Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals

Formation of Iron Oxide Nanoparticles in the Internal Cavity of Ferritin-Like Dps Protein: Studies by Anomalous X-Ray Scattering

DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various types of destructive external factors via in cellulo Dps–DNA co-crystallization. This protective response results in the emergence of bacterial resistance to antibiotics and other drugs. The protective properties of Dps have attracted a significant attention of researchers. However, Dps has another equally important functional role. Being a ferritin-like protein, Dps acts as an iron depot and protects bacterial cells from the oxidative damage initiated by the excess of iron. Here we investigated formation of iron oxide nanoparticles in the internal cavity of the Dps dodecamer. We used anomalous small-angle X-ray scattering as the main research technique, which allows to examine the structure of metal-containing biological macromolecules and to analyze the size distribution of metal nanoparticles formed in them. The contributions of protein and metal components to total scattering were distinguished by varying the energy of the incident X-ray radiation near the edge of the metal atom absorption band (the K-band for iron). We examined Dps specimens containing 50, 500, and 2000 iron atoms per protein dodecamer. Analysis of the particle size distribution showed that, depending on the iron content in the solution, the size of the nanoparticles formed inside the protein molecule was 2 to 4 nm and the growth of metal nanoparticles was limited by the size of the protein inner cavity. We also found some amount of iron ions in the Dps surface layer. This layer is very important for the protein to perform its protective functions, since the surface-located N-terminal domains determine the nature of interactions between Dps and DNA. In general, the results obtained in this work can be useful for the next step in studying the Dps phenomenon, as well as in creating biocompatible and solution-stabilized metal nanoparticles

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages..

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes

Structural investigations of E. Coli dihydrolipoamide dehydrogenase in solution: Small-angle X-ray scattering and molecular docking

Dihydrolipoamide dehydrogenase from Escherichia coli (LpD) is a bacterial enzyme that is involved in the central metabolism and shared in common between the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes. In the crystal structure, E. coli LpD is known to exist as a dimer. The present work is focused on analyzing the solution structure of LpD by small-angle X-ray scattering, molecular docking, and analytical ultracentrifugation. It was shown that in solution LpD exists as an equilibrium mixture of a dimer and a tetramer. The presence of oligomeric forms is determined by the multifunctionality of LpD in the cell, in particular, the required stoichiometry in the complexes

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg$^{2+}$ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe$^{2+}$ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues

Structural peculiarities of the (MHF1–MHF2)4\mathrm{(MHF1–MHF2)_4} octamer provide a long DNA binding patch to anchor the MHF–FANCM complex to chromatin: A solution SAXS study

MHF1 and MHF2 are histone-fold-containing FANCM-associated proteins. FANCM is a Fanconi anemia (FA) complementation group protein. We previously obtained high-resolution structures of MHF1–MHF2 (MHF) and MHF in complex with a fragment of FANCM (MHF–FANCM-F). Here, we use small angle X-ray scattering (SAXS) to investigate the solution behaviors of these protein complexes. In combination with crystallographic data, the results of the SAXS study reveal that a long, positively charged patch exposed on the surface of the MHF complex plays a critical role in double-stranded DNA (dsDNA) binding