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The constantly growing world population leads to increasing demands for food, which challenges modern agriculture manifold. Pests, such as weeds, require the application of agrochemicals to increase crop yield. Due to the environmental impact of these potentially hazardous chemicals, the demand for more efficient formulations is increasing. Promising formulations consist of easily adaptable carriers from which controllable stimuli release the agrochemicals. Here, we investigated poly(N–vinylcaprolactam) (pVCL)-based microgels as a potential carrier for okanin, an inhibitor of the C4 plant key enzyme phosphoenolpyruvate carboxylase, by combining experiments, molecular simulations, and free energy computations. Dynamic light scattering, scanning transmission electron and atomic force microscopy revealed that pVCL microgels collapse and rigidify upon the loading of okanin. The simulations identified loosely adsorbed okanin and tightly bound okanin mediating inter-chain crosslinks. With increasing okanin concentration, stacking interactions of okanin occur with adsorbed and bound okanin. These findings can explain the experimentally observed collapse and the rigidification of the microgels. Based on the atomistic insights, two poly(N–vinylcaprolactam–co–glycidyl methacrylate) microgels were synthesized, for which a doubled loading capacity of okanin was found. Finally, we investigated the triggered release of okanin using the addition of green solvents as a stimulus both in vitro and in planta. This work establishes a basis for the further optimization of pVCL-based microgels as a carrier for the delivery of polyphenolic agrochemicals.

Chemogenetic engineering of metalloproteins emerges as a powerful strategy to generate proteins that catalyze non-natural reactions or convert non-natural substrates. Here, we report on an artificial metalloenzyme (ArM) based on the β-barrel protein nitrobindin (NB) equipped with a manganese protoporphyrin IX catalyst (MnPPIX@NB) for the epoxidation of aromatic alkenes. After exchanging the metal cofactor and two rounds of directed evolution, our ArM shows improved activity (>7-fold increase) with an ee of 20%. The evolution campaign also revealed the importance of the proximal ligand for peroxide activation and subsequent oxygen atom transfer. By utilizing cell adhesion-promoting peptides, a facile strategy is presented to immobilize ArMs on the surface of E. coli cells for on-cell catalysis and chemogenetic engineering of ArMs.

Herein, we present the first spectroscopic characterization of the Cu(I) active site of the plant ethylene receptor ETR1. The X-ray absorption (XAS) and extended X-ray absorption fine structure (EXAFS) spectroscopy presented here establish that ETR1 has a low-coordinate Cu(I) site. The EXAFS resolves a mixed first coordination sphere of N/O and S scatterers at distances consistent with potential histidine and cysteine residues. This finding agrees with the coordination of residues C65 and H69 to the Cu(I) site, which are critical for ethylene activity and well-conserved. Further, the Cu K-edge XAS and EXAFS of ETR1 exhibit spectroscopic changes upon addition of ethylene that are attributed to modifications in the Cu(I) coordination environment, suggestive of ethylene binding. Results from umbrella sampling simulations of the proposed ethylene binding helix of ETR1 at a mixed quantum mechanics/molecular mechanics (QM/MM) level agree with the EXAFS fit distance changes upon ethylene binding, particularly in the increase of the distance between H69 and Cu(I), and yield binding energetics comparable to experimental dissociation constants. The observed changes in the copper coordination environment might be the triggering signal for the transmission of the ethylene response.

The plant ethylene receptor ETR1 is a key player in the perception of the phytohormone and subsequent downstream ethylene signal transmission, crucial for processes such as ripening, senescence and abscission. However, to date, there is sparse structural knowledge about the transmembrane sensor domain (TMD) of ETR1 that is responsible for the binding of the plant hormone and initiates the downstream signal transmission. Sequence information and ab initio modelling suggest that the TMD consists of three transmembrane helices. Here, we combined site-directed spin labelling with electron paramagnetic resonance spectroscopy and obtained distance restraints for liposome-reconstituted ETR1_TMD on the orientation and arrangement of the transmembrane helices. We used these data to scrutinize different computational structure predictions of the TMD. published

