Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules

Hadamard-Zustände für bosonische Quantenfeldtheorie auf global hyperbolischen Raumzeiten

Quantenfeldtheorie auf gekrümmten Raumzeiten ist eine semiklassische Näherung einer Quantentheorie der Gravitation, im Rahmen derer ein Quantenfeld unter dem Einfluss eines klassisch modellierten Gravitationsfeldes, also einer gekrümmten Raumzeit, beschrieben wird. Eine der bemerkenswertesten Vorhersagen dieses Ansatzes ist die Erzeugung von Teilchen durch die gekrümmte Raumzeit selbst, wie zum Beispiel durch Hawkings Verdampfen schwarzer Löcher und den Unruh Effekt. Andererseits deuten diese Aspekte bereits an, dass fundamentale Grundpfeiler der Theorie auf dem Minkowskiraum, insbesondere ein ausgezeichneter Vakuumzustand und damit verbunden der Teilchenbegriff, für allgemeine gekrümmte Raumzeiten keine sinnvolle Entsprechung besitzen. Gleichermaßen benötigen wir eine alternative Implementierung von Kovarianz in die Theorie, da gekrümmte Raumzeiten im Allgemeinen keine nicht-triviale globale Symmetrie aufweisen. Letztere Problematik konnte im Rahmen lokal-kovarianter Quantenfeldtheorie gelöst werden, wohingegen die Abwesenheit entsprechender Konzepte für Vakuum und Teilchen in diesem allgemeinen Fall inzwischen sogar in Form von no-go-Aussagen manifestiert wurde. Beim algebraischen Ansatz für eine Quantenfeldtheorie werden zunächst Observablen eingeführt und erst anschließend Zustände via Zuordnung von Erwartungswerten. Obwohl die Observablen unter physikalischen Gesichtspunkten konstruiert werden, existiert dennoch eine große Anzahl von möglichen Zuständen, von denen viele, aus physikalischen Blickwinkeln betrachtet, nicht sinnvoll sind. Dieses Konzept von Zuständen ist daher noch zu allgemein und bedarf weiterer physikalisch motivierter Einschränkungen. Beispielsweise ist es natürlich, sich im Falle freier Quantenfeldtheorien mit linearen Feldgleichungen auf quasifreie Zustände zu konzentrieren. Darüber hinaus ist die Renormierung von Erwartungswerten für Produkte von Feldern von zentraler Bedeutung. Dies betrifft insbesondere den Energie-Impuls-Tensor, dessen Erwartungswert durch distributionelle Bilösungen der Feldgleichungen gegeben ist. Tatsächlich liefert J. Hadamard Theorie hyperbolischer Differentialgleichungen Bilösungen mit festem singulären Anteil, so dass ein geeignetes Renormierungsverfahren definiert werden kann. Die sogenannte Hadamard-Bedingung an Bidistributionen steht für die Forderung einer solchen Singularitätenstruktur und sie hat sich etabliert als natürliche Verallgemeinerung der für flache Raumzeiten formulierten Spektralbedingung. Seit Radzikowskis wegweisenden Resultaten lässt sie sich außerdem lokal ausdrücken, nämlich als eine Bedingung an die Wellenfrontenmenge der Bilösung. Diese Formulierung schlägt eine Brücke zu der von Duistermaat und Hörmander entwickelten mikrolokalen Analysis, die seitdem bei der Überprüfung der Hadamard-Bedingung sowie der Konstruktion von Hadamard Zuständen vielfach Verwendung findet und rasante Fortschritte auf diesem Gebiet ausgelöst hat. Obwohl unverzichtbar für die Analyse der Charakteristiken von Operatoren und ihrer Parametrizen sind die Methoden und Aussagen der mikrolokalen Analysis ungeeignet für die Analyse von nicht-singulären Strukturen und zentrale Aussagen sind typischerweise bis auf glatte Anteile formuliert. Beispielsweise lassen sich aus Radzikowskis Resultaten nahezu direkt Existenzaussagen und sogar ein konkretes Konstruktionsschema für Hadamard Zustände ableiten, die übrigen Eigenschaften (Bilösung, Kausalität, Positivität) können jedoch auf diesem Wege nur modulo glatte Funktionen gezeigt werden. Es ist das Ziel dieser Dissertation, diesen Ansatz für lineare Wellenoperatoren auf Schnitten in Vektorbündeln über global-hyperbolischen Lorentz-Mannigfaltigkeiten zu vollenden und, ausgehend von einer lokalen Hadamard Reihe, Hadamard Zustände zu konstruieren. Beruhend auf Wightmans Lösung für die d'Alembert-Gleichung auf dem Minkowski-Raum und der Herleitung der avancierten und retardierten Fundamentallösung konstruieren wir lokal Parametrizen in Form von Hadamard-Reihen und fügen sie zu