The crystal structure of PD1, a Haemophilus surface fibril domain

The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis.Peer reviewe

PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function

Fallstudien zu den Auswirkungen der Corona-Krise auf betriebliche Transformationsprozesse: Begleitforschung zur Arbeitsweltberichterstattung im Auftrag des BMAS, Bd. 3

Die Studie untersucht auf der Grundlage von fünf Branchen mit je vier Fallstudien Auswirkungen der Corona-Pandemie auf betriebliche Transformationsprozesse in Organisationen. Dazu wurden pro Fall zwei leitfadengestützte Interviews sowohl mit der Arbeitgeber- als auch mit der Arbeitnehmerseite im Winter 2020/21 und im Frühjahr 2021 im Längsschnitt geführt. Inhaltlich wurden zunächst Ziele, Maßnahmen und Herausforderungen der Transformationsprozesse beleuchtet. Die Transformationsprozesse umfassten dabei IT-bezogene Änderungen bzw. Digitalisierungsprojekte, personalbezogene Änderungen oder Änderungen in der Aufbau- oder Ablauforganisation. In vielen Fällen konnten innovationsförderliche Ressourcen wie Kompetenzen, Organisationsstrukturen und externe Netzwerke für den Prozess aktiviert werden. Anschließend befassen sich die Fallbeschreibungen mit den Auswirkungen der Corona-Pandemie auf die jeweilige Organisation. Dabei wird auf besondere Anpassungsmaßnahmen zum Umgang mit der Pandemie sowie auf die wirtschaftlichen und arbeitsorganisatorischen Folgen eingegangen. In vielen Fällen konnte auf fachliche und soziale Kompetenzen der Beschäftigten zurückgegriffen werden, diese zeigten allerdings auch zunehmend Ermüdungserscheinungen. Schließlich wurden die Einflüsse der Corona-Pandemie auf den Veränderungsprozess betrachtet. Die Pandemie hatte je nach Fall verstärkende oder hemmende Einflüsse; in mehreren Fällen erwiesen sich die Veränderungsprozesse als resistent gegenüber der Krise. Insbesondere Digitalisierungsprozesse wurden durch die Pandemie verstärkt. In einer abschließenden Bewertung legten die Interviewpartner:innen ihre "Lessons Learned" aus der Krise und ihre Erwartungen an die Politik dar.The study examines the effects of the corona pandemic on operational transformation processes in organizations on the basis of five industries with four case studies each. For this purpose, two guideline-based interviews were conducted in each case with both the employers and employees in winter of 2020/21 and in spring of 2021 in a longitudinal section. In terms of content, goals, measures and challenges of the transformation processes were examined first. The transformation processes included IT-related changes or digitization projects, personnel-related changes or changes in the structural or process organization. In many cases, innovation-promoting resources (competencies, organizational structures and external networks) could be activated for the process. The case descriptions then deal with the effects of the corona pandemic on the respective organization. Special adaptation measures for dealing with the pandemic as well as the economic and work-organizational consequences are discussed. In many cases it was possible to fall back on the technical and social skills of the employees, but these also increasingly showed signs of fatigue. Finally, the influences of the corona pandemic on the transformation process were considered. Depending on the case, the pandemic had intensifying or inhibiting influences, and in several cases the change processes proved to be resistant to the crisis. In particular, digitalization processes were intensified by the pandemic. In a final assessment, the case descriptions show the "lessons learned" from the crisis and expectations of politics

Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: An exchange protocol for membrane protein characterisation.

