Toxin release by conditional remodelling of ParDE1 from Mycobacterium tuberculosis leads to gyrase inhibition.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a growing threat to global health, with recent efforts towards its eradication being reversed in the wake of the COVID-19 pandemic. Increasing resistance to gyrase-targeting second-line fluoroquinolone antibiotics indicates the necessity to develop both novel therapeutics and our understanding of M. tuberculosis growth during infection. ParDE toxin-antitoxin systems also target gyrase and are regulated in response to both host-associated and drug-induced stress during infection. Here, we present microbiological, biochemical, structural, and biophysical analyses exploring the ParDE1 and ParDE2 systems of M. tuberculosis H37Rv. The structures reveal conserved modes of toxin-antitoxin recognition, with complex-specific interactions. ParDE1 forms a novel heterohexameric ParDE complex, supported by antitoxin chains taking on two distinct folds. Curiously, ParDE1 exists in solution as a dynamic equilibrium between heterotetrameric and heterohexameric complexes. Conditional remodelling into higher order complexes can be thermally driven in vitro. Remodelling induces toxin release, tracked through concomitant inhibition and poisoning of gyrase activity. Our work aids our understanding of gyrase inhibition, allowing wider exploration of toxin-antitoxin systems as inspiration for potential therapeutic agents.

Structural and biochemical characterisation of the N-carbamoyl-ß-alanine amidohydrolase from Rhizobium radiobacter MDC 8606.

N-carbamoyl-ß-alanine amidohydrolase (CßAA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a CßAA-encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCßAA) showed a specific activity of 14 U·mg-1 using N-carbamoyl-ß-alanine as a substrate with an optimum activity at 55 °C and pH 8.0. In this work, we report also the first prokaryotic CßAA structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCßAA from R. radiobacter MDC 8606 for the industrial production of L-α-, L-ß- and L-γ-amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of CßAAs for high catalytic activity and broad substrate specificity.

Versatile Chemo-Biocatalytic Cascade Driven by a Thermophilic and Irreversible C-C Bond-Forming α-Oxoamine Synthase.

We report a chemo-biocatalytic cascade for the synthesis of substituted pyrroles, driven by the action of an irreversible, thermostable, pyridoxal 5'-phosphate (PLP)-dependent, C-C bond-forming biocatalyst (ThAOS). The ThAOS catalyzes the Claisen-like condensation between various amino acids and acyl-CoA substrates to generate a range of α-aminoketones. These products are reacted with ß-keto esters in an irreversible Knorr pyrrole reaction. The determination of the 1.6 Å resolution crystal structure of the PLP-bound form of ThAOS lays the foundation for future engineering and directed evolution. This report establishes the AOS family as useful and versatile C-C bond-forming biocatalysts.

The time scales of irreversibility in spontaneous brain activity are altered in obsessive compulsive disorder.

We study how obsessive-compulsive disorder (OCD) affects the complexity and time-reversal symmetry-breaking (irreversibility) of the brain resting-state activity as measured by magnetoencephalography (MEG). Comparing MEG recordings from OCD patients and age/sex matched control subjects, we find that irreversibility is more concentrated at faster time scales and more uniformly distributed across different channels of the same hemisphere in OCD patients than in control subjects. Furthermore, the interhemispheric asymmetry between homologous areas of OCD patients and controls is also markedly different. Some of these differences were reduced by 1-year of Kundalini Yoga meditation treatment. Taken together, these results suggest that OCD alters the dynamic attractor of the brain's resting state and hint at a possible novel neurophysiological characterization of this psychiatric disorder and how this therapy can possibly modulate brain function.

Thinking in Stories: Narrative Reasoning of an Occupational Therapist Supporting People with Profound Intellectual Disabilities' Engagement in Occupation.

This article illustrates narrative reasoning using the findings from research into an occupational therapy intervention promoting changes in the ways a staff team facilitated meaningful engagement in occupation. Qualitative critical ethnographic case study research explored a single case over one year of an occupational therapist working with five people with profound intellectual disabilities and their support network. Data were collected using participant observation, interviews and document analysis. Illustrated by an ethnodramatic vignette, the findings demonstrate how the occupational therapist reasoned narratively by eliciting, telling and creating stories and how this supported individualization of her intervention to the specific context. Creation of a prospective story that the support network were invited to share, guided and propelled the intervention toward its hoped-for ending. Narrative reasoning was particularly apparent in opportunities to reflect aloud, supporting occupational therapists' need of opportunities for reflection through story-sharing and story-making. Case study and ethnographic research methodologies may be useful in further clinical reasoning research to better understand narrative reasoning.

A User's Guide to Golden Gate Cloning Methods and Standards.

The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.

Molecular insights into intra-complex signal transmission during stressosome activation.

The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

Characterization of inositol lipid metabolism in gut-associated Bacteroidetes.

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.

PPI-HotspotDB: Database of Protein-Protein Interaction Hot Spots.

Single-point mutations of certain residues (so-called hot spots) impair/disrupt protein-protein interactions (PPIs), leading to pathogenesis and drug resistance. Conventionally, a PPI-hot spot is identified when its replacement decreased the binding free energy significantly, generally by ≥2 kcal/mol. The relatively few mutations with such a significant binding free energy drop limited the number of distinct PPI-hot spots. By defining PPI-hot spots based on mutations that have been manually curated in UniProtKB to significantly impair/disrupt PPIs in addition to binding free energy changes, we have greatly expanded the number of distinct PPI-hot spots by an order of magnitude. These experimentally determined PPI-hot spots along with available structures have been collected in a database called PPI-HotspotDB. We have applied the PPI-HotspotDB to create a nonredundant benchmark, PPI-Hotspot+PDBBM, for assessing methods to predict PPI-hot spots using the free structure as input. PPI-HotspotDB will benefit the design of mutagenesis experiments and development of PPI-hot spot prediction methods. The database and benchmark are freely available at https://ppihotspot.limlab.dnsalias.org.

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo-electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Synthetic biology approaches to copper remediation: bioleaching, accumulation and recycling.

One of the current aims of synthetic biology is the development of novel microorganisms that can mine economically important elements from the environment or remediate toxic waste compounds. Copper, in particular, is a high-priority target for bioremediation owing to its extensive use in the food, metal and electronic industries and its resulting common presence as an environmental pollutant. Even though microbe-aided copper biomining is a mature technology, its application to waste treatment and remediation of contaminated sites still requires further research and development. Crucially, any engineered copper-remediating chassis must survive in copper-rich environments and adapt to copper toxicity; they also require bespoke adaptations to specifically extract copper and safely accumulate it as a human-recoverable deposit to enable biorecycling. Here, we review current strategies in copper bioremediation, biomining and biorecycling, as well as strategies that extant bacteria use to enhance copper tolerance, accumulation and mineralization in the native environment. By describing the existing toolbox of copper homeostasis proteins from naturally occurring bacteria, we show how these modular systems can be exploited through synthetic biology to enhance the properties of engineered microbes for biotechnological copper recovery applications.

Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins.

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Electronic origin of negative thermal expansion in V2OPO4.

Negative volume expansion between 620 and 800 K in V2OPO4 is discovered to be of electronic origin due to the charge ordering transition at 605 K. Domain reorientation and coexistence of the low and high temperature phases close to the transition are observed in X-ray diffraction data from single crystals grown by chemical vapour transport.

Mass spectrometry reveals the assembly pathway of encapsulated ferritins and highlights a dynamic ferroxidase interface.

Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn.

Co-emergence of magnetic order and structural fluctuations in magnetite.

The nature of the Verwey transition occurring at TV ≈ 125 K in magnetite (Fe3O4) has been an outstanding problem over many decades. A complex low temperature electronic order was recently discovered and associated structural fluctuations persisting above TV are widely reported, but the origin of the underlying correlations and hence of the Verwey transition remains unclear. Here we show that local structural fluctuations in magnetite emerge below the Curie transition at TC ≈ 850 K, through X-ray pair distribution function analysis. Around 80% of the low temperature correlations emerge in proportion to magnetization below TC. This confirms that fluctuations in Fe-Fe bonding arising from magnetic order are the primary electronic instability and hence the origin of the Verwey transition. Such hidden instabilities may be important to other spin-polarised conductors and orbitally degenerate materials.

An extracellular acidic cleft confers profound H+-sensitivity to epithelial sodium channels containing the δ-subunit in Xenopus laevis.

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from αßγ- or δßγ-subunit combinations, we demonstrate that Xenopus δßγ-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus αßγ-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the δ-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus δßγ-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.

Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Soluble klotho (sKlotho), the shed ectodomain of α-klotho, protects the heart by down-regulating transient receptor potential canonical isoform 6 (TRPC6)-mediated calcium signaling. Binding to α2-3-sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft-dependent signaling underlies the mechanism. A recent 3-Å X-ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its ß6α6 loop might block access to the proposed binding site for α2-3-sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned ß6α6 loop allows sKlotho to bind α2-3-sialyllactose. Molecular dynamic simulations further show the α2-3-sialyllactose-bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid-recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.-Wright, J. D., An, S.-W., Xie, J., Lim, C., Huang, C.-L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea.

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.

Uncemented Reverse Total Shoulder Arthroplasty as Initial Treatment for Comminuted Proximal Humerus Fractures.

OBJECTIVES: To determine whether uncemented implants would provide similar outcomes while avoiding the complications associated with cement in the treatment of elderly patients with proximal humerus fractures (PHFs) with primary reverse total shoulder arthroplasty (RTSA). DESIGN: Case series. SETTING: A single Level I trauma center. PATIENTS/PARTICIPANTS: A prospectively obtained cohort of 30 patients who underwent uncemented RTSA as initial treatment for a comminuted PHF: 4 male, 26 female; average age 71 ± 11 years. INTERVENTION: Uncemented RTSA. MAIN OUTCOME MEASURES: (1) Radiographic analysis, (2) postoperative clinical range of motion, and (3) functional outcome scores: the American Shoulder and Elbow Surgeons Shoulder score and the Simple Shoulder Test score. RESULTS: Radiographic analysis showed 97% achieved stable humeral stem fixation and 70% had healing of the tuberosities in anatomical position. Average range of motion was 130 ± 31 degrees of forward flexion, 32 ± 18 degrees of external rotation, and internal rotation to the midlumbar spine. Average American Shoulder and Elbow Surgeons Shoulder score was 82.0 ± 13.5 (with an average pain rating of 0.8 ± 1.3), and average Simple Shoulder Test score was 69.4% ± 19.1%. CONCLUSIONS: Our data show that treatment of comminuted PHFs in elderly patients with uncemented RTSA can consistently produce good clinical outcomes with a low rate of complications and suggest that cement may not be necessary for RTSA in the trauma setting. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.