The ABC toxin complex from Yersinia entomophaga can package three different cytotoxic components expressed from distinct genetic loci in an unfolded state: the structures of both shell and cargo.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

RNase HI Depletion Strongly Potentiates Cell Killing by Rifampicin in Mycobacteria.

Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.

Structural characterization of human peptidyl-arginine deiminase type III by X-ray crystallography.

The Ca2+-dependent enzyme peptidyl-arginine deiminase type III (PAD3) catalyses the deimination of arginine residues to form citrulline residues in proteins such as keratin, filaggrin and trichohyalin. This is an important post-translation modification that is required for normal hair and skin formation in follicles and keratocytes. The structure of apo human PAD3 was determined by X-ray crystallography to a resolution of 2.8 Å. The structure of PAD3 revealed a similar overall architecture to other PAD isoforms: the N-terminal and middle domains of PAD3 show sequence and structural variety, whereas the sequence and structure of the C-terminal catalytic domain is highly conserved. Structural analysis indicates that PAD3 is a dimer in solution, as is also the case for the PAD2 and PAD4 isoforms but not the PAD1 isoform.

Structural characterization of a PCP-R didomain from an archaeal nonribosomal peptide synthetase reveals novel interdomain interactions.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.

Collaborative networks enable the rapid establishment of serological assays for SARS-CoV-2 during nationwide lockdown in New Zealand.

BACKGROUND: Serological assays that detect antibodies to SARS-CoV-2 are critical for determining past infection and investigating immune responses in the COVID-19 pandemic. We established ELISA-based immunoassays using locally produced antigens when New Zealand went into a nationwide lockdown and the supply chain of diagnostic reagents was a widely held domestic concern. The relationship between serum antibody binding measured by ELISA and neutralising capacity was investigated using a surrogate viral neutralisation test (sVNT). METHODS: A pre-pandemic sera panel (n = 113), including respiratory infections with symptom overlap with COVID-19, was used to establish assay specificity. Sera from PCR­confirmed SARS-CoV-2 patients (n = 21), and PCR-negative patients with respiratory symptoms suggestive of COVID-19 (n = 82) that presented to the two largest hospitals in Auckland during the lockdown period were included. A two-step IgG ELISA based on the receptor binding domain (RBD) and spike protein was adapted to determine seropositivity, and neutralising antibodies that block the RBD/hACE­2 interaction were quantified by sVNT. RESULTS: The calculated cut-off (>0.2) in the two-step ELISA maximised specificity by classifying all pre-pandemic samples as negative. Sera from all PCR-confirmed COVID-19 patients were classified as seropositive by ELISA ≥7 days after symptom onset. There was 100% concordance between the two-step ELISA and the sVNT with all 7+ day sera from PCR­confirmed COVID-19 patients also classified as positive with respect to neutralising antibodies. Of the symptomatic PCR-negative cohort, one individual with notable travel history was classified as positive by two-step ELISA and sVNT, demonstrating the value of serology in detecting prior infection. CONCLUSIONS: These serological assays were established and assessed at a time when human activity was severely restricted in New Zealand. This was achieved by generous sharing of reagents and technical expertise by the international scientific community, and highly collaborative efforts of scientists and clinicians across the country. The assays have immediate utility in supporting clinical diagnostics, understanding transmission in high-risk cohorts and underpinning longer­term 'exit' strategies based on effective vaccines and therapeutics.

The tryptophan biosynthetic pathway is essential for Mycobacterium tuberculosis to cause disease.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the most significant cause of death from a single infectious agent worldwide. Antibiotic-resistant strains of M. tuberculosis represent a threat to effective treatment, and the long duration, toxicity and complexity of current chemotherapy for antibiotic-resistant disease presents a need for new therapeutic approaches with novel modes of action. M. tuberculosis is an intracellular pathogen that must survive phagocytosis by macrophages, dendritic cells or neutrophils to establish an infection. The tryptophan biosynthetic pathway is required for bacterial survival in the phagosome, presenting a target for new classes of antitubercular compound. The enzymes responsible for the six catalytic steps that produce tryptophan from chorismate have all been characterised in M. tuberculosis, and inhibitors have been described for some of the steps. The innate immune system depletes cellular tryptophan in response to infection in order to inhibit microbial growth, and this effect is likely to be important for the efficacy of tryptophan biosynthesis inhibitors as new antibiotics. Allosteric inhibitors of both the first and final enzymes in the pathway have proven effective, including by a metabolite produced by the gut biota, raising the intriguing possibility that the modulation of tryptophan biosynthesis may be a natural inter-bacterial competition strategy.

Membrane trafficking pathways regulating the epithelial Na+ channel.

Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.

Teneurin Structures Are Composed of Ancient Bacterial Protein Domains.

Pioneering bioinformatic analysis using sequence data revealed that teneurins evolved from bacterial tyrosine-aspartate (YD)-repeat protein precursors. Here, we discuss how structures of the C-terminal domain of teneurins, determined using X-ray crystallography and electron microscopy, support the earlier findings on the proteins' ancestry. This chapter describes the structure of the teneurin scaffold with reference to a large family of teneurin-like proteins that are widespread in modern prokaryotes. The central scaffold of modern eukaryotic teneurins is decorated by additional domains typically found in bacteria, which are re-purposed in eukaryotes to generate highly multifunctional receptors. We discuss how alternative splicing contributed to further diversifying teneurin structure and thereby function. This chapter traces the evolution of teneurins from a structural point of view and presents the state-of-the-art of how teneurin function is encoded by its specific structural features.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in Mycobacterium tuberculosis.

