Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions.

Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

Multicomponent Synthesis of the SARS-CoV-2 Main Protease Inhibitor Nirmatrelvir.

In the wake of the Covid-19 pandemic, it has become clear that global access to efficacious antiviral drugs will be critical to combat future outbreaks of SARS-CoV-2 or related viruses. The orally available SARS-CoV-2 main protease inhibitor nirmatrelvir has proven an effective treatment option for Covid-19, especially in compromised patients. We report a new synthesis of nirmatrelvir featuring a highly enantioselective biocatalytic desymmetrization (>99% ee) and a highly diastereoselective multicomponent reaction (>25:1 dr) as the key steps. Our route avoids the use of transition metals and peptide coupling reagents, resulting in an overall highly efficient and atom-economic process.

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA.

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.

Enantioselective Total Synthesis of (-)-Limaspermidine and (-)-Kopsinine by a Nitroaryl Transfer Cascade Strategy.

We report an intramolecular conjugate addition/Truce-Smiles/E1cb cascade of 2-nitrobenzenesulfonamide-functionalized cyclohexenones as a new entry to the core scaffold of monoterpene indole alkaloids. The method was applied to the asymmetric total synthesis of (-)-limaspermidine, (-)-kopsinilam, and (-)-kopsinine, as well as the framework of the kopsifoline alkaloids, thus highlighting its complementarity to existing approaches involving the use of indole-based starting materials or the interrupted Fischer indole synthesis. Furthermore, we show that the cascade tolerates various substituents on the nitroarene, opening the way to other natural products as well as non-natural analogues.

MMP activation-associated aminopeptidase N reveals a bivalent 14-3-3 binding motif.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3-binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3-binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Bicyclic ß-Sheet Mimetics that Target the Transcriptional Coactivator ß-Catenin and Inhibit Wnt Signaling.

Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.

RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers.

The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light-up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell-permeable small-molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP-tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA-RNA and RNA-protein interactions using split-FLAP and Förster resonance energy transfer (FRET) systems is given.

Constrained Peptides with Fine-Tuned Flexibility Inhibit NF-Y Transcription Factor Assembly.

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.

In†Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme.

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface-exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability.

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function.

The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo- and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B ß-sheet surface including a highly conserved tryptophan (Trp448(mPER1), Trp419(mPER2), Trp359(mPER3)). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.

Redox Modulation of PTEN Phosphatase Activity by Hydrogen Peroxide and Bisperoxidovanadium Complexes.

PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2 O2 ) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions.

Covalent bicyclization of protein complexes yields durable quaternary structures.

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.

Vaccination with a bacterial peptide conjugated to SARS-CoV-2 receptor-binding domain accelerates immunity and protects against COVID-19.

Poor immunogenicity of critical epitopes can hamper vaccine efficacy. To boost immune recognition of non- or low-immunogenic antigens, we developed a vaccine platform based on the conjugation of a target protein to a chimeric designer peptide (CDP) of bacterial origin. Here, we exploited this immune Boost (iBoost) technology to enhance the immune response against the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. Despite its fundamental role during viral infection, RBD is only moderately immunogenic. Immunization studies in mice showed that the conjugation of CDP to RBD induced superior immune responses compared to RBD alone. CDP-RBD elicited cross-reactive antibodies against the variants of concern Delta and Omicron. Furthermore, hamsters vaccinated with CDP-RBD developed potent neutralizing antibody responses and were fully protected from lung lesion formation upon challenge with SARS-CoV-2. In sum, we show that the iBoost conjugate vaccine technology provides a valuable tool for both quantitatively and qualitatively enhancing anti-viral immunity.

Construct optimization for studying protein complexes: obtaining diffraction-quality crystals of the pseudosymmetric PSPC1-NONO heterodimer.

The methodology of protein crystallography provides a number of potential bottlenecks. Here, an approach to successful structure solution of a difficult heterodimeric complex of two human proteins, paraspeckle component 1 (PSPC1) and non-POU domain-containing octamer-binding protein (NONO), that are involved in gene regulation and the structural integrity of nuclear bodies termed paraspeckles is described. With the aid of bioinformatic predictions and systematic screening of a panel of constructs, bottlenecks of protein solubility, crystallization, crystal quality and crystallographic pseudosymmetry were overcome in order to produce crystals that ultimately revealed the structure.

FRET Analysis of RNA -Protein Interactions Using Spinach Aptamers.

The method development to analyze direct RNA-protein interaction is of high importance as not many homogeneous assay formats are available.The discovery of fluorescent light-up aptamers (FLAPs), short RNA aptamers that switch the fluorescence of small, cell-permeable, and nontoxic organic chromophores on, paved the road for their utilization in direct RNA -protein interactions. The combination with fluorescent proteins as biological fluorophores enabled the development of homogeneous assays that are in principle even encodable on genomic level.Here the rules and methods to design a homogeneous in vitro assay for the detection and quantification of a direct RNA -protein interaction will be described. The design and application of a homogeneous assay to observe and quantify the interaction of the Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) with its binding RNA sequence (pp7-RNA) will be shown. For this, the Spinach-DFHBI aptamer as RNA fusion and the red fluorescent mCherry as protein fusion was used.The methods presented here do not require any chemical modification of proteins or RNAs which make them relatively easy to use and to adopt on other systems. As all fluorophores are fusion tags to the according biomolecules, standard cloning strategies and molecular biology technologies are sufficient and make this method available for a broad community of researchers.

Adapting free energy perturbation simulations for large macrocyclic ligands: how to dissect contributions from direct binding and free ligand flexibility.

Large and flexible ligands gain increasing interest in the development of bioactive agents. They challenge the applicability of computational ligand optimization strategies originally developed for small molecules. Free energy perturbation (FEP) is often used for predicting binding affinities of small molecule ligands, however, its use for more complex ligands remains limited. Herein, we report the structure-based design of peptide macrocycles targeting the protein binding site of human adaptor protein 14-3-3. We observe a surprisingly strong dependency of binding affinities on relatively small variations in substituent size. FEP was performed to rationalize observed trends. To account for insufficient convergence of FEP, restrained calculations were performed and complemented with extensive REST MD simulations of the free ligands. These calculations revealed that changes in affinity originate both from altered direct interactions and conformational changes of the free ligand. In addition, MD simulations provided the basis to rationalize unexpected trends in ligand lipophilicity. We also verified the anticipated interaction site and binding mode for one of the high affinity ligands by X-ray crystallography. The introduced fully-atomistic simulation protocol can be used to rationalize the development of structurally complex ligands which will support future ligand maturation efforts.