Funding Information: This work was supported by a grant by the state of North-Rhine Westphalia and the European Fonds for Regional Development EFRE.NRW 2014-2020 to HG. We are grateful for computational support and infrastructure provided by the “Zentrum für Informations- und Medientechnologie” (ZIM) at the Heinrich Heine University Düsseldorf and the computing time provided by the John von Neumann Institute for Computing (NIC) to HG on the supercomputer JUWELS at Jülich Supercomputing Centre (JSC) (user ID: HKF7, VSK33). Financial support by Deutsche Forschungsgemeinschaft (DFG) through funds (INST 208/704-1 FUGG to HG) to purchase the hybrid computer cluster used in this study, an Emmy-Noether- and Heisenberg fellowship to ME (ET 103/2 and ET 103/5), and GRK 2158/2 (project number 270650915) to HG is gratefully acknowledged. Funding Information: This work was supported by a grant by the state of North-Rhine Westphalia and the European Fonds for Regional Development EFRE.NRW 2014-2020 to HG. We are grateful for computational support and infrastructure provided by the “Zentrum für Informations- und Medientechnologie” (ZIM) at the Heinrich Heine University Düsseldorf and the computing time provided by the John von Neumann Institute for Computing (NIC) to HG on the supercomputer JUWELS at Jülich Supercomputing Centre (JSC) (user ID: HKF7, VSK33). Financial support by Deutsche Forschungsgemeinschaft (DFG) through funds (INST 208/704-1 FUGG to HG) to purchase the hybrid computer cluster used in this study, an Emmy-Noether- and Heisenberg fellowship to ME (ET 103/2 and ET 103/5), and GRK 2158/2 (project number 270650915) to HG is gratefully acknowledged. Publisher Copyright: © 2022, The Author(s). Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML. publishersversion published

The synthetic octapeptide NOP-1 inhibits ethylene downstream signalling by disrupting the ETR1- EIN2 interaction, resulting in ripening delay of tomatoes when applied at early ripening stages. In order to better understand the NOP-1 induced ripening delay, we investigated the effect of a repeated surface application of NOP-1 as well as its application at advanced maturity stages of tomatoes. For this, analytic and non-destructive evaluation parameters were used. NOP-1 delayed ripening when applied at the maturity stage breakers (BR) as indicated by colour development. Repeated application had no additional delaying effect and did not reach the level of ripening delay induced by 1-MCP. When applied at maturity stage pink (PI), no ripening delay was observed, indicating that NOP-1 works best when applied at early ripening stages, long before autocatalytic ethylene synthesis takes place. In conclusion, peptide-based impairment of ethylene signalling offers new possibilities for ripening control of climacteric fruits. Thereby, method and timing of peptide application need to be considered.

Background Processes extending the shelf life of climacteric fruit play an important role in terms of a sustainable global food supply. In a previous study, a synthetic octapeptide (NOP-1) was shown to inhibit the interaction between ethylene receptor (ETR) and ethylene insensitive-2 (EIN2), and in consequence delay tomato ripening. We investigated for the first time the effect of NOP-1 on inhibiting the ripening of apples ('Golden Delicious') during postharvest. Results Using purified recombinant proteins from a bacterial expression system, we demonstrate here that EIN2 also interacts tightly (Kd = 136 ± 29 nmol L-1 ) with the corresponding apple ETR MdETR1. In line with previous binding studies on tomato ETRs, the ripening-delaying peptide NOP-1 clearly binds to the purified apple ETR. An NOP-1 solution (1000 µmol L-1 ) was applied with a brush or microdispenser and compared with apples treated with 1-methylcyclopropene (1-MCP) (SmartFresh™, Agrofresh) applied as gaseous treatment or untreated control fruits. NOP-1 inhibited colour development and chlorophyll degradation during shelf life. These effects were more pronounced with the brush application (surface film) than with microdroplets application (mimicking a sprayable formulation). NOP-1 did not alter ethylene release or respiration rate, whereas 1-MCP expectedly strongly suppressed both. There were no differences in quality parameters evaluated. Conclusion Our study shows that NOP-1 binds to MdETR1 which results in delaying of ethylene-dependent ripening developments of skin colour and chlorophyll. Besides application methods, possible reasons for the weak effect of NOP-1 in comparison with previous tomato experiments could be different receptor affinity and penetration differences. © 2019 Society of Chemical Industry.

The plant hormone ethylene was identified as important triggering factor and primary regulator of flower senescence in many species. Consequently, application of chemical inhibitors of ethylene biosynthesis and action is used to extend the longevity of ethylene-sensitive flowers. Here, we show that the peptide NOP-1, a biological derived from the nuclear localization signal of ethylene regulator EIN2 tightly binds to the ethylene receptor of carnation plants - a model to study flower senescence. When applied on cut flowers the peptide biological delays petal senescence similar to previously identified and currently used chemical inhibitors, but offers significant advances to these chemicals in biodegradability, sustainability and ecotoxicity. Our bioinformatic analysis of a wide range of ethylene receptors indicates complete sequence conservation of the anticipated NOP-1 binding site in flower species supporting a widespread use of the peptide on flowering ornamentals to delay senescence and decay in cut flowers. We anticipate our innovative approach to extend flower longevity by a new class of biomolecules such as peptides, peptide analogues and peptide mimetics will significantly advance our technological capability to delay flower senescence and expand vase-life of cut flowers in a sustainable and environmentally friendly manner.

