Biology subjects

Zdorevskyi, O.

Publications and source records attributed to Zdorevskyi, O..

7 recordsLinked to original sources

bioRxiv2026

Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria

Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.

biochemistry↗

bioRxiv2026

Molecular and Energetic Basis of Histidine Switch Dynamics in Respiratory Complex I

The respiratory complex I in mitochondria and bacteria drives the two-electron reduction of quinone to pump protons across the membrane. The molecular basis of this catalytic reaction remains enigmatic despite significant progress in structural characterization of the complex. A highly conserved histidine residue in the distal antiporter-like subunit of its membrane domain has been shown to undergo conformational changes in molecular simulations and cryo-EM structures. However, the function of histidine switch dynamics, and energetics of its conformational transitions remain unclear. Here, by applying enhanced sampling simulations, we evaluate the energetics of the histidine switch dynamics and demonstrate that it is coupled to the charge state of lysine residues ca. 10 [A] apart, which cause hydrogen bond restructuring and stabilize histidine in specific conformations. Hybrid QM/MM metadynamics simulations show that the histidine participates in gated proton transfer and may function as a proton confurcation device in complex I and related proteins. Importance of workProton pumping by the mitochondrial respiratory chain enzymes needs to be highly directional so that the ATP production occurs as efficiently as possible during high energy demands. With the protons being hard to detect by most methods of structural biology, computer simulation approaches as applied in this work offer critical information on how molecular gates within respiratory complexes can function and help elucidate the challenging questions in the field of mitochondrial bioenergetics.

biophysics↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

Structure and functional analysis of CDNF-BiP interaction reveal a role in endoplasmic reticulum proteostasis regulation

Cerebral dopamine neurotrophic factor (CDNF) was identified for ability to rescue midbrain dopamine neurons and as endoplasmic reticulum (ER)-located protein that can be secreted. Structurally homologous to mesencephalic astrocyte-derived neurotrophic factor (MANF), CDNF has been shown to interact with ER-localized chaperone BiP. The ER plays a crucial role in protein synthesis, folding, and quality control, with BiP being a key player in maintaining protein homeostasis. CDNF is protective against ER stress and involved in regulating the unfolded protein response (UPR) signaling and interaction with BiP. Recent studies have shown that CDNF interacts with UPR sensor proteins PERK, IRE1, and ATF6, suggesting an overlap in CDNF binding with UPR sensors and BiP. In rodent models of Parkinsons disease (PD), CDNF protects and restores the function of brain dopamine neurons and was successful in PD phase 1 clinical studies. CDNF has shown therapeutic potential for several neurological diseases, including amyotrophic lateral sclerosis, and ischemic stroke. Despite extensive knowledge on CDNFs impact on cellular function and neuronal degeneration, its detailed molecular mechanism of action in the ER remains unclear. Here, we have characterized the CDNF interaction with BiP both structurally and functionally and solved the crystal structures of CDNF-BiP complexes to 1.5 [A] resolution, complemented with molecular dynamics simulations. Results show CDNFs role as an antagonist of BiP nucleotide exchange, and thus in its chaperone function, binding to the ADP-bound state. Finally, we show its effect on neuroprotection with stem cell-derived human dopamine neurons, highlighting its potential in neurodegenerative disease treatment.

biochemistry↗

bioRxiv2025

Proton transfer through a charged conduit in respiratory complex I - long range effects and conformational gating

Energy coupling processes in respiratory complex I - a large redox-driven proton pump in the inner mitochondrial membrane - remains one of the most enigmatic problems in modern bioenergetics. Recent high-resolution cryo EM structures of complex I revealed extensive hydration in the interior of the protein, including the buried E channel, which is an acidic charged conduit that bridges the quinone binding cavity with the extended membrane domain of the enzyme. Despite the general agreement that E channel participates in proton transfer, absence of proton density in the cryo-EM maps pose a significant challenge to develop viable models of proton pumping. By adhering to the hypothesis that E channel catalyzes transfer of proton(s) from the quinone binding cavity to the membrane-bound proton pumping site(s), we performed hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations using the ~2.4 A cryo-EM structure of mitochondrial complex I from Mus musculus. By combining classical atomistic MD simulations with the hybrid QM/MM free energy calculations, we identify several energetically favorable Grotthuss-competent proton transfer paths in the E channel region. As part of the long-range coupling in complex I, our calculations show that protonation of a single acidic amino acid residue in the distal MM surroundings can alter the dynamics of proton transfer in the E channel region. Additionally, we pinpoint the gating function of a highly conserved tyrosine residue in the E channel, which undergoes conformational flipping to establish an energetically favorable proton transfer path. In the context of the redox-coupled proton pumping mechanism of complex I, we propose a stepping-stone model of proton transfer through the E channel.

biochemistry↗

bioRxiv2024

Catalytic relevance of quinol anion in biological energy conversion by respiratory complex I

Redox chemistry of quinones is an essential component of life on earth. In the mitochondrial electron transport chain, ubiquinone molecule is reduced to ubiquinol by respiratory complex I to drive the synthesis of ATP. By performing both classical and hybrid QM/MM simulations on high-resolution cryo-EM structures, including quantitative free energy calculations, we show that semiquinone species in complex I is anionic in nature and is trapped in the active site chamber for its subsequent reduction. Two-electron reduction of ubiquinone yields a metastable ubiquinol anion, which is electrostatically pushed by 15-20 [A] towards the exit of the ubiquinone binding chamber to drive the proton pump of complex I. As part of the two-electron reduction of ubiquinone, protonic rearrangements take place in the active site in which a highly conserved histidine converts from its one tautomeric state to another. The combined findings provide a detailed and testable mechanistic picture of proton-coupled electron transfer reaction at the active site of complex I in wild-type as well as mutant conditions.

biochemistry↗

bioRxiv2024

Structural basis of respiratory complexes adaptation to cold temperatures

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) was identified with a [~]25{degrees} rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.

cell biology↗