globalen Bilösungen zusammen. Diese besitzen dann die Hadamard-Eigenschaft und wir zeigen anschließend, dass glatte Bischnitte existieren, die addiert werden können, so dass die verbleibenden Bedingungen erfüllt sind.Quantum field theory on curved spacetimes is understood as a semiclassical approximation of some quantum theory of gravitation, which models a quantum field under the influence of a classical gravitational field, that is, a curved spacetime. The most remarkable effect predicted by this approach is the creation of particles by the spacetime itself, represented, for instance, by Hawking's evaporation of black holes or the Unruh effect. On the other hand, these aspects already suggest that certain cornerstones of Minkowski quantum field theory, more precisely a preferred vacuum state and, consequently, the concept of particles, do not have sensible counterparts within a theory on general curved spacetimes. Likewise, the implementation of covariance in the model has to be reconsidered, as curved spacetimes usually lack any non-trivial global symmetry. Whereas this latter issue has been resolved by introducing the paradigm of locally covariant quantum field theory (LCQFT), the absence of a reasonable concept for distinct vacuum and particle states on general curved spacetimes has become manifest even in the form of no-go-theorems. Within the framework of algebraic quantum field theory, one first introduces observables, while states enter the game only afterwards by assigning expectation values to them. Even though the construction of observables is based on physically motivated concepts, there is still a vast number of possible states, and many of them are not reasonable from a physical point of view. We infer that this notion is still too general, that is, further physical constraints are required. For instance, when dealing with a free quantum field theory driven by a linear field equation, it is natural to focus on so-called quasifree states. Furthermore, a suitable renormalization procedure for products of field operators is vitally important. This particularly concerns the expectation values of the energy momentum tensor, which correspond to distributional bisolutions of the field equation on the curved spacetime. J. Hadamard's theory of hyperbolic equations provides a certain class of bisolutions with fixed singular part, which therefore allow for an appropriate renormalization scheme. By now, this specification of the singularity structure is known as the Hadamard condition and widely accepted as the natural generalization of the spectral condition of flat quantum field theory. Moreover, due to Radzikowski's celebrated results, it is equivalent to a local condition, namely on the wave front set of the bisolution. This formulation made the powerful tools of microlocal analysis, developed by Duistermaat and Hörmander, available for the verification of the Hadamard property as well as the construction of corresponding Hadamard states, which initiated much progress in this field. However, although indispensable for the investigation in the characteristics of operators and their parametrices, microlocal analyis is not practicable for the study of their non-singular features and central results are typically stated only up to smooth objects. Consequently, Radzikowski's work almost directly led to existence results and, moreover, a concrete pattern for the construction of Hadamard bidistributions via a Hadamard series. Nevertheless, the remaining properties (bisolution, causality, positivity) are ensured only modulo smooth functions. It is the subject of this thesis to complete this construction for linear and formally self-adjoint wave operators acting on sections in a vector bundle over a globally hyperbolic Lorentzian manifold. Based on Wightman's solution of d'Alembert's equation on Minkowski space and the construction for the advanced and retarded fundamental solution, we set up a Hadamard series for local parametrices and derive global bisolutions from them. These are of Hadamard form and we show existence of smooth bisections such that the sum also satisfies the remaining properties exactly