Interfering peptides (iPs) have been recognized as valuable substances to specifically target protein-protein interactions (PPIs) in senescence and disease. Although the concept of iPs has been validated for several PPIs in medical and pharmaceutical research, little attention so far has been paid to the enormous potential iPs that may provide to target and control plant growth and developmental processes or plant environmental responses. However, recent research on PPIs in the ethylene signaling pathway has identified the synthetic peptide NOP-1 derived from the nuclear localization signal of ethylene regulator EIN2 as an efficient inhibitor of typical ethylene responses such as ripening, aging, and senescence. Biophysical and biochemical studies on purified recombinant proteins of the ethylene receptor family from various plant species demonstrate that the synthetic peptide binds in the nM-μM range at the plant target. Here, we describe methods to evaluate and quantify the effect of the NOP-1 peptide on flower senescence as a typical ethylene response in the intact plant system. This approach will help to systematically advance our technological capability to delay plant ethylene responses and to expand shelf-life or vase life of fruits and flowers.

Interfering peptides (iPs) have been recognized as valuable substances to specifically target protein-protein interactions (PPIs) in senescence and disease. Although the concept of iPs has been validated for several PPIs in medical and pharmaceutical research, little attention so far has been paid to the enormous potential iPs that may provide to target and control plant growth and developmental processes or plant environmental responses. However, recent research on PPIs in the ethylene signaling pathway has identified the synthetic peptide NOP-1 derived from the nuclear localization signal of ethylene regulator EIN2 as an efficient inhibitor of typical ethylene responses such as ripening, aging, and senescence. Biophysical and biochemical studies on purified recombinant proteins of the ethylene receptor family from various plant species demonstrate that the synthetic peptide binds in the nM-μM range at the plant target. Here, we describe methods to evaluate and quantify the effect of the NOP-1 peptide on flower senescence as a typical ethylene response in the intact plant system. This approach will help to systematically advance our technological capability to delay plant ethylene responses and to expand shelf-life or vase life of fruits and flowers.

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein&ndash, protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer’s and Parkinson’s disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability., Author summary Most proteins fold in the cell into stable, compact structures. Nevertheless, many proteins also have the ability to stick together, forming long fibrillar structures that are associated with a wide range of human disorders including Alzheimer’s and Parkinson’s disease. The exact nature of the amyloid-causing stickiness is not well understood, nevertheless amyloid fibrils show some very specific thermodynamic characteristics. Some fibrils even destabilise at low temperatures. In this work we translate hydrophobic theory previously used to model protein folding to fibril formation. We combine this theory with experimental measurements, simulations and meta-data analysis of different types of fibrils. This allowed us to unravel the nature of the stickiness in amyloid fibrils by observing the effect of temperature changes, specifically at low temperatures, on hydrophobicity.

The chloroplast FoF1 ATP synthase—a multi-subunit enzyme located in the plant thylakoid membrane—couples the electrochemical proton gradient built up by photosynthetic electron transport reactions to the synthesis of ATP. Synthesis of this universal cellular energy source in the extra-membranous F1 module is driven by rotary motions in the FoF1 holoenzyme that are powered by proton transport across the membrane-embedded Fo-submodule. Our mechanistic understanding of the ATP synthase has tremendously benefited from detailed insights into the structure and supramolecular architecture of this marvelous molecular machine which have enormously grown in the last decades. This started with low-resolution electron microscopic images of the FoF1 complex, went on with atomic resolution structures of individual subunits or subcomplexes resolved by X-ray crystallography or nuclear magnetic resonance and eventually culminated in a high-resolution molecular model of the complete chloroplast FoF1 holoenzyme obtained by cryo-electron microscopy.

F 1 -ATPase forms the membrane-associated segment of F 0 F 1 -ATP synthase — the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F 1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory e subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 A resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35–40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F 1 subcomplex. Biochemical analyses of the corresponding α 3 β 3 γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α 3 β 3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the e subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F 1 rotation during ATP hydrolysis.

Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8-10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)-stilbene and (+)-nootkatone.

