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Macrophages are an ancient blood cell lineage critical for homeostasis and defence against pathogens. Although their numbers were long thought to be sustained solely by haematopoietic organs, it has recently become clear that their proliferation, or self‐renewal, also plays a major role. In the Drosophila larva, macrophages undergo a phase of rapid self‐renewal, making this an attractive model for elucidating the signals and regulatory mechanisms involved. However, a central self‐renewal pathway has not been identified in this system. Here, we show that the PDGF‐ and VEGF‐receptor related (Pvr) pathway fulfils this role. Our data show that two of the three known Pvr ligands, PDGF‐ and VEGF‐related factor 2 (Pvf2) and Pvf3, are major determinants of overall macrophage numbers, yet they each act in a temporally independent manner and via distinct mechanisms. While Pvf3 is needed prior to the self‐renewal period, we find that Pvf2 is critical specifically for expanding the larval macrophage population. We further show that Pvf2 is a potent macrophage mitogen that is kept at limiting quantities by its transient expression in a remarkably small number of blood cells. Together, these data support a novel mechanism for the regulation of macrophage self‐renewal rates by the dynamic transcriptional control of Pvf2. Given the strong parallels that exist between Drosophila and vertebrate macrophage systems, it is likely that a similar self‐renewal control mechanism is at play across animal phyla. [ABSTRACT FROM AUTHOR]

Protein structure fundamentally underpins the function and processes of numerous biological systems. Fold recognition algorithms offer a sensitive and robust tool to detect structural, and thereby functional, similarities between distantly related homologs. In the era of accurate structure prediction owing to advances in machine learning techniques and a wealth of experimentally determined structures, previously curated sequence databases have become a rich source of biological information. Here, we use bioinformatic fold recognition algorithms to scan the entire AlphaFold structure database to identify novel protein family members, infer function and group predicted protein structures. As an example of the utility of this approach, we identify novel, previously unknown members of various pore-forming protein families, including MACPFs, GSDMs and aerolysin-like proteins. Author summary: Virtually every cellular process in all organisms on Earth is driven by molecular nano-machines known as proteins. The diverse functions of proteins are the result of the unique three-dimensional shape adopted by a given protein molecule. It is therefore important to determine the shape of a given protein, which unlike DNA and our genes, cannot be known from its sequence alone. Since two proteins with similar shapes typically have a similar function, knowing a protein shape provides crucial clues about its function. By virtue of decades of experimental work and advances in artificial intelligence, this complex shape can now be computationally predicted for any protein whose composition is known. Scientists have used these and other methods to produce enormous libraries of protein shapes consisting of nearly a million unique entries. However, these libraries are too large and too complex for researchers to 'read'. We use shape-comparison algorithms to carefully check these shape-libraries to gain insight into the potential function and biological role of previously unknown proteins. Furthermore, we identified new members of protein families using this technique. We show that shape-matching algorithms and computationally generated shape-libraries can be used effectively together to yield new insights and expedite scientific endeavours. [ABSTRACT FROM AUTHOR]

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila, for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing. [ABSTRACT FROM AUTHOR]

Pore Forming Toxins (PFTs) represent a key mechanism for permitting the passage of proteins and small molecules across the lipid membrane. These proteins are typically produced as soluble monomers that self-assemble into ring-like oligomeric structures on the membrane surface. Following such assembly PFTs undergo a remarkable conformational change to insert into the lipid membrane. While many different protein families have independently evolved such ability, members of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) superfamily form distinctive giant β-barrel pores comprised of up to 50 monomers and up to 300 Å in diameter. In this review we focus on recent advances in understanding the structure of these giant MACPF/CDC pores as well as the underlying molecular mechanisms leading to their formation. Commonalities and evolved variations of the pore forming mechanism across the superfamily are discussed. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. [ABSTRACT FROM AUTHOR]

A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin-granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology. [ABSTRACT FROM AUTHOR]

