Structural insights into the iron nitrogenase complex.

Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO, but the molecular architecture facilitating these activities has remained unknown.

Machine Learning-Supported Enzyme Engineering toward Improved CO-Fixation of Glycolyl-CoA Carboxylase.

Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO fixation.

A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.

Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium (AOR) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy.

Purification and structural characterization of the Na-translocating ferredoxin: NAD reductase (Rnf) complex of Clostridium tetanomorphum.

Various microbial metabolisms use H/Na-translocating ferredoxin:NAD reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.

Evolution of increased complexity and specificity at the dawn of form I Rubiscos.

The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present.

Structure and mechanistic features of the prokaryotic minimal RNase P.

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (RNase P) is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various eukarya (there termed PRORPs) and in some bacteria ( and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients.

Molecular and Low-Resolution Structural Characterization of the Na-Translocating Glutaconyl-CoA Decarboxylase From .

Some anaerobic bacteria use biotin-dependent Na-translocating decarboxylases (Bdc) of β-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na across the membrane with the exergonic decarboxylation of glutaconyl-CoA (Δ ' ≈-30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS).

High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease.

Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones.

Structure of the Ty3/Gypsy retrotransposon capsid and the evolution of retroviruses.

Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation.

Gating of Acoustic Transducer Channels Is Shaped by Biomechanical Filter Processes.

Mechanoelectrical transduction of acoustic signals is the fundamental process for hearing in all ears across the animal kingdom. Here, we performed in vivo laser-vibrometric and electrophysiological measurements at the transduction site in an insect ear (Mecopoda elongata) to relate the biomechanical tonotopy along the hearing organ to the frequency tuning of the corresponding sensory cells. Our mechanical and electrophysiological map revealed a biomechanical filter process that considerably sharpens the neuronal response.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

Unlabelled: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear.

An organized co-assembly of clathrin adaptors is essential for endocytosis.

Clathrin-mediated endocytosis, the main trafficking route from the plasma membrane to the cytoplasm, is critical to many fundamental cellular processes. Clathrin, coupled to the membrane by adaptor proteins, is thought to play a major structural role in endocytosis by self-assembling into a cage-like lattice around the forming vesicle. Although clathrin adaptors are essential for endocytosis, little is known about their structural role in this process.

Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation.

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum.

The structure of the COPII transport-vesicle coat assembled on membranes.

Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available.

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions.

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components.

Multibudded tubules formed by COPII on artificial liposomes.

COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation.

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy.

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined.

Coatomer and dimeric ADP ribosylation factor 1 promote distinct steps in membrane scission.

Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle.

A different conformation for EGC stator subcomplex in solution and in the assembled yeast V-ATPase: possible implications for regulatory disassembly.

Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy.

A structural model for duck hepatitis B virus core protein derived by extensive mutagenesis.

Duck hepatitis B virus (DHBV) shares many fundamental features with human HBV. However, the DHBV core protein (DHBc), forming the nucleocapsid shell, is much larger than that of HBV (HBc) and, in contrast to HBc, there is little direct information on its structure. Here we applied an efficient expression system for recombinant DHBc particles to the biochemical analysis of a large panel of mutant DHBc proteins.

The cannabinoid dexanabinol is an inhibitor of the nuclear factor-kappa B (NF-kappa B).

Exogenous and endogenous cannabinoids have been shown to have neuroprotective effects in vitro and in vivo. Although many of the pharmacological effects of cannabinoids have been identified, the mechanism of neuroprotection still represents a controversy. Here we demonstrate for the first time protective effects of the synthetic cannabinoid dexanabinol by inhibiting apoptosis in a neuron-like cell line using nuclear staining and FACS analysis and in primary neurons.

Glutamate activates NF-kappaB through calpain in neurons.

Glutamate induces gene transcription in numerous physiological and pathological conditions. Among the glutamate-responsive transcription factors, NF-kappaB has been mainly implicated in neuronal survival and death. Recent data also suggest a role of NF-kappaB in neural development and memory formation.

The role of interleukin-6 in stress-induced hyperthermia and emotional behaviour in mice.

Psychological stress influences behaviour as well as autonomic functions such as body temperature. The mechanism that induces hyperthermia during stress is unknown. A recent hypothesis suggests that interleukin-6 (IL-6), an endogenous pyrogen, elevates body temperature during stress.

Regulation of body temperature and neuroprotection by endogenous interleukin-6 in cerebral ischemia.

Although the function of fever is still unclear, it is now beyond doubt that body temperature influences the outcome of brain damage. An elevated body temperature is often found in stroke patients and denotes a bad prognosis. However, the pathophysiologic basis and treatment options of elevated body temperature after stroke are still unknown.