Refine
Year of publication
Document Type
- Article (18)
- Part of Periodical (1)
- Preprint (1)
Language
- English (20)
Has Fulltext
- yes (20) (remove)
Is part of the Bibliography
- no (20) (remove)
Keywords
- NMR spectroscopy (4)
- SARS-CoV-2 (4)
- Non-structural protein (3)
- Solution NMR-spectroscopy (3)
- Covid19-NMR (2)
- Protein drugability (2)
- Bacterial Protein Kinases (1)
- Bacterial Protein Phosphatases (1)
- C-mannosylation (1)
- COVID-19 (1)
Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.
Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.
Retournement or turning of the aedeagus about its longitudinal axis through about 180o during development is known in Chrysomeloidea (Coleoptera). This change in the orientation of the organ may be observed during the postembryonic development. This change produces certain morphological effects. By observing these morphological features in the imago the retournement may be inferred. Such morphological features in Curculionidae (Coleoptera) are here recorded. From this it has been inferred not only that retournement of the aedeagus is included in the ontogeny of curculionids, but also that the change of orientation of the organ occurs by the same mechanism as in Chrysomeloidea. These inferences attest the notion of a close phyletic relationship between the superfamilies Curculionoidea and Chrysomeloidea.
Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR‐active nuclei. We report on the incorporation of 13C‐labeled mannose in the C‐mannosylated UNC‐5 thrombospondin repeat. The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of UNC‐5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C‐mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus’ proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.
The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.
Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX5R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.
The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR),[1, 2] a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors.
Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.
The multistep PROTAC (PROteolysis TArgeting Chimeras) degradation process poses challenges for their rational development, as rate limiting steps determining PROTAC efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here we developed cell-based assays using NanoLuciferase and HaloTags, that allow measuring PROTAC induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for degradation of WDR5, the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC induced degradation pathway.
Significance The multistep PROTAC induced degradation process of a POI poses a significant challenge for the rational design of these bifunctional small molecules as critical steps that limit PROTAC efficacy cannot be easily assayed at required throughput. In addition, the cellular location of the POI may pose additional challenges as some cellular compartments, such as the nucleus, may not be easily reached by PROTAC molecules and the targeted E3 ligases may not be present in this cellular compartment. We propose therefore a comprehensive assay panel for PROTACs evaluation in cellular environments using a sensor system that allows continuous monitoring of the protein levels of the endogenous POI. We developed a cell line expressing WDR5 from its endogenous locus in fusion with a small sequence tag (HiBIT) that can be reconstituted to functional NanoLuciferase (NLuc). This system allowed continuous monitoring of endogenous WDR5 levels in cells and together with HaloTag system also the continuous monitoring of ternary complex (E3, WDR5 and PROTAC) formation. As this assay can be run at high throughput, we used this versatile system monitoring three diverse chemical series of WDR5 PROTACs that markedly differ in their degradation properties. Monitoring cell penetration, binary complex formation (PROTAC-WDR5 and PROTAC-VHL) as well as ternary complex formation we found that PROTAC efficiency highly correlated with synergy of ternary complex formation in cells. This study represents a first data set on diverse PROTACs studying this property in cellulo and it outlines a strategy for the rational optimization of PROTACs. It also provided kinetic data on ternary complex assembly and dissociation that may serve as a benchmark for future studies utilizing also kinetic properties for PROTAC development. Comparative structural studies revealed larger PROTAC mediated interaction surfaces for PROTACs that efficiently formed ternary complexes highlighting the utility of structure based optimization of PROTAC induced ternary complexes in the development process.
A toolbox for the generation of chemical probes for Baculovirus IAP Repeat containing proteins
(2022)
E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.
Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10
(2020)
The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.
The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.