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three β-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs. [ABSTRACT FROM AUTHOR]

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding. [ABSTRACT FROM AUTHOR]

Serpins compose the largest superfamily of peptidase inhibitors and are well known as regulators of hemostasis and thrombolysis. Studies using model organisms, from plants to vertebrates, now show that serpins and their unique inhibitory mechanism and conformational flexibility are exploited to control proteolysis in molecular pathways associated with cell survival, development, and host defense. In addition, an increasing number of non-inhibitory serpins are emerging as important elements within a diversity of biological systems by serving as chaperones, hormone transporters, or anti-angiogenic factors. [ABSTRACT FROM AUTHOR]

Members of the serpin (ine rotease hibitor) superfamily fold into a metastable conformation that is crucial for proper function. As a consequence, serpins are susceptible to mutations that cause misfolding and the intracellular accumulation of pathogenic polymers. The mechanism of serpin polymerisation remains to be resolved, however, over the past two decades the ‘loop-sheet’ hypothesis has gained wide acceptance. In this mechanism the reactive centre loop of one serpin monomer inserts into the β-sheet A of another ( in trans), in a manner similar to what is seen for reactive centre loop-cleaved and latent conformations ( in cis). The hypothesis has been refined in response to certain experimental data, but it has proved difficult to assess the various propositions without creating molecular models. Here we evaluate the loop-sheet mechanism by creating models of pentamers of the archetypal serpin α1-antitrypsin. We conclude that an inescapable consequence of the loop-sheet mechanism is polymer compaction and rigidity, properties that are inconsistent with the ‘beads-on-a-string’ morphology of polymers obtained from human tissue. The recent crystal structure of a domain-swapped serpin dimer suggests an alternative mechanism that is consistent with known polymer properties, including the requirement of partial unfolding to induce polymer formation in vitro, and polymerisation from a folding intermediate in vivo. [ABSTRACT FROM AUTHOR]

Maspin (SERPINB5) is a tumor suppressor lost in breast and prostate cancer whose molecular function is unknown. It is a non-inhibitory member of the clade B serpins suggested to play a role in a plethora of intracellular and extracellular settings, yet its normal cellular distribution has never been clarified. Here we investigate the distribution of maspin in non-transformed human epithelial cells. By indirect immuno fluorescence, maspin has a nucleocytoplasmic distribution in breast (MCF10A) and prostate (RWPE-1) cells ands:by immunoblotting and pulse-chase analyses, is neither glycosylated nor secreted. Cell surface biotinylation studies also show that maspin is not present at the cell surface. Differentiation of MCF10A cells into three-dimensional acini results in the redistribution of maspin from the nucleus to the cytoplasm but does not result in secretion. Addition of an efficient conventional signal peptide to maspin directs it into the secretory pathway and results in glycosylation but not secretion. We further show that maspin in the cytoplasm of MCF10A cells is a soluble monomeric protein that is not detectably associated with the cytoskeleton or other extractable components. Taken together, these results suggest that maspin is restricted to an intracellular, possibly nuclear, role in which it influences cell-matrix interactions indirectly. It is probably released only as a consequence of cell damage or necrosis. [ABSTRACT FROM AUTHOR]

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding. [Copyright &y& Elsevier]

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia). [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA[sub cat], the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA[sub cat] or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aα-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bβ-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene. [ABSTRACT FROM AUTHOR]

Examines the role of the F-helix in serpin stability through a singular tryptophan substitution. Indication of the unfolding behavior of the tryptophan variant; Control of conformational changes involved in α&sub1;-antitrypsin (α&sub1;-AT) unfolding of F-helix; Schematic diagram of native α&sub1;-AT.

