Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS

This is the author accepted manuscriptVirulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phophosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility and virulence. GacS is a membraneassociated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S-helix) and the DHp subdomain. The HAMP and S-helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.ANR project REGALADANR project MIdiaZONEVaincre la mucovisicdose - Gregory LemarchalFrench Infrastructures for Integrated Structural BiologyFrench Infrastructures for ProteomicsGIS IBiSARégion AlsaceCNRSAix-Marseille UniversityUniversity of Strasbour

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer–dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer–dimer contacts. We conclude that inducer-dependent dimer–dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members

What Glues a Homodimer Together Systematic Analysis of the Stabilizing Effect of an Aromatic Hot Spot in the Protein Protein Interface of the tRNA Modifying Enzyme Tgt

Shigella bacteria constitute the causative agent of bacillary dysentery, an acute inflammatory disease causing the death of more than one million humans per year. A null mutation in the tgt gene encoding the tRNA modifying enzyme tRNA guanine transglycosylase Tgt was found to drastically decrease the pathogenicity of Shigella bacteria, suggesting the use of Tgt as putative target for selective antibiotics. The enzyme is only functionally active as a homodimer; thus, interference with the formation of its protein protein interface is an attractive opportunity for therapeutic intervention. To better understand the driving forces responsible for the assembly, stability, and formation of the homodimer, we studied the properties of the residues that establish the dimer interface in detail. We performed site directed mutagenesis and controlled shifts in the monomer dimer equilibrium ratio in solution in a concentration dependent manner by native mass spectrometry and used crystal structure analysis to elucidate the geometrical modulations resulting from mutational variations. The wild type enzyme exhibits nearly exclusive dimer geometry. A patch of four aromatic amino acids, embedded into a ring of hydrophobic residues and further stabilized by a network of H bonds, is essential for the stability of the dimer s contact. Accordingly, any perturbance in the constitution of this aromatic patch by nonaromatic residues reduces dimer stability significantly, with some of these exchanges resulting in a nearly exclusively monomeric state. Apart from the aromatic hot spot, the interface comprises an extended loop helix motif that exhibits remarkable flexibility. In the destabilized mutated variants, the loop helix motif adopts deviating conformations in the interface region, and a number of water molecules, penetrating into the interface, are observe

Investigating Ugi/Passerini Multicomponent Reactions for the Site‐Selective Conjugation of Native Trastuzumab

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Functional insights from structural studies of protein arginine methyltransferase 2

FWN – Publicaties zonder aanstelling Universiteit Leide

Use of virtual screening for discovering antiretroviral compounds interacting with the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NC) is considered as an emerging drug target for the therapy of AIDS. Several studies have highlighted the crucial role of NC within the viral replication cycle. However, although NC inhibition has provided in vitro and in vivo antiretroviral activity, drug-candidates which interfere with NC functions are still missing in the therapeutic arsenal against HIV. Based on previous studies, where the dynamic behavior of NC and its ligand binding properties have been investigated by means of computational methods, here we used a virtual screening protocol for discovering novel antiretroviral compounds which interact with NC. The antiretroviral activity of virtual hits was tested in vitro, whereas biophysical studies elucidated the direct interaction of most active compounds with NC(11-55), a peptide corresponding to the zinc finger domain of NC. Two novel antiretroviral small molecules capable of interacting with NC are presented here

Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly

International audienceDespite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reductionoxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG 1), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays)