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.

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.

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.

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.

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.

Introduction: Protein Oligomerization and the Formation of Macromolecular Assemblies.

The ability of biomolecules to link together to form higher order assemblies underlies much of cellular structure and function. Here we emphasise protein oligomerisation and discuss some of the principles of molecular interaction, from early considerations through to the present day. A few protein examples are presented, selected from our research interests, to highlight assembly features, ranging from the hemoglobins, the hemocyanins to the peroxiredoxins, collagen, the encapsulins and ferritins.

The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair.

The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe-4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.

Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Structure and Function of the Stressosome Signalling Hub.

The stressosome is a multi-protein signal integration and transduction hub found in a wide range of bacterial species. The role that the stressosome plays in regulating the transcription of genes involved in the general stress response has been studied most extensively in the Gram-positive model organism Bacillus subtilis. The stressosome receives and relays the signal(s) that initiate a complex phosphorylation-dependent partner switching cascade, resulting in the activation of the alternative sigma factor σB. This sigma factor controls transcription of more than 150 genes involved in the general stress response. X-ray crystal structures of individual components of the stressosome and single-particle cryo-EM reconstructions of stressosome complexes, coupled with biochemical and single cell analyses, have permitted a detailed understanding of the dynamic signalling behaviour that arises from this multi-protein complex. Furthermore, bioinformatics analyses indicate that genetic modules encoding key stressosome proteins are found in a wide range of bacterial species, indicating an evolutionary advantage afforded by stressosome complexes. Interestingly, the genetic modules are associated with a variety of signalling modules encoding secondary messenger regulation systems, as well as classical two-component signal transduction systems, suggesting a diversification in function. In this chapter we review the current research into stressosome systems and discuss the functional implications of the unique structure of these signalling complexes.

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

Structural evidence for the covalent modification of FabH by 4,5-dichloro-1,2-dithiol-3-one (HR45).

We use mass spectrometry analysis and molecular modelling to show the established antimicrobial inhibitor 4,5-dichloro-1,2-dithiol-3-one (HR45) acts by forming a covalent adduct with the target ß-ketoacyl-ACP synthase III (FabH). The 5-chloro substituent directs attack of the essential active site thiol (C112) via a Michael-type addition elimination reaction mechanism.

Charge order and three-site distortions in the Verwey structure of magnetite.

The mineral magnetite (Fe(3)O(4)) undergoes a complex structural distortion and becomes electrically insulating at temperatures less than 125 kelvin. Verwey proposed in 1939 that this transition is driven by a charge ordering of Fe(2+) and Fe(3+) ions, but the ground state of the low-temperature phase has remained contentious because twinning of crystal domains hampers diffraction studies of the structure. Recent powder diffraction refinements and resonant X-ray studies have led to proposals of a variety of charge-ordered and bond-dimerized ground-state models. Here we report the full low-temperature superstructure of magnetite, determined by high-energy X-ray diffraction from an almost single-domain, 40-micrometre grain, and identify the emergent order. The acentric structure is described by a superposition of 168 atomic displacement waves (frozen phonon modes), all with amplitudes of less than 0.24 ångströms. Distortions of the FeO(6) octahedra show that Verwey's hypothesis is correct to a first approximation and that the charge and Fe(2+) orbital order are consistent with a recent prediction. However, anomalous shortening of some Fe-Fe distances suggests that the localized electrons are distributed over linear three-Fe-site units, which we call 'trimerons'. The charge order and three-site distortions induce substantial off-centre atomic displacements and couple the resulting large electrical polarization to the magnetization. Trimerons may be important quasiparticles in magnetite above the Verwey transition and in other transition metal oxides.

Identifying RNA-binding residues based on evolutionary conserved structural and energetic features.

