Elastin-derived peptides (EDPs) interact with elastin-binding protein (EBP), a key component of the elastin receptor complex, modulating cellular processes such as protease activation, apoptosis, and chemotaxis via regulation of NEU-1 sialidase activity. Despite their therapeutic relevance, the structural basis of EDP-EBP interactions remains poorly understood. Here, we present the first full-length homology model of EBP (residues 29-516), constructed and validated through duplicate 1 microsecond long molecular dynamics (MD) simulations. Molecular docking of VGVAPG (as a representative EDP) and lactose (as a representative galactosugar) followed by MD simulations, allowed us to identify key binding residues: D98, E99, S104, and S136 for VGVAPG; and E99, S101, S104, N116, Q124, E137, and Y139 for lactose. These data reveal a shared binding region within residues 83-139, suggesting a competitive binding between EDPs and galactosugars. Furthermore, EBP residues P233 and I234 were implicated in potential interactions with PPCA or NEU-1, contributing to ERC assembly. These findings offer new insights into the molecular recognition mechanisms of EBP and provide a foundation for the design of EBP-targeted elastin peptide antagonists.

We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW=39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37%) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of T. thermophilus phages: φYS40 (87%) and φTMA (88%). The enzyme exhibits lytic activity mainly against bacteria of the genus Thermus and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (Tm=98.3°C). It retains∼50% of its lytic activity after 90min of incubation at 99°C. Crystallographic analysis, at 2.2Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40% of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn2+ ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of E. coli peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein's thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.

Isoprenoids represent one of the largest and functionally diverse class of natural products, playing essential roles in cellular processes across all domains of life. Unlike humans, many pathogenic organisms such as bacteria and protozoa produce their isoprenoid precursors through the 2-C-methylerythritol phosphate (MEP) pathway. 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of this pathway. Despite its biological importance and potential as a drug target, structural studies on DXPS were limited due to its intrinsic flexibility and difficulties in crystallisation. Recent advances, including the development of more crystallisation-friendly constructs and the application of single-particle cryo-electron microscopy (cryo-EM), have significantly expanded our structural understanding of DXPS. This review provides a comprehensive overview of the structural insights gained over the past decades, focusing on the overall architecture of DXPS, its catalytic mechanism, and emerging relevance in structure-based drug discovery.

Cryogenic correlative light and electron microscopy (cryo-CLEM) is an imaging strategy that integrates specific molecular labeling and molecular resolution structural information. However, there is a resolution gap of more than two orders of magnitude between diffraction-limited fluorescence microscopy and electron microscopy (EM). Single-molecule localization microscopy (SMLM) performed at cryogenic temperatures promises to bridge this resolution gap. Nevertheless, the high excitation laser powers required for SMLM risk the devitrification of frozen biological samples, leading to perturbation of their native-like state. Here, we investigate how base cooling temperature, immersion medium, and EM grid support materials influence sample devitrification. Using finite element simulations and experimental validation, we show that a cryo-immersion medium enhances heat dissipation for carbon supports, while metallic supports in a cold nitrogen gas medium tolerate higher laser intensities due to lower base temperatures. Gold supports illuminated at 640nm exhibit markedly high laser thresholds, similar to silver-coated grids. Additionally, metallic supports maintain efficient heat dissipation in vacuum-based cryostats. Our findings provide quantitative insights that aid in optimization of cryo-SMLM setups for improved cryo-CLEM imaging.