Biology subjects

Bottcher, B.

Publications and source records attributed to Bottcher, B..

5 recordsLinked to original sources

bioRxiv2024

A filamentous scaffold for gene regulation

Proteins of the Drosophila behaviour/human splicing (DBHS) family are involved in many aspects of gene regulation and maintenance like transcription, splicing and DNA repair. The three known members of this family in humans, Non-POU domain-containing octamer-binding protein (NONO), splicing factor proline/glutamine rich (SFPQ), and paraspeckle protein component 1 (PSPC1), form homo- and heterodimers to fulfil these functions by mediating contacts between RNA, DNA, and other protein factors. The dimers can further dynamically oligomerise through -helical coiled-coils to larger aggregates, which is crucial for many functions of DBHS proteins. While the atomic structures of the dimers are established, the native arrangement in higher oligomers was unknown. Here we present the structure of a filamentous NONO/SFPQ heterooligomer from Cricetulus griseus resolved by cryo-EM. Globular heterodimer domains are alternating on both sides of a strand that is stabilized by an interdigitating network of coiled-coil interactions. Two of these strands assemble into a double strand with only few interactions between them. The globular domains of SFPQ face the counter strand and form a groove while those of NONO face outwards. The different environments of NONO and SFPQ in the filament provide the basis for a differential functionality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/617013v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@19af4caorg.highwire.dtl.DTLVardef@3d04daorg.highwire.dtl.DTLVardef@9bdc7org.highwire.dtl.DTLVardef@18a6f88_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Lymphostatin: Structure of a large multi-functional virulence factor

Enteropathogenic and Enterohaemorrhagic Escherichia coli are enteric pathogens of global importance and human infections can be life-threatening. Lymphostatin is a key virulence factor of these bacteria, being required for intestinal colonisation and a potent inhibitor of the mitogen- and antigen-activated proliferation of lymphocytes and proinflammatory responses. In some strains, it also mediates adherence to host cells and influences actin nucleation at sites of attachment. This 365 kDa protein requires glycosyltransferase and cysteine protease motifs for activity against lymphocytes, but high-resolution structural information has proven elusive and the molecular mechanisms by which it acts remain unclear. Here, we describe the structure of lymphostatin from the prototype O127:H6 enteropathogenic E. coli strain determined by electron cryo-microscopy. Our results reveal two glycosyltransferase domains, one PaTox-like protease domain, an ADP-ribosyltransferase domain, and a delivery domain. Long linkers act to hold these domains together. These linkers occlude the catalytic sites of the N-terminal glycosyltransferase and protease domains. In this dormant state, lymphostatin binds to HEK-293T cells, where it forms large clusters before being taken up and sequestered into cytosolic foci. With more functional domains than any other known large bacterial toxin, lymphostatin can be regarded as the multifunctional Swiss army knife of pathogenic Escherichia coli, enabling complex interactions with the host cells in different environments.

biophysics↗

bioRxiv2023

Discovery of a functionally selective serotonin 5-HT1A receptor agonist for the treatment of pain

The G protein-coupled serotonin receptor 5-HT1AR mediates antinociception and may serve as a valuable target for the treatment of pain. Starting from a chemical library, ST171, a bitopic chemotype activating 5-HT1AR was evolved. In vitro pharmacological investigations of ST171 revealed potent and selective Gi activation (EC50 = 0.3 nM), with marginal Gs and {beta}-arrestin recruitment. Preclinical studies in mice showed that ST171 was effective in acute and chronic (inflammatory and neuropathic) pain models, without causing sedation. Comparison of cryo-EM structures of a 5-HT1AR-Gi complex bound to the functionally biased agonist ST171, with a structure bound to the functionally balanced agonist befiradol, showed that both ligands bind to the same orthosteric site, but address different exo-sites. The individual poses are associated with ligand-specific helical dispositions and rearrangements of microdomains. Complementation of these studies with molecular dynamics simulations allowed us to derive structural features associated with ST171s functional selectivity, a phenomenon that may be crucial to the discovery of more effective and safe GPCR drugs.

pharmacology and toxicology↗

bioRxiv2023

Immediate targeting of host ribosomes by jumbo phage encoded proteins

Bacteriophages must seize control of the host gene expression machinery to promote their own protein synthesis. Since the bacterial hosts are armed with numerous anti-phage defence systems, it is essential that mechanisms of host take-over act immediately upon infection. Although individual proteins that modulate components of the bacterial gene expression apparatus have been described in several different phages, systematic approaches which capture the phages arsenal for immediate targeting of host transcription and translation processes have been lacking. In particular, there are no known phage factors that associate directly with host ribosomes to modulate protein synthesis. Here, we take an integrative high-throughput approach to uncover numerous new proteins encoded by the jumbo phage {Phi}KZ that target the gene expression machinery of the Gram-negative human pathogen Pseudomonas aeruginosa immediately upon infection. By integrating biochemical and structural analyses, we identify a conserved phage factor that associates with the large ribosomal subunit by binding the 5S ribosomal RNA. This highly abundant factor is amongst the earliest {Phi}KZ proteins expressed after infection and stays bound to ribosomes during the entire translation cycle. Our study provides a general strategy to decipher molecular components of phage-mediated host take-over and argues that phage genomes represent a large discovery space for proteins that modulate the host gene expression machinery.

microbiology↗

bioRxiv2016

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state the DNA binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavourable alignment of C-terminal DNA binding region in solution contributes to an auto-inhibited state.

molecular biology↗