Biology subjects

Lorenzen, K.

Publications and source records attributed to Lorenzen, K..

10 recordsLinked to original sources

bioRxiv2025

X-ray Spectroscopy Meets Native Mass Spectrometry: Probing Gas-phase Protein Complexes

Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.

biochemistry↗

bioRxiv2023

Light-induced Trpin/Metout switching during BLUF domain activation in ATP-bound photoactivatable adenylate cyclase OaPAC

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in a non-productive conformation. However, FTIR spectroscopic experiments confirm that the non-productive conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the initial events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Initial changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180{degrees} rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via -helix 3 and 4 in the linker region between sensor and effector domain is discussed.

biochemistry↗

bioRxiv2023

Convolutional neural network approach for the automated identification of in cellulo crystals

In cellulo crystallization is a rarely occurring event in nature. Recent advances, making use of heterologous overexpression, can promote the intracellular formation of protein crystals, but new tools are required to detect and to characterize these targets in the complex cell environment. In the present work we make use of Mask R-CNN, a Convolutional Neural Network (CNN) based instance segmentation method, for the identification of either single or multi-shaped crystals growing in living insect cells, using conventional bright field images. The algorithm can be rapidly adapted to recognize different targets, with the aim to extract relevant information to support a semi-automated screening pipeline, with the purpose to aid in the development of the intracellular protein crystallization approach.

biophysics↗

bioRxiv2022

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↗

bioRxiv2022

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that - in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 [A] resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae from Anopheles stephensi, Aedes albopictus and Culex tarsalis - substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained using Culex cell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components. Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 [A]. Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity. We expand the known target range to include three more mosquito species: Anopheles stephensi, Aedes albopictus and Culex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

biochemistry↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

bioRxiv2020

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

biophysics↗

bioRxiv2020

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

bioRxiv2020

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

biophysics↗

bioRxiv2017

The Fes tyrosine kinase guides CD19 receptor fate in B-cells by shaping regulatory Src phosphorylation networks

The c-Fes protein tyrosine kinase is a proto-oncogene that can also act as a tumor suppressor. We implemented an unbiased phosphoproteomics-based analysis that establishes cognate kinase-substrate associations, and revealed that c-Fes directly phosphorylates Dok1, Ptpn18 and Sts1, facilitating recruitment of the Src inhibitory kinase Csk to these substrates. These interactions resulted in modulation of Src signaling following B-cell receptor (BCR) stimulation and subsequent alteration of the protein levels of CD19, a membrane-localized BCR co-receptor and emerging key protein affecting the development of B- and plasma cell-lymphoma. Strikingly, manipulating c-Fes expression levels drove opposing biological outcomes. Low-level exogenous c-Fes expression led to a strong increase in CD19 protein levels while high c-Fes expression abolished CD19 protein levels. Thus, we propose that a balance of c-Fes and Src signaling can regulate receptor maintenance, which may influence cellular outcome such as tumorigenesis or tumor suppression.

systems biology↗