Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.
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http://dx.doi.org/10.1021/acschembio.1c00296 | DOI Listing |
Viruses
January 2025
Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization.
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December 2024
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
Background: Marek's disease (MD) is a pathology affecting chickens caused by Marek's disease virus (MDV), an acute transforming alphaherpesvirus of the genus . MD is characterized by paralysis, immune suppression, and the rapid formation of T-cell (primarily CD4+) lymphomas. Over the last 50 years, losses due to MDV infection have been controlled worldwide through vaccination; however, these live-attenuated vaccines are non-sterilizing and potentially contributed to the virulence evolution of MDV field strains.
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January 2025
Cancer Microenvironment Branch, Division of Cancer Biology, Research Institute, National Cancer Center, Goyang-si 10408, Republic of Korea.
As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf-MEK-ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners.
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January 2025
Centre for Surface Chemistry and Catalysis-Characterization and Application Team (COK-KAT), KU Leuven, Celestijnenlaan 200F Box 2461, 3001 Heverlee, Belgium.
This work describes the synthesis of ordered 3D siloxane-silsesquioxane reticular materials with silicate D4R cubes (SiO), harvested from a sacrificial tetrabutylammonium cyclosilicate hydrate (TBA-CySH) precursor, interlinked with octyl and dicyclopentyl (Cp) hydrocarbon functionalities in a one-step synthesis with organodichlorosilanes. Advanced solid-state NMR spectroscopy allowed us to unravel the molecular order of the nodes and their interconnection by the silicone linkers. In the case of octyl-methyl silicone linkers, changing the silane-to-silicate ratio in the synthesis allowed for tuning the length of the linker between the nodes.
View Article and Find Full Text PDFLife (Basel)
January 2025
Chemistry Department, Lomonosov Moscow State University, 119991 Moscow, Russia.
Inorganic pyrophosphatases, or PPases, are ubiquitous enzymes whose activity is necessary for a large number of biosynthetic reactions. The catalytic function of PPases is dependent on certain conformational changes that have been previously characterized based on the comparison of the crystal structures of various complexes. The current work describes the conformational dynamics of a structural model of human mitochondrial pyrophosphatase hPPA2 using molecular dynamics simulation, all-atom principal component analysis, and coarse-grained normal mode analysis.
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