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of both the C4 photosynthetic pathway and cellular energy metabolism of some bacteria and unicellular protists. In C4 plants, it catalyzes the ATP- and Pi -dependent formation of phosphoenolpyruvate (PEP) while in bacteria and protozoa the ATP-forming direction is used. PPDK is composed out of three distinct domains and exhibits one of the largest single domain movements known today during its catalytic cycle. However, little information about potential intermediate steps of this movement was available. A recent study resolved a discrete intermediate step of PPDK's swiveling movement, shedding light on the details of this intriguing mechanism. Here we present an additional structural intermediate that possibly represents another crucial step in the catalytic cycle of PPDK, providing means to get a more detailed understanding of PPDK's mode of function.

The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient in vivo assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C4 plant Bienertia sinuspersici to identify and characterize inhibitors of C4 photosynthesis. This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphat dikinase (PPDK) inhibitor bisindolymaleimide IV (BIM4) and phosphoenolpyruvat carboxylase (PEPC) inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations (IC50) in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors

Ovule development in Arabidopsis thaliana involves pattern formation which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, how a regular spacing of ovules is achieved is not known. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signalling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1 and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signalling results in shorter fruits and irregular spacing of ovules or even ovule twinning. The EPFL2 expression pattern between ovules is under negative-feedback regulation by auxin, which accumulates in the arising ovule primordia. We propose that the auxin-EPFL2 signalling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which serves to minimise competition between developing seeds. Together, EPFL2 and EPFL9 coordinate ovule patterning and thereby seed number with fruit growth through a set of shared receptors.

The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor. Nonetheless, molecular processes and structures controlling assembly and integration of the metal into the functional plant hormone receptor are still unknown. Here, we have explored the molecular pathways of copper transfer from the plant cytosol to the ethylene receptor family by analyzing protein–protein interactions of receptors with soluble and membrane-bound plant copper carriers. Our results suggest that receptors primarily acquire their metal cofactor from copper transporter RESPONSIVE-TO-ANTAGONIST-1 (RAN1) which has been loaded with the transition metal beforehand by soluble copper carriers of the ATX1-family. In addition, we found evidence for a direct interaction of ETRs with soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH) raising the possibility of a direct copper exchange between soluble chaperones and receptors.

Scientific reports 9(1), 8869 (2019). doi:10.1038/s41598-019-45189-w, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Background Small molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified. Methods Three peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the KD of the peptides towards the C-terminal domain of Hsp90. Results MD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC50 values of 2.08 μM for peptide H2 and 8.96 μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a KD of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a KD of 1.02 μM and 1.46 μM, respectively. Conclusions These results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity. General significance These results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.

The plant hormone ethylene is a key regulator of growth, development and stress adaptation at all stages of the plant life cycle. Signal perception and response to the plant hormone are mediated by a family of receptor kinases localized at the ER-Golgi network which gain their high affinity and specificity for the chemically simple ethylene molecule by an essential copper cofactor bound at their transmembrane domain. Transfer of this cofactor from the plant plasma membrane to the ER-localized receptors requires secured cellular transport of the reactive transition metal. In a recent study, we disclosed the transport proteins involved in the copper transfer to the receptors and identified that cytoplasmic chaperones of the ATX1-family and a membrane-bound P-type ATPase are involved in copper routing. Strictly speaking, our data show that receptors can acquire their copper load by different routes and adopt the metal ion from the plasma membrane either by sequential transfer from soluble chaperones of the ATX1-family via the ER-bound copper-transporting ATPase RAN1 or by direct transfer from the soluble chaperones. Here, we have studied the properties of the soluble plant copper chaperone isoforms, ATX1 and CCH, in more detail. Our data support different cellular functions of these isoforms on copper mobilization.

F

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein–protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 – the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.

The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38−) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)–resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and P i -dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Circular dichroism (CD) spectroscopy is an invaluable technique to analyze secondary structure and functional folding of recombinant purified proteins. CD spectroscopy can also be applied to detect changes in protein secondary structure related to the pH or redox conditions found in different cellular compartments or to the interaction with other molecules. Another biophysical technique to monitor conformational changes and interaction with small molecule ligands or biological macromolecules is protein fluorescence spectroscopy making use of the aromatic amino acid tryptophan as a sensitive intrinsic fluorescent probe. Here, we describe the application of CD and tryptophan fluorescence spectroscopy to study soluble and membrane proteins of the ethylene signaling pathway.

Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a “denaturation temperature” of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.

The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode.

