Sébastien Campagne

Structure-Function Analysis of the THAP Zinc Finger of THAP1, a Large C2CH DNA-binding Module Linked to Rb/E2F Pathways

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of ∼80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel β-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.

Role of Sphingomonas sp. Strain Fr1 PhyR-NepR-σEcfG Cascade in General Stress Response and Identification of a Negative Regulator of PhyR

The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor σ(EcfG), whose activity is regulated by its anti-sigma factor NepR. The response regulator PhyR, in turn, regulates NepR activity in a partner-switching mechanism according to which phosphorylation of PhyR triggers sequestration of NepR by the sigma factor-like effector domain of PhyR. Although genes encoding predicted histidine kinases can often be found associated with phyR, little is known about their role in modulation of PhyR phosphorylation status. We demonstrate here that the PhyR-NepR-σ(EcfG) cascade is important for multiple stress resistance and competitiveness in the phyllosphere in a naturally abundant plant epiphyte, Sphingomonas sp. strain Fr1, and provide evidence that the partner switching mechanism is conserved. We furthermore identify a gene, designated phyP, encoding a predicted histidine kinase at the phyR locus as essential. Genetic epistasis experiments suggest that PhyP acts upstream of PhyR, keeping PhyR in an unphosphorylated, inactive state in nonstress conditions, strictly depending on the predicted phosphorylatable site of PhyP, His-341. In vitro experiments show that Escherichia coli inner membrane fractions containing PhyP disrupt the PhyR-P/NepR complex. Together with the fact that PhyP lacks an obvious ATPase domain, these results are in agreement with PhyP functioning as a phosphatase of PhyR, rather than a kinase.

Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria.

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σEcfG, was identified. σEcfG is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σEcfG sigma factor-like output domain (PhyRSL). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyRSL–NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyRSL binding, NepR forms two helices that extend over the surface of the PhyRSL subdomains. Homology modeling and comparative analysis of NepR, PhyRSL, and σEcfG mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σEcfG–NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σEcfG. Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

Structural basis for −10 promoter element melting by environmentally induced sigma factors

Bacterial transcription is controlled by sigma factors, the RNA polymerase subunits that act as initiation factors. Although a single housekeeping sigma factor enables transcription from thousands of promoters, environmentally induced sigma factors redirect gene expression toward small regulons to carry out focused responses. Using structural and functional analyses, we determined the molecular basis of −10 promoter element recognition by Escherichia coli σE, which revealed an unprecedented way to achieve promoter melting. Group IV sigma factors induced strand separation at the −10 element by flipping out a single nucleotide from the nontemplate-strand DNA base stack. Unambiguous selection of this critical base was driven by a dynamic protein loop, which can be substituted to modify specificity of promoter recognition. This mechanism of promoter melting explains the increased promoter-selection stringency of environmentally induced sigma factors.

Extra Cytoplasmic Function sigma factors, recent structural insights into promoter recognition and regulation

Bacterial transcription initiation is controlled by sigma factors, the RNA polymerase (RNAP) subunits responsive for promoter specificity. While the primary sigma factor ensures the bulk of transcription during growth, a major strategy used by bacteria to regulate gene expression consists of modifying the RNAP promoter specificity by means of alternative sigma factors. Among these factors, Extra Cytoplasmic Function sigma factors (σ(ECF)) constitute the most abundant group and are generally kept inactive by specific anti-sigma factors that are directly or indirectly sensitive to environmental stimuli. When activated by anti-sigma factor release, σ(ECF) turn on the transcription of dedicated regulons, which trigger adaptive responses for the survival of the cell. Recent structural studies have deciphered the molecular basis for σ(ECF) promoter recognition and original regulatory mechanisms.

Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1

The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease.

NMR studies of a new family of DNA binding proteins: the THAP proteins

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.Small molecules correcting the splicing deficit of the survival of motor neuron 2 (SMN2) gene have been identified as having therapeutic potential. Here, the authors provide evidence that SMN2 mRNA forms a ribonucleoprotein complex that can be specifically targeted by these small molecules.

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa

Abstract In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria

Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous system from an obligate intracellular (rickettsial) pathogen. The NMR solution structure of the zinc-finger (ZnR) domain from the bifunctional and bipolar ZitP pilus assembly/motility regulator revealed conserved interaction determinants for PopZ, a bipolar matrix protein that anchors the ParB centromere-binding protein and other regulatory factors at the poles. We show that ZitP regulates cytokinesis and the localization of ParB and PopZ, targeting PopZ independently of the previously known binding sites for its client proteins. Through heterologous localization assays with rickettsial ZitP and PopZ orthologs, we document the shared ancestries, activities and structural determinants of a (bi-)polarization system encoded in free-living and obligate intracellular α-proteobacteria. DOI: http://dx.doi.org/10.7554/eLife.20640.001