-80 condensates on the scale of hundreds of nanometers starting from individual protein conformers captured in simulations. The simulation-derived fractal dimensions of condensates of Lge11-80 and its mutants agree with their in vitro morphologies. The presented framework enables a multiscale description of biomolecular condensates and embeds their study in a wider context of colloid self-organization.

biophysics↗

bioRxiv2021

Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the cytoplasmic and membrane domains suggests an uncoupled conformation of the complex. Based on these structural data we suggest a new simple and testable coupling mechanism for the molecular machine.

biochemistry↗