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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An Integrative Approach Combining Noncovalent Mass Spectrometry, Enzyme Kinetics and X ray Crystallography to Decipher Tgt Protein Protein and Protein RNA Interaction

The tRNA modifying enzyme tRNA guanine transglycosylase Tgt is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used i to confirm the dimeric oligomerization state of Tgt in solution and ii to evidence the binding stoichiometry of the complex formed between Tgt and its full length substrate tRNA. To further investigate the importance of Tgt protein protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein protein interaction, which could provide a new way to inhibit Tg

Online collision-induced unfolding of therapeutic monoclonal antibody glyco-variants through direct hyphenation of cation exchange chromatography with native ion mobility-mass spectrometry

Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX-CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX-CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX-CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.</p

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