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One function of Mediator complex subunit MED23 is to mediate transcriptional activation by the phosphorylated transcription factor Elk-1, in response to the Ras-MAPK signaling pathway. Using cryogenic electron microscopy, we solve a 3.0 Å structure of human MED23 complexed with the phosphorylated activation domain of Elk-1. Elk-1 binds to MED23 via a hydrophobic sequence PSIHFWSTLS P P containing one phosphorylated residue (S383 p ), which forms a tight turn around the central Phenylalanine. Binding of Elk-1 induces allosteric changes in MED23 that propagate to the opposite face of the subunit, resulting in the dynamic behavior of a 19-residue segment, which alters the molecular surface of MED23. We design a specific MED23 mutation (G382F) that disrupts Elk­-1 binding and consequently impairs Elk-1-dependent serum-induced activation of target genes in the Ras-Raf-MEK-ERK signaling pathway. The structure provides molecular details and insights into a Mediator subunit-transcription factor interface. The Mediator complex subunit MED23 contributes to transcriptional activation by the phosphorylated transcription factor Elk-1, in response to Ras-MAPK signalling. Here, the authors determine a cryo-EM structure of human MED23 with the phosphorylated activation domain of Elk-1 providing insights into the Mediator subunit-transcription factor interface.

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin. Haemophilus influenzae requires haem, and acquires it from host haemoproteins including haemopexin. Here, the authors examine the haem transport system consisting of HxuA, HxuB and HxuC via the structures of HxuA in complex with haemopexin.

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

In Gram-negative bacteria and eukaryotic organelles, β-barrel proteins of the outer membrane protein 85-two-partner secretion B (Omp85-TpsB) superfamily are essential components of protein transport machineries. The TpsB transporter FhaC mediates the secretion of Bordetella pertussis filamentous hemagglutinin (FHA). We report the 3.15 Å crystal structure of FhaC. The transporter comprises a 16-stranded β barrel that is occluded by an N-terminal α helix and an extracellular loop and a periplasmic module composed of two aligned polypeptide-transport-associated (POTRA) domains. Functional data reveal that FHA binds to the POTRA 1 domain via its N-terminal domain and likely translocates the adhesin-repeated motifs in an extended hairpin conformation, with folding occurring at the cell surface. General features of the mechanism obtained here are likely to apply throughout the superfamily.

The Mediator complex transduces regulatory information from enhancers to promoters and performs essential roles in the initiation of transcription in eukaryotes. Human Mediator comprises 26 subunits forming three modules termed Head, Middle and Tail. Here we present the 2.8 Å crystal structure of MED23, the largest subunit from the human Tail module. The structure identifies 25 HEAT repeats-like motifs organized into 5 α-solenoids. MED23 adopts an arch-shaped conformation, with an N-terminal domain (Nter) protruding from a large core region. In the core four solenoids, motifs wrap on themselves, creating triangular-shaped structural motifs on both faces of the arch, with extended grooves propagating through the interfaces between the solenoid motifs. MED23 is known to interact with several specific transcription activators and is involved in splicing, elongation, and post-transcriptional events. The structure rationalizes previous biochemical observations and paves the way for improved understanding of the cross-talk between Mediator and transcriptional activators. Mediator is a large multi-subunits complex essential to the regulation of transcription by RNA pol II. Here the authors report the crystal structure of MED23—one of the largest subunits of the complex together with MED1 and MED14—revealing a complex architecture and filling an important gap in the structural characterization of Mediator.

Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

Background The Ets‐1 transcription factor plays a primordial role in regulating the expression of numerous genes implicated in cancer progression. In a previous study, we revealed that poly(ADP‐ribose) polymerase‐1 (PARP‐1) inhibition by PJ‐34 results in Ets‐1 level increase in cells, which is related with cell death of Ets‐1‐expressing cancer cells. Aims The mechanism of the antitumor effect of PARP‐1 inhibition was investigated in the Ets‐1‐expressing MDA‐MB‐231 breast cancer cells. Methods and Results We tested the effects of four PARP inhibitors (PARPi) (PJ‐34, Veliparib, Olaparib, and Rucaparib). We first demonstrated that PARPi reduced cells growth through G2/M cell cycle arrest. Next, we evaluated PARP‐1 inhibition effect on oxidative DNA damage in Ets‐1‐overexpressing and Ets‐1‐non‐expressing breast cancer cells and we showed that PARPi led only Ets‐1‐overexpressing cells to accumulate it, which triggers the DNA damage response as revealed by the increase in the level of a panel of DNA damage‐related proteins. Importantly, we demonstrated that PARPi increased reactive oxygen species (ROS), only in Ets‐1‐overexpressing cells and this is accompanied by upregulation of p47phox expression, a subunit of the NAPDH oxidase (NOX). Conclusion These preliminary findings correlate PARPi‐induced oxidative DNA damage/oxidative stress to Ets‐1 expression in breast cancer cells.

Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cought agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria. FHA is secreted by means of the two-partner secretion (TPS) pathway. Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion. A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outer-membrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7-Å structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a β-helix, with three extrahelical motifs, a β-hairpin, a four-stranded β-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain. The structure thus explains why the TPS domain is able to initiate folding of the β-helical motifs that form the central domain of the adhesin, because it is itself a β-helical scaffold. It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter. This structure is representative of the TPS domains found so far in > 100 secreted proteins from pathogenic bacteria. It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.

Two-component sensory transduction systems control important bacterial programs. In Bordetella pertussis, expression of the virulence regulon is controlled by the unorthodox BvgAS two-component system. BvgS is the prototype of a family of sensor-kinases that harbor periplasmic domains homologous to bacterial solute-binding proteins. Although BvgAS is active under laboratory conditions, no activating signal has been identified, only negative modulators. Here we show that the second periplasmic domain of BvgS interacts with modulators and adopts a Venus flytrap (VFT) fold. X-ray crystallography reveals that the two lobes of VFT2 delimitate a ligand-binding cavity enclosing fortuitous ligands. Most substitutions of putative ligand-binding residues in the VFT2 cavity keep BvgS active, and alteration of the cavity's electrostatic potential affects responsiveness to modulation. The crystal structure of this VFT2 variant conferring constitutive kinase activity to BvgS shows a closed cavity with another nonspecific ligand. Thus, VFT2 is closed and active without a specific agonist ligand, in contrast to typical VFTs. Modulators are antagonists of VFT2 that interrupt signaling. BvgAS is active for most of the B. pertussis infectious cycle, consistent with the proposed mechanism.