Increasing numbers of protein structures are solved each year, but many of these structures belong to proteins whose sequences are homologous to sequences in the Protein Data Bank. Nevertheless, the structures of homologous proteins belonging to the same family contain useful information because functionally important residues are expected to preserve physico-chemical, structural and energetic features. This information forms the basis of our method, which detects RNA-binding residues of a given RNA-binding protein as those residues that preserve physico-chemical, structural and energetic features in its homologs. Tests on 81 RNA-bound and 35 RNA-free protein structures showed that our method yields a higher fraction of true RNA-binding residues (higher precision) than two structure-based and two sequence-based machine-learning methods. Because the method requires no training data set and has no parameters, its precision does not degrade when applied to 'novel' protein sequences unlike methods that are parameterized for a given training data set. It was used to predict the 'unknown' RNA-binding residues in the C-terminal RNA-binding domain of human CPEB3. The two predicted residues, F430 and F474, were experimentally verified to bind RNA, in particular F430, whose mutation to alanine or asparagine nearly abolished RNA binding. The method has been implemented in a webserver called DR_bind1, which is freely available with no login requirement at http://drbind.limlab.ibms.sinica.edu.tw.

Deletion of membrane-associated Asp23 leads to upregulation of cell wall stress genes in Staphylococcus aureus.

With about 25 000 molecules per cell, Asp23 is one of the most abundant proteins in Staphylococcus aureus. Asp23 has been characterized as a protein that, following an alkaline shock, accumulates in the soluble protein fraction. Transcription of the asp23 gene is exclusively regulated by the alternative sigma factor σ(B) , which controls the response of the bacterium to environmental stress. Sequence analysis identified Asp23 as a member of the widely distributed Pfam DUF322 family, precluding functional predictions based on its sequence. Using fluorescence microscopy we found that Asp23 colocalized with the cell membrane of Staphylococcus aureus. Since Asp23 has no recognizable transmembrane spanning domains, we initiated a search for proteins that link Asp23 to the cell membrane. We identified SAOUHSC_02443 as the Asp23 membrane anchor and have renamed it AmaP (Asp23 membrane anchoring protein). Deletion of the asp23 gene led to an upregulation of the cell wall stress response. In summary, we have identified Asp23 as a membrane-associated protein and we suggest a function for Asp23 in cell envelope homoeostasis.

A widespread family of bacterial cell wall assembly proteins.

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains.

The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated ß-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed ß-propeller domain and a C-terminal ß-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall.

GeoPCA: a new tool for multivariate analysis of dihedral angles based on principal component geodesics.

The GeoPCA package is the first tool developed for multivariate analysis of dihedral angles based on principal component geodesics. Principal component geodesic analysis provides a natural generalization of principal component analysis for data distributed in non-Euclidean space, as in the case of angular data. GeoPCA presents projection of angular data on a sphere composed of the first two principal component geodesics, allowing clustering based on dihedral angles as opposed to Cartesian coordinates. It also provides a measure of the similarity between input structures based on only dihedral angles, in analogy to the root-mean-square deviation of atoms based on Cartesian coordinates. The principal component geodesic approach is shown herein to reproduce clusters of nucleotides observed in an η-θ plot. GeoPCA can be accessed via http://pca.limlab.ibms.sinica.edu.tw.

DR_bind: a web server for predicting DNA-binding residues from the protein structure based on electrostatics, evolution and geometry.

DR_bind is a web server that automatically predicts DNA-binding residues, given the respective protein structure based on (i) electrostatics, (ii) evolution and (iii) geometry. In contrast to machine-learning methods, DR_bind does not require a training data set or any parameters. It predicts DNA-binding residues by detecting a cluster of conserved, solvent-accessible residues that are electrostatically stabilized upon mutation to Asp(-)/Glu(-). The server requires as input the DNA-binding protein structure in PDB format and outputs a downloadable text file of the predicted DNA-binding residues, a 3D visualization of the predicted residues highlighted in the given protein structure, and a downloadable PyMol script for visualization of the results. Calibration on 83 and 55 non-redundant DNA-bound and DNA-free protein structures yielded a DNA-binding residue prediction accuracy/precision of 90/47% and 88/42%, respectively. Since DR_bind does not require any training using protein-DNA complex structures, it may predict DNA-binding residues in novel structures of DNA-binding proteins resulting from structural genomics projects with no conservation data. The DR_bind server is freely available with no login requirement at http://dnasite.limlab.ibms.sinica.edu.tw.