Agrivoltaics: The Environmental Impacts of Combining Food Crop Cultivation and Solar Energy Generation

The demand for food and renewable energy is increasing significantly, whereas the availability of land for agricultural use is declining. Agrivoltaic systems (AVS), which combine agricultural production with solar energy generation on the same area, are a promising opportunity with the potential to satisfy this demand while avoiding land-use conflicts. In the current study, a Consequential Life-Cycle Assessment (CLCA) was conducted to holistically assess the environmental consequences arising from a shift from single-use agriculture to AVS in Germany. The results of the study show that the environmental consequences of the installation of overhead AVS on agricultural land are positive and reduce the impacts in 15 of the 16 analysed impact categories especially for climate change, eutrophication and fossil resource use, as well as in the single score assessment, mainly due to the substitution of the marginal energy mix. It was demonstrated that, under certain conditions, AVS can contribute to the extension of renewable energy production resources without reducing food production resources. These include maintaining the agricultural yields underneath the photovoltaic (PV) modules, seeking synergies between solar energy generation and crop production and minimising the loss of good agricultural land

Agrivoltaics: The Environmental Impacts of Combining Food Crop Cultivation and Solar Energy Generation

The demand for food and renewable energy is increasing significantly, whereas the availability of land for agricultural use is declining. Agrivoltaic systems (AVS), which combine agricultural production with solar energy generation on the same area, are a promising opportunity with the potential to satisfy this demand while avoiding land-use conflicts. In the current study, a Consequential Life-Cycle Assessment (CLCA) was conducted to holistically assess the environmental consequences arising from a shift from single-use agriculture to AVS in Germany. The results of the study show that the environmental consequences of the installation of overhead AVS on agricultural land are positive and reduce the impacts in 15 of the 16 analysed impact categories especially for climate change, eutrophication and fossil resource use, as well as in the single score assessment, mainly due to the substitution of the marginal energy mix. It was demonstrated that, under certain conditions, AVS can contribute to the extension of renewable energy production resources without reducing food production resources. These include maintaining the agricultural yields underneath the photovoltaic (PV) modules, seeking synergies between solar energy generation and crop production and minimising the loss of good agricultural land

Development of Nurr1 agonists from amodiaquine by scaffold hopping and fragment growing

Abstract The neuroprotective transcription factor nuclear receptor-related 1 (Nurr1) has shown great promise as a therapeutic target in Parkinson’s and Alzheimer’s disease as well as multiple sclerosis but high-quality chemical tools for pharmacological target validation of Nurr1 are rare. We have employed the weak Nurr1 modulator amodiaquine (AQ) and AQ-derived fragments as templates to design a new Nurr1 agonist chemotype by scaffold hopping and fragment growing strategies. Systematic structural optimization of this scaffold yielded Nurr1 agonists with nanomolar potency and binding affinity. Comprehensive in vitro profiling revealed efficient cellular target engagement and compliance with the highest probe criteria. In human midbrain organoids bearing a Parkinson-driving LRRK2 mutation, a novel Nurr1 agonist rescued tyrosine hydroxylase expression highlighting the potential of the new Nurr1 modulator chemotype as lead and as a chemical tool for biological studies

Structure-guided design of a highly potent RXR agonist with superior physicochemical properties

Retinoid X receptors (RXR, NR2B1-3) hold therapeutic potential in oncology, neurodegeneration and metabolic diseases but traditional RXR agonists mimicking the natural ligand 9-cis retinoic acid exhibit poor physicochemical properties, pharmacokinetics and safety profiles. Improved RXR ligands are needed to exploit RXR modulation as a promising therapeutic concept in various indications beyond its current role in second-line cancer treatment. Here we report the co-crystal structure of RXR in complex with a novel pyrimidine-based ligand and the structure-informed optimization of this scaffold to highly potent and highly soluble RXR agonists. Rigidization resulted in significantly improved potency and focused structure-activity relationship elucidation indicated potential avenues to RXRα preference. We obtained an optimized RXR agonist with low nanomolar potency, no cytotoxic activity and very favorable physicochemical properties highlighting this promising scaffold for the development of next-generation RXR agonist drugs

Structure-Guided Design of a Highly Potent Partial RXR Agonist with Superior Physicochemical Properties

Retinoid X receptors (RXRs, NR2B1–3) hold therapeutic potential in oncology, neurodegeneration, and metabolic diseases, but traditional RXR agonists mimicking the natural ligand 9-cis retinoic acid exhibit poor physicochemical properties, pharmacokinetics, and safety profiles. Improved RXR ligands are needed to exploit RXR modulation as a promising therapeutic concept in various indications beyond its current role in second-line cancer treatment. Here, we report the co-crystal structure of RXR in complex with a novel pyrimidine-based ligand and the structure-informed optimization of this scaffold to highly potent and highly soluble RXR agonists. Focused structure–activity relationship elucidation and rigidization resulted in a substantially optimized partial RXR agonist with low nanomolar potency, no cytotoxic activity, and very favorable physicochemical properties highlighting this promising scaffold for the development of next-generation RXR targeting drugs