• C1-inhibitor plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. • The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate • Co factor binding site of the serpin was mapped and shown to be vital for rate enhancement • Findings provide new insight into the mechanism of interaction of co factors with C1-inhibitor. The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules. [ABSTRACT FROM AUTHOR]

The author comments on the study by Lectenberg and colleagues, which explores the structure of coagulation. He states that the study determines the structure of re-engineered prothrombinase complex, pseutarin C, from proteins obtained from the venom of the Australian eastern brown snake (Pseudonaja textilis). He says that the study offers a compelling and adaptable ways to determine the complete ternary complex between prothrombin and prothrombinase.

The article presents a study of intermediate states of aeromonas hydrophila toxin aerolysin and its route to pore formation within membranes. It mentions that researchers used single particle cryo-Electron Microscopy to detect three intermediate structures on the pathway including an initial heptameric prepore state, post-prepore structure and a 14 stranded membrane-inserted beta-barrel. Results indicate a marked rotation of the core aerolysin machinery to penetrate membrane-spanning regions.

The article discusses that polymers of serpin proteins underlie diseases, including neurodegenerative disorders. It is said that Alois Alzheimer has offered a description on the abnormal aggregates of the amyloid protein in the brain of a patient who has died of dementia. The known conformational diseases include prion diseases, Parkinson's disease, Huntington's disease and several disorders known as serpinopathies. It is mentioned that the serpin polymer is correctly folded and resembles the relaxed serpin, which does not evoke protective signalling pathway.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg–RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes. [ABSTRACT FROM AUTHOR]

Presents information on a study which used SH3 domain of the tyrosine kinase to identify prokaryote sequences. Pattern of similarity in the sequences suggested; Evidence provided by threading calculations and model building; Effect of the invasion of eukaryotic cells by pathogenic bacteria.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway. [ABSTRACT FROM AUTHOR]

The rapid and orderly folding of epithelial tissue during developmental processes such as gastrulation requires the precise coordination of changes in cell shape. Here, we report that the perforin-like protein Torso-like (Tsl), the key extracellular determinant for Drosophila embryonic terminal patterning, also functions to control epithelial morphogenesis. We find that tsl null mutants display a ventral cuticular hole phenotype that is independent of the loss of terminal structures, and arises as a consequence of mesoderm invagination defects. We show that the holes are caused by uncoordinated constriction of ventral cell apices, resulting in the formation of an incomplete ventral furrow. Consistent with these data, we find that loss of tsl is sensitive to gene dosage of RhoGEF2, a critical mediator of Rho1-dependent ventral cell shape changes during furrow formation, suggesting that Tsl may act in this pathway. In addition, loss of tsl strongly suppressed the effects of ectopic expression of Folded Gastrulation (Fog), a secreted protein that promotes apical constriction. Taken together, our data suggest that Tsl controls Rho1-mediated apical constriction via Fog. Therefore, we propose that Tsl regulates extracellular Fog activity to synchronize cell shape changes and coordinate ventral morphogenesis in Drosophila. Identifying the Tsl-mediated event that is common to both terminal patterning and morphogenesis will be valuable for our understanding of the extracellular control of developmental signaling by perforin-like proteins. [ABSTRACT FROM AUTHOR]

No abstract available [ABSTRACT FROM AUTHOR]

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11. The cryo-EM structure of monoclonal antibody aE11 bound to polyC9 reveals that the binding site within the Membrane Attack Complex is a quaternary discontinuous epitope formed by two separate surfaces of the oligomeric C9 periphery. [ABSTRACT FROM AUTHOR]

To control infections phagocytes can directly kill invading microbes. Macrophage‐expressed gene 1 (Mpeg1), a pore‐forming protein sometimes known as perforin‐2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68‐positive endolysosomal compartment, and that it exists predominantly as a processed, two‐chain disulfide‐linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response. [ABSTRACT FROM AUTHOR]

