Molecular basis of the final step of cell division in Streptococcus pneumoniae

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.We thank the staff from the ALBA synchrotron facilities for their help during crystallographic data collection. We gratefully thank Pedro Garcia (CIB, Madrid, Spain) for providing us with the plasmid allowing overproduction of GFP-LytB. This work was supported by grants from the CNRS, the University of Lyon, the Agence National de la Recherche (ANR-18-CE11-0017-02 and ANR-19-CE15-0011-01), and the Bettencourt Schueller Foundation to C.G. The work in Spain was supported by grants BFU2017-90030-P and PID2020-115331GB-100 to J.A.H., funded by MCIN/AEI/10.13039/501100011033. The work in the United States was supported by a grant from the National Institutes of Health (GM131685). J.A.H. and C.G. supervised this work and share last authorship.Peer reviewe

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

Cloning, purification, crystallization and preliminary X-ray analysis of a bacterial GABA receptor with a Venus flytrap fold

A 1.35 Å resolution data set was collected from a crystal of the periplasmic GABA receptor Atu2422 from A. tumefaciens. Atu2422 adopts a closed Venus flytrap conformation

X-ray structure of a domain-swapped dimer of Ser46-phosphorylated Crh from Bacillus subtilis

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NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

AbstractNfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD+ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H2O2 produced during the oxidative process or added exogenously.Structured summaryMINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystallography (MI:0114

Nucleotide-free Crystal Structure of Nucleotide-binding Domain 1 from Human ABCC1 Supports a ‘General-Base Catalysis’ Mechanism for ATP Hydrolysis

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HPr kinase/phosphorylase, a Walker motif A-containing bifunctional sensor enzyme controlling catabolite repression in Gram-positive bacteria

International audienceCarbon catabolite repression (CCR) in Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase (HprK/P). This enzyme catalyses the ATP- as well as the pyrophosphate-dependent phosphorylation of Ser-46 in HPr, a phosphocarrier protein of a sugar transport and phosphorylation system. HprK/P also catalyses the pyrophosphate-producing, inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr (P-Ser-HPr). P-Ser-HPr functions as catabolite co-repressor by interacting with the LacI/GalR-type repressor, catabolite control protein A (CcpA), and allowing it to bind to operator sites preceding catabolite-regulated transcription units. HprK/P thus indirectly controls the expression of about 10% of the genes of Gram-positive bacteria. The two antagonistic activities of HprK/P are regulated by intracellular metabolites, which change their concentration in response to the absence or presence of rapidly metabolisable carbon sources (glucose, fructose, etc.) in the growth medium. Biochemical and structural studies revealed that HprK/P exhibits no similarity to eukaryotic protein kinases and that it contains a Walker motif A (or P-loop) as nucleotide binding site. Interestingly, HprK/P has a structural fold resembling that in kinases phosphorylating certain low molecular weight substrates such as nucleosides, nucleotides or oxaloacetate. The structures of the complexes of HprK/P with HPr and P-Ser-HPr have also been determined, which allowed proposing a detailed mechanism for the kinase and phosphorylase functions of HprK/P

Structural analysis of the bacterial HPr kinase/phosphorylase V267F mutant gives insights into the allosteric regulation mechanism of this bifunctional enzyme.

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phosphocarrier protein HPr, which regulates the utilization of carbon sources in gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of P-Ser46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of P-loop proteins. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the X-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6 A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide-binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P