An intermolecular electrostatic interaction controls the prepore-to-pore transition in a cholesterol-dependent cytolysin.

ß-Barrel pore-forming toxins (ßPFTs) form an obligatory oligomeric prepore intermediate before the formation of the ß-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for ßPFTs. Using the archetype ßPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant ß-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼ 19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a ßPFT.

The structural basis for membrane binding and pore formation by lymphocyte perforin.

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.

N-Sulfonylphenoxazines as neuronal calcium ion channel blockers.

Neuropathic pain is a type of chronic pain, usually caused by nerve damage, that responds poorly to traditional pain therapies. The N-type calcium channel (CaV2.2) is a well-validated pharmacological target to treat this condition. In order to further improve the inhibition of the N-type calcium channel relative to previously described inhibitors, and also address their problematic instability in blood plasma, the development of N-sulfonylphenoxazines as new calcium channel inhibitors was pursued. A series of N-sulfonylphenoxazines bearing ammonium side chains were synthesised and tested for their ability to inhibit both CaV2.2 and CaV3.2 (T-type) neuronal ion channels. Compounds with low micromolar activity in CaV2.2 were identified, equivalent to the most effective reported for this class of bioactive, and calculations based on their physical and chemical characteristics suggest that the best performing compounds have a high likelihood of being able to penetrate the blood-brain barrier. Representative N-sulfonylphenoxazines were tested for their stability in rat plasma and were found to be much more resilient than the previously reported N-acyl analogues. These compounds were also found to be relatively stable in an in vitro liver microsome metabolism model, the first time that this has been investigated for this class of compound. Finally, molecular modelling of the CaV2.2 channel was used to gain an understanding of the mode of action of these inhibitors at a molecular level. They appear to bind in a part of the channel, in and above its selectivity filter, in a way that hinders its ability to undergo the conformational changes required to open and allow calcium ions to pass through.

Brain computer tomography in critically ill patients--a prospective cohort study.

BACKGROUND: Brain computer tomography (brain CT) is an important imaging tool in patients with intracranial disorders. In ICU patients, a brain CT implies an intrahospital transport which has inherent risks. The proceeds and consequences of a brain CT in a critically ill patient should outweigh these risks. The aim of this study was to critically evaluate the diagnostic and therapeutic yield of brain CT in ICU patients. METHODS: In a prospective observational study data were collected during one year on the reasons to request a brain CT, expected abnormalities, abnormalities found by the radiologist and consequences for treatment. An "expected abnormality" was any finding that had been predicted by the physician requesting the brain CT. A brain CT was "diagnostically positive", if the abnormality found was new or if an already known abnormality was increased. It was "diagnostically negative" if an already known abnormality was unchanged or if an expected abnormality was not found. The treatment consequences of the brain CT, were registered as "treatment as planned", "treatment changed, not as planned", "treatment unchanged". RESULTS: Data of 225 brain CT in 175 patients were analyzed. In 115 (51%) brain CT the abnormalities found were new or increased known abnormalities. 115 (51%) brain CT were found to be diagnostically positive. In the medical group 29 (39%) of brain CT were positive, in the surgical group 86 (57%), p 0.01. After a positive brain CT, in which the expected abnormalities were found, treatment was changed as planned in 33%, and in 19% treatment was changed otherwise than planned. CONCLUSIONS: The results of this study show that the diagnostic and therapeutic yield of brain CT in critically ill patients is moderate. The development of guidelines regarding the decision rules for performing a brain CT in ICU patients is needed.

Inhibition of N-type calcium ion channels by tricyclic antidepressants - experimental and theoretical justification for their use for neuropathic pain.

A number of tricyclic antidepressants (TCAs) are commonly prescribed off-label for the treatment of neuropathic pain. The blockade of neuronal calcium ion channels is often invoked to partially explain the analgesic activity of TCAs, but there has been very limited experimental or theoretical evidence reported to support this assertion. The N-type calcium ion channel (CaV2.2) is a well-established target for the treatment of neuropathic pain and in this study a series of eleven TCAs and two closely related drugs were shown to be moderately effective inhibitors of this channel when endogenously expressed in the SH-SY5Y neuroblastoma cell line. A homology model of the channel, which matches closely a recently reported Cryo-EM structure, was used to investigate via docking and molecular dynamics experiments the possible mode of inhibition of CaV2.2 channels by TCAs. Two closely related binding modes, that occur in the channel cavity that exists between the selectivity filter and the internal gate, were identified. The TCAs are predicted to position themselves such that their ammonium side chains interfere with the selectivity filter, with some, such as amitriptyline, also appearing to hinder the channel's ability to open. This study provides the most comprehensive evidence to date that supports the notion that the blockade of neuronal calcium ion channels by TCAs is at least partially responsible for their analgesic effect.

At Least Three Doses of Leading Vaccines Essential for Neutralisation of SARS-CoV-2 Omicron Variant.