Glycolate oxidase (GOX) is a crucial enzyme of plant photorespiration. The encoding gene is thought to have originated from endosymbiotic gene transfer between the eukaryotic host and the cyanobacterial endosymbiont at the base of plantae. However, animals also possess GOX activities. Plant and animal GOX belong to the gene family of (L)-2-hydroxyacid-oxidases ((L)-2-HAOX). We find that all (L)-2-HAOX proteins in animals and archaeplastida go back to one ancestral eukaryotic sequence; the sole exceptions are green algae of the chlorophyta lineage. Chlorophyta replaced the ancestral eukaryotic (L)-2-HAOX with a bacterial ortholog, a lactate oxidase that may have been obtained through the primary endosymbiosis at the base of plantae; independent losses of this gene may explain its absence in other algal lineages (glaucophyta, rhodophyta, and charophyta). We also show that in addition to GOX, plants possess (L)-2-HAOX proteins with different specificities for medium- and long-chain hydroxyacids (lHAOX), likely involved in fatty acid and protein catabolism. Vertebrates possess lHAOX proteins acting on similar substrates as plant lHAOX; however, the existence of GOX and lHAOX subfamilies in both plants and animals is not due to shared ancestry but is the result of convergent evolution in the two most complex eukaryotic lineages. On the basis of targeting sequences and predicted substrate specificities, we conclude that the biological role of plantae (L)-2-HAOX in photorespiration evolved by co-opting an existing peroxisomal protein.

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Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

The plant hormone ethylene is involved in the regulation of several processes with high importance for agricultural applications, e.g. ripening, aging and senescence. Previous work in our group has identified a small peptide (NOP-1) derived from the nuclear localization signal of the Arabidopsis ethylene regulator ETHYLENE INSENSITIVE-2 (EIN2) C-terminal part as efficient inhibitor of ethylene responses. Here, we show that NOP-1 is also able to efficiently disrupt EIN2-ETR1 complex formation in tomato, indicating that the NOP-1 inhibition mode is conserved across plant species. Surface application of NOP-1 on green tomato fruits delays ripening similar to known inhibitors of ethylene perception (MCP) and ethylene biosynthesis (AVG). Fruits treated with NOP-1 showed similar ethylene production as untreated controls underlining that NOP-1 blocks ethylene signaling by targeting an essential interaction in this pathway, while having no effect on ethylene biosynthesis.

The gaseous phytohormone ethylene C 2 H 4 mediates numerous aspects of growth and development. Genetic analysis has identified a number of critical elements in ethylene signaling, but how these elements interact biochemically to transduce the signal from the ethylene receptor complex at the endoplasmic reticulum (ER) membrane to transcription factors in the nucleus is unknown. To close this gap in our understanding of the ethylene signaling pathway, the challenge has been to identify the target of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) Raf-like protein kinase, as well as the molecular events surrounding ETHYLENE-INSENSITIVE2 (EIN2), an ER membrane-localized Nramp homolog that positively regulates ethylene responses. Here we demonstrate that CTR1 interacts with and directly phosphorylates the cytosolic C-terminal domain of EIN2. Mutations that block the EIN2 phosphorylation sites result in constitutive nuclear localization of the EIN2 C terminus, concomitant with constitutive activation of ethylene responses in Arabidopsis . Our results suggest that phosphorylation of EIN2 by CTR1 prevents EIN2 from signaling in the absence of ethylene, whereas inhibition of CTR1 upon ethylene perception is a signal for cleavage and nuclear localization of the EIN2 C terminus, allowing the ethylene signal to reach the downstream transcription factors. These findings significantly advance our understanding of the mechanisms underlying ethylene signal transduction.

Ethylene insensitive 2 (EIN2), an integral membrane protein of the ER network, has been identified as the central regulator of the ethylene signaling pathway. Still, the mechanism by which the ethylene signal is transferred from the receptors to EIN2 has not been solved yet. Here, we show that protein phosphorylation is a key mechanism to control the interaction of EIN2 and the receptors. In vivo and in vitro fluorescence studies reveal that the kinase domain of the receptors is essential for the interaction. Cyanide, an ethylene agonist, which is known to reduce auto-phosphorylation of the ethylene receptor ethylene resistant 1 (ETR1) or a mutation in the kinase domain of ETR1 that prevents auto-phosphorylation (H353A), increases the affinity of the receptors for EIN2. On the other hand, mimicking permanent auto-phosphorylation of ETR1 as in the mutant H353E releases the EIN2-ETR1 interaction from the control by the plant hormone. Based on our data, we propose a novel model on the integration of EIN2 in the ethylene signaling cascade.