Biology subjects

Flemming, D.

Publications and source records attributed to Flemming, D..

5 recordsLinked to original sources

bioRxiv2026

Small-molecule activators of the Staphylococcus aureus ClpC/ClpP AAA+ protease

Summary/AbstractThe central AAA+ ClpC/ClpP protease in Gram-positive bacteria is crucial for virulence and stress resistance and has been recognized as drug target. Natural cyclic peptides deregulate the essential Mycobacterium tuberculosis ClpC1 and cause cell death. Similarly, overactivated mutants of the non-essential Staphylococcus aureus ClpC homologue cause uncontrolled proteolysis and severe toxicity in vivo. However, no chemical modulators of S. aureus ClpC have been described. Here, using a biochemical high-throughput screen we identify eight chemically distinct bona fide small molecules that robustly stimulate ClpC ATPase and proteolytic activity in vitro. Structural, computational, and mutational analyses define two ligandable regulatory sites within the ClpC N-terminal domain (NTD) as compound targets: a conserved hydrophobic groove and an allosteric pArg1 pocket, both engaged in substrate recognition. These findings establish S. aureus ClpC as chemically targetable and provide mechanistic insight into its regulatory architecture, enabling future development and optimization of chemical probes to deregulate AAA+ protease control.

biochemistry↗

bioRxiv2026

Buoyancy-driven sorting of synthetic cells for nanopore activity

The development of sorting strategies that directly report on functional activity remains a bottleneck in synthetic cell research. Current methodologies typically rely on sequential label-dependent probing, which limits throughput. Here, we introduce a label-free, buoyancy-driven selection strategy in which the mode of separation and the mode of decision-making are intrinsically linked, coupling pore activity directly to the synthetic vesicles internal density in a one-pot assay. In this system, sorting emerges intrinsically: Giant unilamellar vesicles (GUVs) that contain a dense medium sediment by default, while only those with functional transmembrane pores undergo solute exchange, leading to density equilibration and flotation. We exploit this principle to separate pore-active from non-functional GUVs without external markers or imaging-based readouts. Using protein pores and DNA origami and DNA tile nanopores, we demonstrate that buoyancy-driven separation enables parallel functional assessment of heterogeneous populations and supports flow-based enrichment of highly active synthetic cells. By directly linking molecular transport performance to GUV buoyancy, this approach collapses decision-making into the physical separation process itself, providing a scalable platform for screening, sorting, and evolving membrane pores in synthetic cell systems.

biophysics↗

bioRxiv2024

Structures of aberrant spliceosome intermediates on their way to disassembly

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans1. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses2,3, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-Bact spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5 exon leading to a single nucleotide insertion at the 5splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

biochemistry↗

bioRxiv2024

H/ACA snoRNP guides ribosomal RNA subdomain folding in a satellite particle before joining the core 90S pre-ribosome

During synthesis, modification and maturation of the ribosomal RNA, correct subdomain folding without additional guidance poses a major challenge. An essential H/ACA snoRNP, snR30, was observed to be outsourced from the core of the 90S, the earliest known pre-ribosome, to form a "satellite particle". This particle interacts with the so-called 18S rRNA platform subdomain, guiding its independent folding through localized structural interactions. Once this subdomain achieves its proper conformation, it is integrated into the core pre-ribosome, contributing to the overall maturation of the 18S rRNA. By temporarily segregating the folding process within a specialized satellite particle, the correct assembly of individual components is facilitated, thereby reducing the risk of errors during the complex process of ribosome assembly. In a broader sense, molecular outsourcing in cellular processes, where transient subcomplexes handle specific tasks before rejoining the main assembly line, can enhance efficiency and accuracy of complex molecular processes like ribosome biogenesis.

biochemistry↗

bioRxiv2023

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

biochemistry↗