Highlights: [•] Plasminogen initially binds C-terminal lysine containing receptors through kringle 1. [•] Plasminogen conformational change is triggered through lysine binding to kringle 5. [•] Glycosylation patterns change plasminogen shape, stability and biological function. [•] Bacterial virulence factors evade the mechanism that regulates plasminogen activation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Recombinant DNA technologies are leading the rapid expansion of bispecific antibody formats. [•] The therapeutic potential of bispecific antibodies is being realized through creative design. [•] Bispecific antibodies are potentially underutilized reagents for diagnostics. [ABSTRACT FROM AUTHOR]

Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism. [ABSTRACT FROM AUTHOR]

Motivation: The β-barrel is a ubiquitous fold that is deployed to accomplish a wide variety of biological functions including membrane-embedded pores. Key influences of β-barrel lumen diameter include the number of β-strands (n) and the degree of shear (S), the latter value measuring the extent to which the β-sheet is tilted within the β-barrel. Notably, it has previously been reported that the shear value for small antiparallel β-barrels (n≤24) typically ranges between n and 2n. Conversely, it has been suggested that the β-strands in giant antiparallel β-barrels, such as those formed by pore forming cholesterol-dependent cytolysins (CDC), are parallel relative to the axis of the β-barrel, i.e. S=0. The S=0 arrangement, however, has never been observed in crystal structures of small β-barrels. Therefore, the structural basis for how CDCs form a β-barrel and span a membrane remains to be understood.Results: Through comparison of molecular models with experimental data, we are able to identify how giant CDC β-barrels utilize a ‘near parallel’ arrangement of β-strands where S=n/2. Furthermore, we show how side-chain packing within the β-barrel lumen is an important limiting factor with respect to the possible shear values for small β-barrels (n≤24 β-strands). In contrast, our models reveal no such limitation restricts the shear value of giant β-barrels (n>24 β-strands). Giant β-barrels can thus access a different architecture compared with smaller β-barrels.Contact: michelle.dunstone@monash.eduSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM PUBLISHER]

Background: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here we characterize the origins and evolution of perforin. Results: We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1gene, and that it shares a common ancestor with the functionally related complement proteins. Conclusions: The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism. [ABSTRACT FROM AUTHOR]

As an organism that has evolved to live in environments ranging from soil to the cytosol of mammalian cells, Listeria monocytogenes must regulate the secretion and activity of protein products that promote survival within these habitats. The post-translocation chaperone PrsA2 has been adapted to assist in the folding and activity of L. monocytogenes secreted proteins required for bacterial replication within host cells. Here we present the first structure/function investigation of the contributions of PrsA2 to protein secretion and activity as well as to bacterial virulence. Domain swap experiments with the closely related L. monocytogenes PrsA1 protein combined with targeted mutagenesis indicate distinct functional roles for the PrsA2 peptidyl-prolyl isomerase (PPIase) and the N- and C-terminal domains in pathogenesis. In contrast to other PrsA-like proteins described thus far in the literature, an absolute in vivo requirement for PrsA2 PPIase activity is evident in mouse infection models. This work illustrates the diversity of function associated with L. monocytogenes PrsA2 that serves to promote bacterial life within the infected host. [ABSTRACT FROM AUTHOR]

Abstract: Our aim was to ascertain structural determinants of autoantigenicity based on the model of the diabetes autoantigen glutamic acid decarboxylase 65kDa isoform (GAD65) in comparison with that of the non-autoantigenic isoform GAD67. This difference exists despite the two isoforms having the same fold and high sequence identity. Autoantibodies to GAD65 precede the development of type 1 diabetes and are clinical markers of this and certain neural autoimmune diseases. To date, epitope mapping has been based on particular amino acid differences between the two isoforms, and there is no explanation as to why autoantibodies that react with GAD65 only infrequently cross-react with GAD67. To characterize each isoform of the enzyme and gain insights into their contrasting autoantigenic properties, we have used the recently determined crystal structures of GAD65 and GAD67 to compare their structure, hydrophobicity, electrostatics, flexibility and physiochemical properties. The results revealed striking differences which appear almost exclusively at the C-terminal domain of the isoforms. Whereas GAD65 displayed a highly charged and flexible C-terminal domain containing numerous patches of high electrostatic and solvation energies, these characteristics were absent in the GAD67 molecule. Additionally, analysis indicated potential N-terminal and PLP domain binding sites surrounding the C-terminal domain of GAD65, a major region of autoantigenic activity, but not of GAD67. These features agree with good accuracy with published epitope-mapping data. Our analysis suggests that the high flexibility and charge of GAD65 in the C-terminal domain is coupled with the mobility of its catalytic loop, a property that is absolutely required for its enzymatic function. Thus, the structural features that distinguish GAD65 from GAD67 as a B cell autoantigen are related to functional requirements for its enzymatic mechanism. This could well apply to the various other enzyme autoantigens and, if so, these features could be used as the basis of future predictive strategies. [Copyright &y& Elsevier]

The pore-forming protein perform is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perform cytotoxicity with cancer susceptibility in humans. Here, we examined perform function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their per-form (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast. truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perform-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perform function is a predictor of FHL severity. [ABSTRACT FROM AUTHOR]

SerpinB6 (Proteinase inhibitor 6/PI-6) is an intracellular serpin produced by leukocytes, platelets, endothelial cells, keratinocytes and other epithelial cells. It is a potent cathepsin G inhibitor thought to protect monocytes, neutrophils and bystander cells from ectopic cathepsin G during inflammation. Here we show that serpinB6 also inhibits the human serine protease kallikrein-8 (hK8) and that in human and mouse skin, serpinB6 and kallikrein-8 co-localize in differentiated keratinocytes. SerpinB6 inhibits hK8 with an association rate constant (kass) of 1.8 ± 0.2 × 105 M−1s−1 compared to 3.4 ± 0.2 × 106 M−1 s−1 for the interaction between the mouse orthologue of serpinB6 (SPI3/serpinb6a) and mouse kallikrein-8 (mK8). Molecular modelling suggested that the lower efficiency of the serpinB6/hK8 interaction is partly due to the bulkier P2 methionine residue of serpinB6 compared to the smaller P2 valine in SPI3. Taken together, these results suggest that serpinB6 is a physiologically relevant inhibitor of hK8 in skin. We postulate that serpinB6 protects the intracellular compartment of keratinocytes from ectopic hK8. [ABSTRACT FROM PUBLISHER]

Ataxin-3 belongs to the family of polyglutamine proteins, which are associated with nine different neurodegenerative disorders. Relatively little is known about the structural and functional properties of ataxin-3, and only recently have these aspects of the protein begun to be explored. We have performed a preliminary investigation into the conserved N-terminal domain of ataxin-3, termed Josephin. We show that Josephin is a monomeric domain which folds into a globular conformation and possesses ubiquitin protease activity. In addition, we demonstrate that the presence of the polyglutamine region of the protein does not alter the structure of the protein. However, its presence destabilizes the Josephin domain. The implications of these data in the pathogenesis of polyglutamine repeat proteins are discussed. [Copyright &y& Elsevier]

Many gene superfamilies have hundreds or thousands of members and hence pose a significant challenge when performing a large-scale phylogenetic analysis. Derivation of the most accurate alignment possible and inference of evolutionary relationships (with an appropriate measure of confidence) are significant “bottlenecks” in the process. A generally applicable strategy is outlined for identifying and aligning sequences, performing simple analysis of the resulting alignment, and inferring evolutionary relationships. Reference is made to the serpin superfamily. The ‘partition cluster’ method, a relatively rapid technique for extracting underlying associations from phylogenetic bootstrap trees, is also presented. [Copyright &y& Elsevier]

