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Dorner, K.

Publications and source records attributed to Dorner, K..

4 recordsLinked to original sources

A bacterial nucleolus for cold adaptation

Compartmentalisation enables cells to spatially organise essential biochemical processes. This is exemplified by the eukaryotic nucleolus, which concentrates the machinery for ribosome biogenesis. Bacteria are generally thought to lack an equivalent compartment. Here we discover a nucleolus-like condensate in Escherichia coli that emerges at rDNA loci during cold adaptation to spatially organise and promote ribosome biogenesis. Low temperatures, which stabilise RNA secondary structures, induce accumulation of the RNA-remodelling enzyme CsdA. This raises the cellular CsdA concentration above its condensation threshold, driving condensate formation at rDNA loci when rRNA transcription is active. CsdA compartments enrich the rRNA transcription machinery and ribosome-biogenesis factors, spatially linking rRNA synthesis to downstream processing and assembly. Disrupting CsdA condensation impairs ribosome maturation and reduces bacterial adaptation to cold temperature, demonstrating functional importance of condensate formation. Comparative genomics suggests that analogous condensate-forming RNA remodelling enzymes have evolved repeatedly across diverse bacterial lineages. Our findings establish biomolecular condensates as a unifying organisational principle of ribosome biogenesis across the tree of life and suggest that adaptive compartmentalisation can emerge when environmental conditions challenge the efficiency of essential biochemical processes.

cell biology↗

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. SynopsisStructures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

biophysics↗

Mix-and-extrude using 3D printed nozzles for time-resolved membrane protein crystallography

Time-resolved crystallography enabled the visualization of protein molecular motion during reaction. While light is commonly used to initiate reactions in time-resolved crystallography, only a small number of proteins can in fact be activated by light. However, many biological reactions can be triggered by the interaction of proteins with ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D printed microchannels in conjunction with a micronozzle to study the dynamics of samples in viscous media that can be triggered by diffusive mixing. The device design allows for mixing of ligands and protein crystals in a time window of 2 to 20 seconds. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within the lipidic cubic phase, can be mixed efficiently. The potential use of this approach for time-resolved membrane protein crystallography to support in the development of new drugs is also discussed. Synopsis3D printed mixing-HVE devices address time-resolved membrane protein crystallography challenges via compact dual-flow LCP injection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/517685v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12f6f68org.highwire.dtl.DTLVardef@13b198eorg.highwire.dtl.DTLVardef@10abe5eorg.highwire.dtl.DTLVardef@5810ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