Biology subjects

Klopprogge, B.

Publications and source records attributed to Klopprogge, B..

3 recordsLinked to original sources

bioRxiv2024

SARS-CoV-2 methyltransferase nsp10-16 in complex with natural and drug-like purine analogs for guiding structure-based drug discovery

Non-structural protein 10 (nsp10) and non-structural protein 16 (nsp16) are part of the RNA synthesis complex, which is crucial for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nsp16 exhibits 2-O-methyltransferase activity during viral messenger RNA capping and is active in a heterodimeric complex with enzymatically inactive nsp10. It has been shown that inactivation of the nsp10-16 protein complex interferes severely with viral replication, making it a highly promising drug target. As information on ligands binding to the nsp10-16 complex (nsp10-16) is still scarce, we screened the active site for potential binding of drug-like and fragment-like compounds using X-ray crystallography. The screened set of 234 compounds consists of derivatives of the natural substrate S-adenosyl methionine (SAM) and adenine derivatives, of which some have been described previously as methyltransferase inhibitors and nsp16 binders. A docking study guided the selection of many of these compounds. Here we report structures of binders to the SAM site of nsp10-16 and for two of them, toyocamycin and sangivamycin, we present additional crystal structures in the presence of a second substrate, Cap0-analog/Cap0-RNA. The identified hits were tested for binding to nsp10-16 in solution and antiviral activity in cell culture. Our data provide important structural information on various molecules that bind to the SAM substrate site which can be used as novel starting points for selective methyltransferase inhibitor designs.

biochemistry↗

bioRxiv2023

Engineering orthogonal ribosomes for real-time monitoring using fluorescence

A promising route to tackle the trade-off in cellular resources between synthetic protein production and cellular growth is to use a separate dedicated pool of orthogonal ribosomes to produce synthetic proteins. However, the optimisation of strains containing two ribosomal pools - native for the host cells proteome and orthogonal for synthetic proteins - has yet to be thoroughly explored. Here, we address this by creating orthogonal ribosomes that fluoresce by inserting fluorescent RNA aptamers into tethered orthogonal ribosomal RNA (TO-rRNA). To study the tolerance of the engineered ribosomes to aptamer insertion, we assembled and screened a library of candidate insertion sites, identifying several sites in both the 16S and 23S TO-rRNA that enables ribosome labelling with minimal effect on translation activity. Serendipitously, we identify one site in 23S TO-rRNA, where insertion appears to not only be tolerated but to enhance orthogonal ribosome activity, across multiple bacterial strains and RNA insertions. Using bulk and single cell assays, we demonstrate that this variant allows us to label orthogonal ribosomes for dynamic tracking and across populations, making it a promising tool for optimising orthogonal translation in engineered cells. Ribosome engineering offers great potential, both for the development of next-generation microbial cell factories, as well as a tool to expand our understanding of ribosome function in living cells. This work provides a window into the assembly, localisation and function of these molecular machines to meet these aims.

synthetic biology↗

bioRxiv2022

Tailored synthetic sRNAs dynamically tune multilayer genetic circuits

Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose a rational design of de novo small RNAs (sRNAs) to dynamically modulate gene expression within a broad range, from high, medium to low repression, and implemented them in Escherichia coli. We designed multiple multilayer genetic circuits, in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein. Our approach harnesses the intrinsic RNA-interference pathway in E. coli to exert dynamic and modular control of the multilayer genetic circuits. The sRNAs were designed to target TFs instead of the reporter gene, which allowed for wide range of expression modulation of the reporter protein, including the most difficult to achieve dynamic switch to an OFF state. Out work provides a frame for achieving independent modulation of gene expression, by only including an independent control circuit expressing synthetic sRNAs, without altering the structure of existing genetic circuits.

synthetic biology↗