The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases Clr and Cls. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for Cls, C2 and C4. These studies showed that the P[SUB1]'-P[SUB4]' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When Cls was in complex with Clq and Clr, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C 1 s for prime side amino acids by examining a substrate library in which each of the P[SUB1]'-P[SUB4]' positions had been substituted by different classes of amino acids. This revealed that the Pi' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P[SUB1]'-P[SUB4]' positions abolished the allosteric behavior, indicating that contact residues at these positions in the Cls enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates. [ABSTRACT FROM AUTHOR]

MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wildtype MENT with nucleosome arrays caused their tight folding and self. association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-37 is a member of the IL-1 family of cytokines. Although its broad anti-inflammatory properties are well described, the effects of IL-37 on inflammasome function remain poorly understood. Performing gene expression analyses, ASC oligomerization/speck assays and caspase-1 assays in bone marrow-derived macrophages (BMDM), and employing an in vivo endotoxemia model, we studied how IL-37 affects the expression and maturation of IL-1β and IL-18, inflammasome activation, and pyroptosis in detail. IL-37 inhibited IL-1β production by NLRP3 and AIM2 inflammasomes, and IL-18 production by the NLRP3 inflammasome. This inhibition was partially attributable to effects on gene expression: whereas IL-37 did not affect lipopolysaccharide (LPS)-induced mRNA expression of Il18 or inflammasome components, IL-37-transgenic BMDM displayed an up to 83% inhibition of baseline and LPS-stimulated Il1b compared to their wild-type counterparts. Importantly, we observed that IL-37 suppresses nigericin- and silica-induced ASC oligomerization/speck formation (a step in inflammasome activation and subsequent caspase-1 activation), and pyroptosis (−50%). In mice subjected to endotoxemia, IL-37 inhibited plasma IL-1β (−78% compared to wild-type animals) and IL-18 (−61%). Thus, our study adds suppression of inflammasome activity to the portfolio of anti-inflammatory pathways employed by IL-37, highlighting this cytokine as a potential tool for treating inflammasome-driven diseases. [ABSTRACT FROM AUTHOR]

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands. [Display omitted] • A fragment screen was used to identify binders of the cytolytic protein perforin. • Structure-guided design enabled optimisation to yield ligands with nanomolar potency in cells. • Target engagement of ligands with perforin was demonstrated using 19F NMR and SPR. • These ligands have superior physicochemical properties compared to previous inhibitors. • Further cellular studies will be undertaken to define the mechanism of action of these novel ligands. [ABSTRACT FROM AUTHOR]

The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction. [ABSTRACT FROM AUTHOR]

• Solution structural determination by high-resolution NMR of the A-domain of PAM reveals occupancy by two molecules of K2 hPg on opposite faces of the A-domain helix. • K2 hPg binds to PAM through repeating sequences (a1a2 -repeats) in the A domain of PAM and uses the same lysine binding site for its interaction with each of the two a -repeats. • K2 hPg binds more tightly to the a2 -region than the a1 -region due to different sets of exosite interactions. VEK50 is a truncated peptide from a Streptococcal pyogenes surface human plasminogen (hPg) binding M-protein (PAM). VEK50 contains the full A-domain of PAM, which is responsible for its low nanomolar binding to hPg. The interaction of VEK50 with kringle 2, the PAM-binding domain in hPg (K2 hPg), has been studied by high-resolution NMR spectroscopy. The data show that each VEK50 monomer in solution contains two tight binding sites for K2 hPg , one each in the a1- (RH1; R17H18) and a2- (RH2; R30H31) repeats within the A-domain of VEK50. Two mutant forms of VEK50, viz., VEK50[RH1/AA] (VEK50ΔRH1) and VEK50[RH2/AA] (VEK50ΔRH2), were designed by replacing each RH with AA, thus eliminating one of the K2 hPg binding sites within VEK50, and allowing separate study of each binding site. Using 13C- and 15N-labeled peptides, NMR-derived solution structures of VEK50 in its complex with K2 hPg were solved. We conclude that the A-domain of PAM can accommodate two molecules of K2 hPg docked within a short distance of each other, and the strength of the binding is slightly different for each site. The solution structure of the VEK50/K2 hPg , complex, which is a reductionist model of the PAM/hPg complex, provides insights for the binding mechanism of PAM to a host protein, a process that is critical to S. pyogenes virulence. [ABSTRACT FROM AUTHOR]

Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly. Unlabelled Image • PAM is a major virulence factor of GAS and a receptor for human Plg. • PAM contains two Plg KR2 binding motifs (a1 and a2). • Co-crystal structure shows VEK75 binds to two KR2 via the a1 and a2 motifs. • Mutational studies reveal that a2 has higher affinity for Plg. • This study provides new insights into the high affinity interaction between PAM and Plg [ABSTRACT FROM AUTHOR]

Plasminogen (Plg) is the zymogen form of the serine protease plasmin (Plm), and it plays a crucial role in fibrinolysis as well as wound healing, immunity, tissue remodeling and inflammation. Binding to the targets via the lysine-binding sites allows for Plg activation by plasminogen activators (PAs) present on the same target. Cellular uptake of fibrin degradation products leads to apoptosis, which represents one of the pathways for cross-talk between fibrinolysis and tissue remodeling. Therapeutic manipulation of Plm activity plays a vital role in the treatments of a range of diseases, whereas Plm inhibitors are used in trauma and surgeries as antifibrinolytic agents. Plm inhibitors are also used in conditions such as angioedema, menorrhagia and melasma. Here, we review the rationale for the further development of new Plm inhibitors, with a particular focus on the structural studies of the active site inhibitors of Plm. We compare the binding mode of different classes of inhibitors and comment on how it relates to their efficacy, as well as possible future developments. [ABSTRACT FROM AUTHOR]

• Cross-platform and cross-manufacturer API support for FIB-SEM instruments. • Enabling the rapid of automated pipelines through high-level abstractions. • Extended features such as advanced movements and image post-processing. Standardised data handling and image/metadata format. • Standardised and re-usable user interface components using napari. • Integrated modules for U-Net-based image segmentation and annotation. This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem). [ABSTRACT FROM AUTHOR]

In Drosophila, key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila. We previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like, the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torsolike from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila. [ABSTRACT FROM AUTHOR]

Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to <15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signaling. These include twins (tws), which encodes an integral subunit of the protein phosphatase 2A complex, and α-tubulin at 84B (aTub84B), a major constituent of the microtubule network, suggesting that these may play an important part in terminal patterning. Together, these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signaling controls the initiation of metamorphosis. [ABSTRACT FROM AUTHOR]

Cytotoxic lymphocytes play a key role in immune homeostasis through elimination of virally-infected and transformed target cells. They do this by employing the potent pore-forming protein, perforin, a molecule that permits cytotoxic proteases, such as granzyme B, to enter the target cell cytoplasm. The synergistic activities of perforin and granzymes bring about the destruction of target cells in a process that is now more clearly understood as a result of structural and cellular biology. These data are helping the development of new classes of immunosuppressive molecules for use in treating immune driven disease and in enhancing the success of transplant therapies. This review focuses on structural and biological aspects of perforin function. [ABSTRACT FROM AUTHOR]

Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications. [ABSTRACT FROM AUTHOR]

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens , the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C. perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus. [Display omitted] • Crystal structures of TcpA (with and without ATP) were determined • TcpA shares most structural similarity with the bacterial segregation protein FtsK • The unstructured C-terminal domain of TcpA might be an anchor point for the relaxosome Conjugative plasmids have the capacity to move themselves from one bacterium to another. Traore et al. determined the crystal structure of TcpA, a key protein involved in conjugation in the pathogen Clostridium perfringens. This study suggests that conjugative transfer in C. perfringens might have evolved from the bacterial cell division machinery. [ABSTRACT FROM AUTHOR]