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function

Struktur und Funktion der ersten bakteriellen Chalconisomerase und einer (R)-selektiven Amin-Transaminase

Die in dieser Arbeit durchgeführten Kristallstrukturanalysen der ersten bakteriellen Chalconisomerase (CHI) bilden die Grundlage für das strukturelle Verständnis der Flavonoiddegradation von Eubacterium ramulus. Das Enzym zeigt eine offene und eine geschlossene Lid-Konformation, die das aktive Zentrum vollständig vom Solvens abgrenzt. Durch SAXS-Messungen konnte gezeigt werden, dass sich diese beiden Konformationen im Solvens in einem dynamischen Gleichgewicht befinden und nur eine geringe Energiebarriere zur Schließung überwunden werden muss. Die Lokalisation des aktiven Zentrums konnte durch Cokristallisation mit dem Substrat (2S)-Naringenin bewiesen werden. Der Reaktionsmechanismus konnte durch Mutagenese-Studien und spezifischen 1H/2H-Austausch durch NMR bewiesen werden. Trotz jeglicher fehlender funktionaler Verwandtschaft zeigt die Tertiärstruktur der bakteriellen CHI große Ähnlichkeiten zu der ferredoxin-like Faltung der Chloritdismutase aus Dechloromonas aromatica und dem mit Stress verbundenen Protein SP1 aus Populus tremula. Ein Vergleich der bakteriellen CHI mit der pflanzlichen CHI von Medicago sativa zeigt, dass deren 3D-Struktur in keinem verwandtschaftlichen Verhältnis steht. Dies suggeriert eine konvergente Evolution der beiden Chalconisomerasen ausgehend von unterschiedlichen Vorläuferproteinen. Anhand von Strukturaufklärungen der (R)-selektiven Amin-Transaminase aus Aspergillus fumigatus konnten erste Informationen über die strukturellen Voraussetzungen zur (R)-Selektivität dieser neuen Enzymklasse gewonnen werden. Die in silico Experimente zeigen, dass ähnlich zu den BCATs und D-ATAs das aktive Zentrum der (R)-ATA in eine große und eine kleine Bindetasche unterteilt ist. Dies konnte strukturell über den Inhibitorkomplex verifiziert werden. Die De-/Protonierung des Substrates durch das katalytische aktive Lys179 kann ausschließlich von der si-Seite erfolgen, sodass es zur Bildung des (R)-Enantiomers kommt. Der Mechanismus zur Bindung polarer Substrate (dual substrate recognition) wurde durch einen kovalenten Inhibitorkomplex und Mutagenese-Studien belegt und ist auf ein konserviertes Arginin im active site loop zurückzuführen.The first determination of the protein structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism for the isomerase reaction is verified by a specific 1H /2H isotope exchange observed by 1H NMR experiments and further supported by mutagenesis studies. The active site is shielded by a flexible lid whose varying structure could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals unrelated folds, suggesting that the enzyme activity evolved convergent from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1. The crystal structure of the (R)-selective amine transaminase elucidated here for the enzyme from Aspergillus fumigatus provides essential information and insights into the understanding how the substrate recognition occurs in (R)-selective amine transaminases and distinguishes them from other enzymes of the fold class IV. In addition, a crystal structure with the bound inhibitor gabaculine was solved. The orientation and binding of the carboxylate of the inhibitor is facilitated by Arg126 via water molecules

Structural Basis for (2R,3R)-Taxifolin Binding and Reaction Products to the Bacterial Chalcone Isomerase of Eubacterium ramulus

The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by a reverse Michael addition. The overall fold and the constitution of the active site of the enzyme completely differ from the well-characterised chalcone isomerase of plants. For (+)-taxifolin, CHI catalyses the intramolecular ring contraction to alphitonin. In this study, Fwe perform crystal structure analyses of CHI and its active site mutant His33Ala in the presence of the substrate taxifolin at 2.15 and 2.8 Å resolution, respectively. The inactive enzyme binds the substrate (+)-taxifolin as well defined, whereas the electron density maps of the native CHI show a superposition of substrate, product alphitonin, and most probably also the reaction intermediate taxifolin chalcone. Evidently, His33 mediates the stereospecific acid-base reaction by abstracting a proton from the flavonoid scaffold. The stereospecificity of the product is discussed

PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function