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

"But Mouse, You Are Not Alone": On Some Severe Acute Respiratory Syndrome Coronavirus 2 Variants Infecting Mice.

In silico predictions combined with in vitro, in vivo, and in situ observations collectively suggest that mouse adaptation of the severe acute respiratory syndrome 2 virus requires an aromatic substitution in position 501 or position 498 (but not both) of the spike protein's receptor binding domain. This effect could be enhanced by mutations in positions 417, 484, and 493 (especially K417N, E484K, Q493K, and Q493R), and to a lesser extent by mutations in positions 486 and 499 (such as F486L and P499T). Such enhancements, due to more favorable binding interactions with residues on the complementary angiotensin-converting enzyme 2 interface, are, however, unlikely to sustain mouse infectivity on their own based on theoretical and experimental evidence to date. Our current understanding thus points to the Alpha, Beta, Gamma, and Omicron variants of concern infecting mice, whereas Delta and "Delta Plus" lack a similar biomolecular basis to do so. This paper identifies 11 countries (Brazil, Chile, Djibouti, Haiti, Malawi, Mozambique, Reunion, Suriname, Trinidad and Tobago, Uruguay, and Venezuela) where targeted local field surveillance of mice is encouraged because they may have come in contact with humans who had the virus with adaptive mutation(s). It also provides a systematic methodology to analyze the potential for other animal reservoirs and their likely locations.

Live Virus Neutralisation of the 501Y.V1 and 501Y.V2 SARS-CoV-2 Variants following INO-4800 Vaccination of Ferrets.

The ongoing COVID-19 pandemic has resulted in significant global morbidity and mortality on a scale similar to the influenza pandemic of 1918. Over the course of the last few months, a number of SARS-CoV-2 variants have been identified against which vaccine-induced immune responses may be less effective. These "variants-of-concern" have garnered significant attention in the media, with discussion around their impact on the future of the pandemic and the ability of leading COVID-19 vaccines to protect against them effectively. To address concerns about emerging SARS-CoV-2 variants affecting vaccine-induced immunity, we investigated the neutralisation of representative 'G614', '501Y.V1' and '501Y.V2' virus isolates using sera from ferrets that had received prime-boost doses of the DNA vaccine, INO-4800. Neutralisation titres against G614 and 501Y.V1 were comparable, but titres against the 501Y.V2 variant were approximately 4-fold lower, similar to results reported with other nucleic acid vaccines and supported by in silico biomolecular modelling. The results confirm that the vaccine-induced neutralising antibodies generated by INO-4800 remain effective against current variants-of-concern, albeit with lower neutralisation titres against 501Y.V2 similar to other leading nucleic acid-based vaccines.

Experimental and in silico evidence suggests vaccines are unlikely to be affected by D614G mutation in SARS-CoV-2 spike protein.

The 'D614G' mutation (Aspartate-to-Glycine change at position 614) of the SARS-CoV-2 spike protein has been speculated to adversely affect the efficacy of most vaccines and countermeasures that target this glycoprotein, necessitating frequent vaccine matching. Virus neutralisation assays were performed using sera from ferrets which received two doses of the INO-4800 COVID-19 vaccine, and Australian virus isolates (VIC01, SA01 and VIC31) which either possess or lack this mutation but are otherwise comparable. Through this approach, supported by biomolecular modelling of this mutation and the commonly-associated P314L mutation in the RNA-dependent RNA polymerase, we have shown that there is no experimental evidence to support this speculation. We additionally demonstrate that the putative elastase cleavage site introduced by the D614G mutation is unlikely to be accessible to proteases.

PConPy--a Python module for generating 2D protein maps.

UNLABELLED: PConPy is an open-source Python module for generating protein contact maps, distance maps and hydrogen bond plots. These maps can be generated in a number of publication-quality vector and raster image formats. Contact maps can be annotated with secondary structure and hydrogen bond assignments. PConPy offers a more flexible choice of contact definition parameters than existing toolkits, most notably a greater choice of inter-residue distance metrics. PConPy can be used as a stand-alone application or imported into existing source code. A web-interface to PConPy is also available for use. AVAILABILITY: The PConPy web-interface and source code can be accessed from its website at http://www.csse.unimelb.edu.au/~hohkhkh1/pconpy/. CONTACT: hohkhkh1@csse.unimelb.edu.au

The MACPF/CDC family of pore-forming toxins.

Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane beta-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development.

The Structural Basis for a Transition State That Regulates Pore Formation in a Bacterial Toxin.

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning ß-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other ß-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning ß-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large ß-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma.

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51CIn vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984-98. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Quigley et al., p. 999See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins.

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.

The biological function of an insect antifreeze protein simulated by molecular dynamics.

Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs-Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs-Thomson law.

Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust.

The ecdysone receptor(1), which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

Structural studies of Streptococcus pyogenes streptolysin O provide insights into the early steps of membrane penetration.

Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation-pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.