Biology subjects

Stetsenko, A.

Publications and source records attributed to Stetsenko, A..

6 recordsLinked to original sources

bioRxiv2025

Determination of the water network surrounding the type I pilus from E. coli by cryo-electron microscopy

Type 1 pili are protein filamentous surface structures of Gram-negative bacteria that mediate adhesion to host and play a crucial role in infection. Here, we report the cryogenic electron microscopy structure of the type 1 pilus from E. coli K-12 comprising 15 subunits of the major protein pilin FimA. The final resolution of EM reconstruction was estimated to be in the range from 2.09 to 2.30 [A], which is higher than that of the previously published structure. This improvement in the resolution enabled us to refine side-chain conformations to reliably determine the distances between the side-chain residues participating in the intersubunit interactions, and determine a network of water molecules surrounding the pilus rod. The analysis revealed that water contributes to intersubunit stabilization both through discrete bridging interactions and through extended hydrogen-bonded clusters, thereby supporting both the rigidity and flexibility of the filament. Comparison with a homologous high-resolution pilus model from enterotoxigenic E. coli showed that nearly all "conserved" water molecules i.e., those that are present at equivalent positions in different subunits of our model occupy also equivalent positions across the two structures, under-scoring their functional relevance. At the same time, sequence-specific differences in hydration patterns were observed. These findings highlight the structural and functional importance of water in pilus architecture and provide a more detailed molecular framework for understanding bacterial adhesion. SynopsisThe improvement in the resolution of the Cryo-EM reconstruction for type I pilus from E. coli made it possible to determine the positions of water molecules surrounding the pilus rod and reveal a more detailed picture of interactions between different subunits of the rod.

molecular biology↗

bioRxiv2025

Unveiling the Molecular Architecture of Candida auris Ribosome

Candida auris is an emerging multidrug-resistant fungal pathogen causing life-threatening invasive candidiasis and bloodstream infections (candidemia), posing significant global health challenges. Despite its importance, the protein translation in pathogenic fungi is poorly characterized. Using cryo-electron microscopy and single-particle reconstruction, we resolved high-resolution structures of the 80S ribosome from C. auris in its vacant state and in complexes with three inhibitors: cycloheximide (CHX), blasticidin-S (BLS), and geneticin (G418). We uncovered a unique substitution of a key nucleotide in the P-site of the small ribosomal subunit (C1160 in C. auris), which may influence ribosome-tRNA interactions and translation fidelity. Comparative analysis of ribosome inhibitor interactions showed that resistance to CHX was observed in only two Candida species examined, while BLS binding displayed no significant differences between C. auris and S. cerevisiae, although C. auris was more sensitive to it. We identified that G418 exhibits promiscuous binding across multiple nonspecific sites, yet its primary interaction site at the decoding center remains highly conserved among Candida species. These findings provide a previously uncharacterized structure of the C. auris ribosome, highlighting novel features that may be leveraged for the development of targeted antifungal therapies to combat multidrug resistance. These insights not only enhance our understanding of ribosomal inhibitor interactions but also suggest potential biomarkers for predicting antifungal susceptibility in clinical applications.

molecular biology↗

bioRxiv2025

Condition-dependent, amorphous protein agglomerates control cytoplasmic rheology

Molecular crowding in the bacterial cytoplasm restricts the diffusion of large molecules, impacting cellular processes. However, how nutrient availability influences cytoplasmic rheology is not well understood. With single-particle tracking in Escherichia coli, we observed a threefold variation in the diffusion of a 40-nm particle across exponential growth conditions. Previously suggested determinants of rheology did not account for this variation; instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins, persisting upon genetic perturbations and showing that lower diffusion is associated with increased viscoelasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins could form amorphous agglomerates at physiological concentrations in vitro, likely driven by their low intrinsic disorder, high compactness and hydropathy score. These findings show that protein agglomerates regulate cytoplasmic rheology in a condition-dependent manner, suggesting an underappreciated level of cytoplasmic organization. HighlightsO_LIDiffusion of 40-nm particles varies threefold across growth conditions in E. coli C_LIO_LICytoplasmic diffusion inversely correlates with COG-E protein abundance COG-E proteins form agglomerates that increase cytoplasmic viscoelasticity C_LIO_LIProtein compactness and hydrophobicity predict condition-dependent crowding effects C_LI

biophysics↗

bioRxiv2025

Structural basis for no retinal binding in flotillin-associated rhodopsins

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins were reported to lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins) were identified and suggested to lack the typical retinal binding pocket despite preserving the lysine residue in many members of the group. Here we present cryo-EM structures of paralog FArhodopsin and proteorhodopsin from marine bacteria Pseudothioglobus. The structures revealed pentameric assemblies of both proteins similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Thus, our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of the FArhodopsin clade.

biophysics↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗

bioRxiv2023

A novel subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

biophysics↗