Hybrid histidine kinase activation by cyclic di-GMP-mediated domain liberation

Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In; Caulobacter crescentus; , the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses, and kinetic modeling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudoreceiver domain Rec1, thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically invoke a downstream effect. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand and show that autophosphorylation is a reversible process

Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography

We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis

High-throughput screening of BAM inhibitors in native membrane environment

Abstract The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin had a tenfold higher potency than in synthetic membranes, highlighting the need for native conditions in antibiotics development. The assay is successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of antibiotics targeting BAM

The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis; 1-3; . Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets; 4,5; . The natural compound darobactin targets the bacterial insertase BamA; 6; -the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins; 7-13; . BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel

The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis; 1-3; . Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets; 4,5; . The natural compound darobactin targets the bacterial insertase BamA; 6; -the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins; 7-13; . BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel

Integrating data from multidisciplinary Management of Malignant Pleural Mesothelioma: a cohort study

none17Malignant pleural mesothelioma (MPM) is a rare and aggressive malignancy that most commonly affects the pleural layers. MPM has a strong association with asbestos, mainly caused by exposure to its biopersistent fibers in at least 80% of cases. Individuals with a chronic exposure to asbestos might develop disease with a 20-40-year latency with few or no symptoms. Such has been the case in the Italian regions of Piedmont and Lombardy, where industrial production of materials laden with asbestos, mainly cements, has been responsible for the onset of a large epidemic. Since 2018, a multidisciplinary team at San Matteo hospital in Pavia has been collecting data on over 100 patients with MPM. The main goal of this project is to define and describe an integrated profile for each MPM case at diagnosis by using data mining and partition analysis.restrictedSaracino, Laura; Bortolotto, Chandra; Tomaselli, Stefano; Fraolini, Elia; Bosio, Matteo; Accordino, Giulia; Agustoni, Francesco; Abbott, David M; Pozzi, Emma; Eleftheriou, Dimitrios; Morbini, Patrizia; Rinaldi, Pietro; Primiceri, Cristiano; Lancia, Andrea; Comoli, Patrizia; Filippi, Andrea R; Stella, Giulia MSaracino, Laura; Bortolotto, Chandra; Tomaselli, Stefano; Fraolini, Elia; Bosio, Matteo; Accordino, Giulia; Agustoni, Francesco; Abbott, David M; Pozzi, Emma; Eleftheriou, Dimitrios; Morbini, Patrizia; Rinaldi, Pietro; Primiceri, Cristiano; Lancia, Andrea; Comoli, Patrizia; Filippi, Andrea R; Stella, Giulia