The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalyzed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis. M. tuberculosis strains auxotrophic for tryptophan, an essential amino acid in the human diet, are avirulent. Thus, tryptophan synthesis in M. tuberculosis has been suggested as a potential drug target, and it has been reported that fluorinated anthranilate is lethal to the bacillus. Two mechanisms that could explain the cellular toxicity were tested: (1) the inhibition of tryptophan biosynthesis by a fluorinated intermediate or (2) formation of fluorotryptophan and its subsequent effects. Here, M. tuberculosis mc2 6230 cultures were treated with anthranilates fluorinated at positions 4, 5, and 6. These compounds inhibited bacterial growth on tryptophan-free media with 4-fluoroanthranilate being more potent than 5-fluoroanthranilate or 6-fluoroanthranilate. LC-MS based analysis of extracts from bacteria treated with these compounds did not reveal accumulation of any of the expected fluorinated intermediates in tryptophan synthesis. However, in all cases, significant levels of fluorotryptophan were readily observed, suggesting that the enzymes involved in the conversion of fluoro-anthranilate to fluorotryptophan were not being inhibited. Inclusion of tryptophan in cultures treated with the fluoro-anthranilates obviated the cellular toxicity. Bacterial growth was also inhibited in a dose-dependent manner by exposure to tryptophan substituted with fluorine at positions 5 or 6. Thus, the data suggest that fluorotryptophan rather than fluoro-anthranilate or intermediates in the synthesis of fluorotryptophan causes the inhibition of M. tuberculosis growth.

Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The Km values for UQ-1 using recombinant MenJ from Mycobacterium smegmatis or M. tuberculosis without saturating concentrations of FAD were found to be 52 ± 9.6 and 44 ± 4.8 µM, respectively, while the KmNADH values were determined to be 59 ± 14 and 64 ± 15 µM. The Km for MK-1, the menaquinone analogue of UQ-1, using recombinant MenJ from M. tuberculosis without saturating concentrations of FAD but in the presence of 0.5% Tween 80 was shown to be 30 ± 2.9 µM. Thus, this is the first report of a kinetic characterization of a member of the geranylgeranyl reductase family of enzymes.

Structural insights into the architecture and membrane interactions of the conserved COMMD proteins.

The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.

Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization.

Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs. As a preliminary step towards their biophysical and structural characterization, we have expressed and purified a HS KAP (KAP11.1) and a HGT KAP (KAP6.1). The expression and purification of KAPs is challenging because they are cysteine-rich proteins with unusual amino acid compositions, they tend to be insoluble in isolation and are prone to forming aggregates in solution. Here we describe the high yield production of pure, soluble KAPs in a chaotrope- and detergent-free buffer. This method has the potential to be used for the overproduction of other KAPs and similar cysteine-rich proteins with high isoelectric points.

Anthranilate phosphoribosyltransferase: Binding determinants for 5'-phospho-alpha-d-ribosyl-1'-pyrophosphate (PRPP) and the implications for inhibitor design.

Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a "bianthranilate"-like moiety are inhibitors with IC50 values of 10-60µM, and Ki values of 1.3-15µM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops.

Datasets, processing and refinement details for Mtb-AnPRT: inhibitor structures with various space groups.

There are twenty-five published structures of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase (Mtb-AnPRT) that use the same crystallization protocol. The structures include protein complexed with natural and alternative substrates, protein:inhibitor complexes, and variants with mutations of substrate-binding residues. Amongst these are varying space groups (i.e. P21, C2, P21212, P212121). This article outlines experimental details for 3 additional Mtb-AnPRT:inhibitor structures. For one protein:inhibitor complex, two datasets are presented - one generated by crystallization of protein in the presence of the inhibitor and another where a protein crystal was soaked with the inhibitor. Automatic and manual processing of these datasets indicated the same space group for both datasets and thus indicate that the space group differences between structures of Mtb-AnPRT:ligand complexes are not related to the method used to introduce the ligand.

Revealing the Inter-Module Interactions of Multi-Modular Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetase (NRPS) enzymes are large modular proteins involved in the biosynthesis of many important pharmaceuticals such as penicillin and cyclosporin. In this issue of Structure, Tarry et al. (2017) use X-ray crystallography and electron microscopy to shed light on the inter-module interactions that underpin the complex function of these enzymes.

Combining cross-crystal averaging and MRSAD to phase a 4354-amino-acid structure.

The B and C proteins from the ABC toxin complex of Yersinia entomophaga form a large heterodimer that cleaves and encapsulates the C-terminal toxin domain of the C protein. Determining the structure of the complex formed by B and the N-terminal region of C was challenging owing to its large size, the non-isomorphism of different crystals and their sensitivity to radiation damage. A native data set was collected to 2.5 Šresolution and a non-isomorphous Ta6Br12-derivative data set was collected that showed strong anomalous signal at low resolution. The tantalum-cluster sites could be found, but the anomalous signal did not extend to a high enough resolution to allow model building. Selenomethionine (SeMet)-derivatized protein crystals were produced, but the high number (60) of SeMet sites and the sensitivity of the crystals to radiation damage made phasing using the SAD or MAD methods difficult. Multiple SeMet data sets were combined to provide 30-fold multiplicity, and the low-resolution phase information from the Ta6Br12 data set was transferred to this combined data set by cross-crystal averaging. This allowed the Se atoms to be located in an anomalous difference Fourier map; they were then used in Auto-Rickshaw for multiple rounds of autobuilding and MRSAD.

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis.

Cholesterol can be a major carbon source forMycobacterium tuberculosisduring infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, includingkstR, are either induced during infection or are essential for survival ofM. tuberculosis in vivo In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50for ligand = 25 nm). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release.

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site.

Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²âº-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile ß2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The ß2-α6 loop moves sequentially between a "folded" conformation, partially occluding the anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the ß2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the ß2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme.