Structural and biochemical characterization establishes a detailed understanding of KEAP1-CUL3 complex assembly

KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function

Molecular architecture of Streptococcus pneumoniae surface thioredoxin-fold lipoproteins crucial for extracellular oxidative stress resistance and maintenance of virulence.

19 pags, 8 figs, tabsThe respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we character ize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their r edox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the w ay for the design of novel a ntimicrobialsThe authors thank the PXIII beamline at SLS and the ESRF beamline ID14‐1 for access to synchrotron radiation. We are also grateful to Kristine Sievert‐Giermann, Nadine Gotzmann and Melanie Skibbe (Department of Genetics, University of Greifswald, Germany) for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG HA3125/4‐2 (to S.H.), DFG AN746/3‐1 (to H.A.), BFU2011‐25326 and S2010/BMD‐2457 (to J.A.H.) and EU FP7 CAREPNEUMO Grant EU‐CP223111 from the European Union (to J.A.H. and S.H.

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

Target highlights in CASP9: Experimental target structures for the critical assessment of techniques for protein structure prediction

15 pags, 9 figsOne goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae. © 2011 Wiley-Liss, Inc.Grant sponsor: Spanish Ministry of Education and Science; Grant number: BFU2008-01588; Grant sponsor: European Commission; Grant number: NMP4-CT-2006-033256; Grant sponsor: Spanish Ministry of Education and Science (José Castillejo fellowship); Grant sponsor: Xunta de Galicia (Angeles Alvariño fellowship); Grant sponsor: National Institutes of Health; Grant numbers: K22-CA124517 (D.E.C.); R01-GM090161 (C.K.) GM074942; GM094585; Grant sponsor: U. S. Department of Energy, Office of Biological and Environmental Research; Grant number: DE-AC02-06CH11357 (to A.J.); Grant sponsor: Foundation for Polish Science (to K.M.); Grant sponsor: NSF; Grant number: DBI 0829586

Two-chaperone assisted soluble expression and purification of the bacteriophage T4 long tail fibre protein gp37

Bacteriophage T4 recognises its host cells through its long tail fibre protein gene product (gp) 37. Gp37 is a protein containing 1026 amino acids per monomer, forming a fibrous parallel homotrimer at the distal end of the long tail fibres. The other distal half-fibre protein, gp36, is much smaller, forming a trimer of 221 amino acids per monomer. Functional and structural studies of gp37 have been hampered by the inability to produce suitable amounts of it. We produced soluble gp37 by co-expression with two bacteriophage T4-encoded chaperones in a two-vector system; co-expression with each chaperone separately did not lead to good amounts of correctly folded, trimeric protein. An expression vector for the bacteriophage T4 fibrous protein chaperone gp57 was co-transformed into bacteria with a compatible bi-cistronic expression vector containing bacteriophage T4 genes 37 and 38. A six-histidine tag is encoded amino-terminal to the gp37 gene. Recombinant trimeric gp37, containing the histidine tag and residues 12-1026 of gp37, was purified from lysed bacteria by subsequent nickel-affinity, size exclusion and strong anion exchange column chromatography. Yields of approximately 4 mg of purified protein per litre of bacterial culture were achieved. Electron microscopy confirmed the protein to form fibres around 63 nm long, presumably gp36 makes up the remaining 11 nm in the intact distal half-fibre. Purified, correctly folded, gp37 will be useful for receptor-binding studies, high-resolution structural studies and for specific binding and detection of bacteria. © 2009 Elsevier Inc. All rights reserved.This research was sponsored by the European Commission Sixth Framework Programme under contract grant number NMP4-CT-2006-033256 and, as part of the European Science Foundation EUROCORES Programme EuroSCOPE, by contract ERAS-CT-2003-980409.Peer Reviewe

Pneumococcal surface proteins: when the whole is greater than the sum of its parts

25 pags., 5 figs., 4 tabs.Surface-exposed proteins of pathogenic bacteria are considered as potential virulence factors through their direct contribution to host-pathogen interactions. Four families of surface proteins decorate the cell surface of the human pathogen Streptococcus pneumoniae. Besides lipoproteins and LPXTG proteins, also present in other gram-positive bacteria, the pneumococcus presents the choline-binding protein (CBP) family and the non-classical surface proteins (NCSPs). The CBPs present specific structural features that allow their anchorage to the cell envelope through non-covalent interaction with choline residues of lipoteichoic acid and teichoic acid. NCSP is an umbrella term for less characterized proteins displaying moonlighting functions on the pneumococcal surface that lack a leader peptide and membrane-anchor motif. Considering the unceasing evolution of microbial species under the selective pressure of antibiotic use, detailed understanding of the interaction between pathogen and the host cells is required for the development of novel therapeutic strategies to combat pneumococcal infections. This article reviews recent progress in the investigation of the three-dimensional structures of surface-exposed pneumococcal proteins. The modular nature of some of them produces a great versatility and sophistication of the virulence functions that, in most cases, cannot be deduced by the structural analysis of the isolated modules.This work was supported by agrant from the Spanish Ministry of Economy and Competitiveness (BFU2011-25326), EU-CP223111(CAREPNEUMO, European Union), and the biomedicine program of the government of the autonomous community of Madrid (S2010/BMD-2457). IP-D isfunded by a FEBS long-term fellowship.Peer reviewe

Crystal structure of enoylpyruvate transferase in Streptococcus pneumoniae

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Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

43 p.-5 fig.Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled–coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.This work was supported by grants BFU2011-25326 and S2010/BMD-2457 and grants from the Research Council of Norway.Peer reviewe