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Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors. Fluorescence proximity sensing enables high throughput determination of binding affinities and kinetics of peptide inhibitors with varying valency and multivalent architecture.

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements. The comprehensive bioanalysis of proteins usually requires multi-step surface and mobile phase measurements. Here, the authors use chips functionalized with dynamically actuated nanolevers—DNA strands that can be switched in an electric field—to obtain motional dynamic measurements of proteins on a chip.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of β2-glycoprotein I (β2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of β2-GPI to the cardiolipin surface. Using the switchSENSE sensor, β2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between β2-GPI-dependent autoimmune- and β2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Objectives We set out to investigate the benefit of distal main or side branch treatment with a DCB compared to POBA in coronary bifurcation lesions. Background The standard treatment of bifurcation lesions is application of a DES to the main branch with provisional side branch stenting. While this resulted in considerable improvement in overall MACE rate suboptimal side branch results remained a problem. Methods The study was performed from 2011 to 2013 in six German centers. Native bifurcation lesions were included if side branch vessel diameter was ≥2 and ≤3.5 mm and no proximal main branch lesions was found. After successful predilatation randomization was performed to either DCB application or no further treatment. Follow-up angiograms for QCA analysis were done after 9 months. Primary endpoint was late lumen loss (LLL). Results 64 patients were successfully randomized. Minimal lumen diameter and grade of stenosis were equal in both groups. Only five stents were used as bail out. Angiographic follow-up was achieved in 75 % of patients. No patient died. There was one NSTEMI in the POBA group. Restenosis rate was 6 % in the DCB group vs 26 % in the POBA group ( p  = 0.045). TLR was necessary in one patient of the DCB group vs three patients of the POBA. The primary endpoint LLL was 0.13 mm in the DCB vs 0.51 mm in the POBA group ( p  = 0.013). Conclusion In bifurcation lesions that show only class A or B dissection and recoil not beyond 30 % the use of DCBs is a sound strategy.

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m 7 G‐cap‐dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m 7 G‐cap is hypermethylated to the 2,2,7‐trimethylguanosine (m 3 G)‐cap. The import adaptor snurportin1 recognises the m 3 G‐cap and facilitates the nuclear import of the UsnRNPs by binding to importin‐β. Here we report the crystal structure of the m 3 G‐cap‐binding domain of snurportin1 with bound m 3 GpppG at 2.4 Å resolution, revealing a structural similarity to the mRNA‐guanyly‐transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m 7 G‐cap‐binding proteins Cap‐binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m 3 G‐cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m 7 G‐capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap‐binding activity tests using several snurportin1 mutants.

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease 1 . Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella -infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes. Inhibition of BCL-XL eliminates Legionella infection, suggesting that host-directed BH3-mimetic therapy may be effective against intracellular pathogens that inhibit host cell protein synthesis.

A series of 51 5-HT 2A partial agonistic arylethylamines (primary or benzylamines) from different structural classes (indoles, methoxybenzenes, quinazolinediones) was investigated by fragment regression analysis (FRA), docking and 3D-QSAR approaches. The data, pEC 50 values and intrinsic activities (E max ) on rat arteries, show high variability of pEC 50 from 4 to 10 and of E max from 15 to 70%. FRA indicates which substructures affect potency or intrinsic activity. The high contribution of halogens in para position of phenethylamines to pEC 50 points to a specific hydrophobic pocket. Other results suggest the significance of hydrogen bonds of the aryl moiety for activation and the contrary effect of benzyl groups on affinity (increasing) and intrinsic activity (decreasing). Results from fragment regression and data on all available mutants were considered to derive a common binding site at the rat 5-HT 2A receptor. After generation and MD simulations of a receptor model based on the β 2 -adrenoceptor structure, typical derivatives were docked, leading to the suggestion of common interactions, e.g., with serines in TM3 and TM5 and with a cluster of aromatic amino acids in TM5 and TM6. The whole series was aligned by docking and minimization of the complexes. The pEC 50 values correlate well with Sybyl docking energies and hydrophobicity of the aryl moieties. With this alignment, CoMFA and CoMSIA approaches based on a training set of 36 and a test set of 15 compounds were performed. The correlation of pEC 50 with steric, electrostatic, hydrophobic and H-bond acceptor fields resulted in sufficient fit ( q 2 : 0.75–0.8, r 2 : 0.92–0.95) and predictive power ( r pred 2 : 0.85–0.88). The important interaction regions largely reflect the patterns provided by the putative binding site. In particular, the fit of the aryl moieties and benzyl substituents to two hydrophobic pockets is evident.