Cell layer-specific expression of the homeotic MADS-box transcription factor PhDEF contributes to modular petal morphogenesis in petunia.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development.

MADS transcription factors are master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily of MADS transcription factors is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. SEPALLATAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro. To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes is sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization--impaired SEPALLATA mutant proteins. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS (AG) tetramerization domain, structure-based mutagenesis and complementation experiments in Arabidopsis (Arabidopsis thaliana) sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers can bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild-type binding sites genome-wide, tetramerization is required not only for floral meristem determinacy, but also for floral organ identity in the second, third and fourth whorls.

The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.

Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

BioSAXS at European Synchrotron Radiation Facility - Extremely Brilliant Source: BM29 with an upgraded source, detector, robot, sample environment, data collection and analysis software.

As part of its Extremely Brilliant Source (EBS) upgrade project, the ESRF's BM29 BioSAXS beamline was subject to a significant upgrade and refurbishment. In addition to the replacement of the beamline's original bending magnet source by a two-pole wiggler, leading to an increase in brilliance by a factor of 60, the sample environment of the beamline was almost completely refurbished: a vacuum-compatible Pilatus3 X 2M with a sensitive area of 253.7 mm × 288 mm and frame rates up to 250 Hz was installed, increasing the active area available and thus the q-scaling of scattering images taken; the sample changer was replaced with an upgraded version, allowing more space for customizable sample environments and the installation of two new sample exposure units; the software associated with the beamline was also renewed. In addition, the layout and functionality of the BSXCuBE3 (BioSAXS Customized Beamline Environment) data acquisition software was redesigned, providing an intuitive `user first' approach for inexperienced users, while at the same time maintaining more powerful options for experienced users and beamline staff. Additional features of BSXCuBE3 are queuing of samples; either consecutive sample changer and/or SEC-SAXS (size-exclusion chromatography small-angle X-ray scattering) experiments, including column equilibration were also implemented. Automatic data processing and analysis are now managed via Dahu, an online server with upstream data reduction, data scaling and azimuthal integration built around PyFAI (Python Fast Azimuthal Integration), and data analysis performed using the open source FreeSAS. The results of this automated data analysis pipeline are displayed in ISPyB/ExiSAXS. The upgraded BM29 has been in operation since the post-EBS restart in September 2020, and here a full description of its new hardware and software characteristics together with examples of data obtained are provided.

Molecular mechanisms of Evening Complex activity in Arabidopsis.

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing.

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3-AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3-AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3-AG tetramer. SEP3 and SEP3-AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3-AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3-AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

Unraveling the role of MADS transcription factor complexes in apple tree dormancy.

A group of MADS transcription factors (TFs) are believed to control temperature-mediated bud dormancy. These TFs, called DORMANCY-ASSOCIATED MADS-BOX (DAM), are encoded by genes similar to SHORT VEGETATIVE PHASE (SVP) from Arabidopsis. MADS proteins form transcriptional complexes whose combinatory composition defines their molecular function. However, how MADS multimeric complexes control the dormancy cycle in trees is unclear. Apple MdDAM and other dormancy-related MADS proteins form complexes with MdSVPa, which is essential for the ability of transcriptional complexes to bind to DNA. Sequential DNA-affinity purification sequencing (seq-DAP-seq) was performed to identify the genome-wide binding sites of apple MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained using a glucocorticoid receptor fusion system, and RNA-seq data related to apple dormancy. We describe a gene regulatory network (GRN) formed by MdSVPa-containing complexes, which regulate the dormancy cycle in response to environmental cues and hormonal signaling pathways. Additionally, novel molecular evidence regarding the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae is presented. MdSVPa sequentially forms complexes with the MADS TFs that predominate at each dormancy phase, altering its DNA-binding specificity and, therefore, the transcriptional regulation of its target genes.

Brassicaceae-specific Gretchen Hagen 3 acyl acid amido synthetases conjugate amino acids to chorismate, a precursor of aromatic amino acids and salicylic acid.

To modulate responses to developmental or environmental cues, plants use Gretchen Hagen 3 (GH3) acyl acid amido synthetases to conjugate an amino acid to a plant hormone, a reaction that regulates free hormone concentration and downstream responses. The model plant Arabidopsis thaliana has 19 GH3 proteins, of which 8 have confirmed biochemical functions. One Brassicaceae-specific clade of GH3 proteins was predicted to use benzoate as a substrate and includes AtGH3.7 and AtGH3.12/PBS3. Previously identified as a 4-hydroxybenzoic acid-glutamate synthetase, AtGH3.12/PBS3 influences pathogen defense responses through salicylic acid. Recent work has shown that AtGH3.12/PBS3 uses isochorismate as a substrate, forming an isochorismate-glutamate conjugate that converts into salicylic acid. Here, we show that AtGH3.7 and AtGH3.12/PBS3 can also conjugate chorismate to cysteine and glutamate, which act as precursors to aromatic amino acids and salicylic acid, respectively. The X-ray crystal structure of AtGH3.12/PBS3 in complex with AMP and chorismate at 1.94 Å resolution, along with site-directed mutagenesis, revealed how the active site potentially accommodates this substrate. Examination of Arabidopsis knockout lines indicated that the gh3.7 mutants do not alter growth and showed no increased susceptibility to the pathogen Pseudomonas syringae, unlike gh3.12 mutants, which were more susceptible than WT plants, as was the gh3.7/gh3.12 double mutant. The findings of our study suggest that GH3 proteins can use metabolic precursors of aromatic amino acids as substrates.

Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis.

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.

Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis.

In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A thaliana This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants.

CircRNAs in Plants.

Circular RNAs (circRNAs) are covalently closed, single-stranded transcripts that are ubiquitously expressed in all eukaryotes and even prokaryotic archaea. Although once regarded as splicing artifacts, circRNAs are a novel class of regulatory molecules with diverse biological functions, including regulation of transcription, modulation of alternative splicing, and binding of miRNAs and proteins. The majority of studies of circRNAs have been performed in animals with a focus on the biogenesis, function, and mechanistic characterization of these molecules. In contrast, the study of circRNAs in plants is just emerging. Interestingly, recent circRNA profiling studies in model plant systems show distinct features of plant circRNAs compared with those from animals, including putative roles in stress response, differences in expression patterns, and novel biogenesis mechanisms. This provides a great opportunity to broaden our knowledge of circRNAs using plant model systems, such as Arabidopsis and rice, which are ideal for phenotypic characterization and genetic studies. In this review, we summarize current knowledge of plant circRNAs, discuss their identification and biogenesis, describe potential functions, and propose future perspectives for plant circRNA study.

Pioneer Factors in Animals and Plants-Colonizing Chromatin for Gene Regulation.

Unlike most transcription factors (TF), pioneer TFs have a specialized role in binding closed regions of chromatin and initiating the subsequent opening of these regions. Thus, pioneer TFs are key factors in gene regulation with critical roles in developmental transitions, including organ biogenesis, tissue development, and cellular differentiation. These developmental events involve some major reprogramming of gene expression patterns, specifically the opening and closing of distinct chromatin regions. Here, we discuss how pioneer TFs are identified using biochemical and genome-wide techniques. What is known about pioneer TFs from animals and plants is reviewed, with a focus on the strategies used by pioneer factors in different organisms. Finally, the different molecular mechanisms pioneer factors used are discussed, highlighting the roles that tertiary and quaternary structures play in nucleosome-compatible DNA-binding.

Structural basis for the oligomerization of the MADS domain transcription factor SEPALLATA3 in Arabidopsis.

In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.

Intracellular Signaling and Desmoglein 2 Shedding Triggered by Human Adenoviruses Ad3, Ad14, and Ad14P1.

UNLABELLED: We recently discovered that desmoglein 2 (DSG2) is a receptor for human adenovirus species B serotypes Ad3, Ad7, Ad11, and Ad14. Ad3 is considered to be a widely distributed human pathogen. Ad3 binding to DSG2 triggers the transient opening of epithelial junctions. Here, we further delineate the mechanism that leads to DSG2-mediated epithelial junction opening in cells exposed to Ad3 and recombinant Ad3 fiber proteins. We identified an Ad3 fiber knob-dependent pathway that involves the phosphorylation of mitogen-activated protein (MAP) kinases triggering the activation of the matrix-metalloproteinase ADAM17. ADAM17, in turn, cleaves the extracellular domain of DSG2 that links epithelial cells together. The shed DSG2 domain can be detected in cell culture supernatant and also in serum of mice with established human xenograft tumors. We then extended our studies to Ad14 and Ad14P1. Ad14 is an important research and clinical object because of the recent appearance of a new, more pathogenic strain (Ad14P1). In a human epithelial cancer xenograft model, Ad14P1 showed more efficient viral spread and oncolysis than Ad14. Here, we tested the hypothesis that a mutation in the Ad14P1 fiber knob could account for the differences between the two strains. While our X-ray crystallography studies suggested an altered three-dimensional (3D) structure of the Ad14P1 fiber knob in the F-G loop region, this did not significantly change the fiber knob affinity to DSG2 or the intracellular signaling and DSG2 shedding in epithelial cancer cells. IMPORTANCE: A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 allows the virus not only to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus-triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber knob domain and triggers intracellular signaling that culminates in the cleavage of the extracellular domain of DSG2, thereby disrupting DSG2 homodimers between epithelial cells. We confirmed this pathway with a second DSG2-interacting serotype, Ad14, and its recently emerged strain Ad14P1. These new insights in basic adenovirus biology can be employed to develop novel drugs to treat adenovirus infection as well as be used as tools for gene delivery into epithelial tissues or epithelial tumors.

Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.

Structural and functional studies on the interaction of adenovirus fiber knobs and desmoglein 2.

Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. Our findings have practical relevance for cancer therapy. We have recently reported that an Ad3 fiber knob-containing recombinant protein (JO-1) is able to trigger opening of junctions between epithelial cancer cells which, in turn, greatly improved the intratumoral penetration and efficacy of therapeutic agents (I. Beyer, et al., Clin. Cancer Res. 18:3340-3351, 2012; I. Beyer, et al., Cancer Res. 71:7080-7090, 2011). Here, we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models.

Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase.

It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.