Sal-type ABC-F proteins: intrinsic and common mediators of pleuromutilin resistance by target protection in staphylococci.

The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that-by analogy to other proteins of this type-may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.

A missense variant in specificity protein 6 (SP6) is associated with amelogenesis imperfecta.

Amelogenesis is the process of enamel formation. For amelogenesis to proceed, the cells of the inner enamel epithelium (IEE) must first proliferate and then differentiate into the enamel-producing ameloblasts. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective or absent tooth enamel. We identified a 2 bp variant c.817_818GC>AA in SP6, the gene encoding the SP6 transcription factor, in a Caucasian family with autosomal dominant hypoplastic AI. The resulting missense protein change, p.(Ala273Lys), is predicted to alter a DNA-binding residue in the first of three zinc fingers. SP6 has been shown to be crucial to both proliferation of the IEE and to its differentiation into ameloblasts. SP6 has also been implicated as an AI candidate gene through its study in rodent models. We investigated the effect of the missense variant in SP6 (p.(Ala273Lys)) using surface plasmon resonance protein-DNA binding studies. We identified a potential SP6 binding motif in the AMBN proximal promoter sequence and showed that wild-type (WT) SP6 binds more strongly to it than the mutant protein. We hypothesize that SP6 variants may be a very rare cause of AI due to the critical roles of SP6 in development and that the relatively mild effect of the missense variant identified in this study is sufficient to affect amelogenesis causing AI, but not so severe as to be incompatible with life. We suggest that current AI cohorts, both with autosomal recessive and dominant disease, be screened for SP6 variants.

Selective Affimers Recognise the BCL-2 Family Proteins BCL-xL and MCL-1 through Noncanonical Structural Motifs*.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-xL and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.

Stapled Peptides as HIF-1α/p300 Inhibitors: Helicity Enhancement in the Bound State Increases Inhibitory Potency.

Protein-protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide-based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia-inducible factor 1 (HIF-1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF-1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α-helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α-helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra.

Tula orthohantavirus nucleocapsid protein is cleaved in infected cells and may sequester activated caspase-3 during persistent infection to suppress apoptosis.

The family Hantaviridae mostly comprises rodent-borne segmented negative-sense RNA viruses, many of which are capable of causing devastating disease in humans. In contrast, hantavirus infection of rodent hosts results in a persistent and inapparent infection through their ability to evade immune detection and inhibit apoptosis. In this study, we used Tula hantavirus (TULV) to investigate the interplay between viral and host apoptotic responses during early, peak and persistent phases of virus infection in cell culture. Examination of early-phase TULV infection revealed that infected cells were refractory to apoptosis, as evidenced by the complete lack of cleaved caspase-3 (casp-3C) staining, whereas in non-infected bystander cells casp-3C was highly abundant. Interestingly, at later time points, casp-3C was abundant in infected cells, but the cells remained viable and able to continue shedding infectious virus, and together these observations were suggestive of a TULV-associated apoptotic block. To investigate this block, we viewed TULV-infected cells using laser scanning confocal and wide-field deconvolution microscopy, which revealed that TULV nucleocapsid protein (NP) colocalized with, and sequestered, casp-3C within cytoplasmic ultrastructures. Consistent with casp-3C colocalization, we showed for the first time that TULV NP was cleaved in cells and that TULV NP and casp-3C could be co-immunoprecipitated, suggesting that this interaction was stable and thus unlikely to be solely confined to NP binding as a substrate to the casp-3C active site. To account for these findings, we propose a novel mechanism by which TULV NP inhibits apoptosis by spatially sequestering casp-3C from its downstream apoptotic targets within the cytosol.

Genetic Analysis of Physcomitrella patens Identifies ABSCISIC ACID NON-RESPONSIVE, a Regulator of ABA Responses Unique to Basal Land Plants and Required for Desiccation Tolerance.

The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. We propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.

Structure and Function of the Human Respiratory Syncytial Virus M2-1 Protein.

Human respiratory syncytial virus (HRSV) is a non-segmented negative stranded RNA virus and is recognized as the most important viral agent of lower respiratory tract infection worldwide, responsible for up to 199,000 deaths each year. The only FDA-approved regime to prevent HRSV-mediated disease is pre-exposure administration of a humanized HRSV-specific monoclonal antibody, which although being effective, is not in widespread usage due to its cost. No HRSV vaccine exists and so there remains a strong need for alternative and complementary anti-HRSV therapies. The HRSV M2-1 protein is a transcription factor and represents an attractive target for the development of antiviral compounds, based on its essential role in the viral replication cycle. To this end, a detailed analysis of M2-1 structure and functions will aid in identifying rational targets for structure-based antiviral drug design that can be developed in future translational research. Here we present an overview of the current understanding of the structure and function of HRSV M2-1, drawing on additional information derived from its structural homologues from other related viruses.

Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle.

UNLABELLED: The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease. IMPORTANCE: Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance.

Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation.

α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Probing Protein Surfaces: QSAR Analysis with Helix Mimetics.

α-Helix-mediated protein-protein interactions (PPIs) are important targets for small-molecule inhibition; however, generic approaches to inhibitor design are in their infancy and would benefit from QSAR analyses to rationalise the noncovalent basis of molecular recognition by designed ligands. Using a helix mimetic based on an oligoamide scaffold, we have exploited the power of a modular synthesis to access compounds that can readily be used to understand the noncovalent determinants of hDM2 recognition by this series of cell-active p53/hDM2 inhibitors.

Synthesis of highly functionalized oligobenzamide proteomimetic foldamers by late stage introduction of sensitive groups.

α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation.

The crystal structure of the Hazara virus nucleocapsid protein.

BACKGROUND: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target. RESULTS: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P212121 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N- and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers. CONCLUSIONS: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

Bespoke RNA recognition by Pumilios.

Pumilio is an RNA-binding protein originally identified in Drosophila, with a Puf domain made up of eight Puf repeats, three helix bundles arranged in a rainbow architecture, where each repeat recognizes a single base of the RNA-binding sequence. The eight-base recognition sequence can therefore be modified simply via mutation of the repeat that recognizes the base to be changed and this is understood in detail via high-resolution crystal structures. The binding mechanism is also altered in a variety of homologues from different species, with bases flipped out from the binding site to regenerate a consensus sequence. Thus Pumilios can be designed with bespoke RNA recognition sequences and can be fused to nucleases, split GFP, etc. as tools in vitro and in cells.

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization.

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid.

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.

Selective and potent proteomimetic inhibitors of intracellular protein-protein interactions.

Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.

Small-molecule proteomimetic inhibitors of the HIF-1α-p300 protein-protein interaction.

The therapeutically relevant hypoxia inducible factor HIF-1α-p300 protein-protein interaction can be orthosterically inhibited with α-helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C-terminal helix of the HIF-1α C-TAD (C-terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side-chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF-1α-p300 PPI inhibitors and the first examples of small-molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF-1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42-residue HIF-1α C-TAD from which it is derived.

Probing bunyavirus N protein oligomerisation using mass spectrometry.

RATIONALE: Bunyaviruses have become a major threat to both humans and livestock in Europe and the Americas. The nucleocapsid (N) protein of these viruses is key to the replication cycle and knowledge of the N oligomerisation state is central to understanding the viral lifecycle and for development of therapeutic strategies. METHODS: Bunyamwera virus and Schmallenberg virus N proteins (BUNV-N and SBV-N) were expressed recombinantly in E. coli as hexahistidine-SUMO-tagged fusions, and the tag removed subsequently. Noncovalent nano-electrospray ionisation mass spectrometry was conducted in the presence and absence of short RNA oligonucleotides. Instrumental conditions were optimised for the transmission of intact protein complexes into the gas phase. The resulting protein-protein and protein-RNA complexes were identified and their stoichiometries verified by their mass. Collision-induced dissociation tandem mass spectrometry was used in cases of ambiguity. RESULTS: Both BUNV-N and SBV-N proteins reassembled into N-RNA complexes in the presence of RNA; however, SBV-N formed a wider range of complexes with varying oligomeric states. The N:RNA oligomers observed were consistent with a model of assembly via stepwise addition of N proteins. Furthermore, upon mixing the two proteins in the presence of RNA no heteromeric complexes were observed, thus revealing insights into the specificity of oligomerisation. CONCLUSIONS: Noncovalent mass spectrometry has provided the first detailed analysis of the co-populated oligomeric species formed by these important viral proteins and revealed insights into their assembly pathways. Using this technique has also enabled comparisons to be made between the two N proteins.

Kinked ß-strands mediate high-affinity recognition of mRNA targets by the germ-cell regulator DAZL.

A defect in germ-cell (sperm and oocyte) development is the leading cause of male and female infertility. Control of translation through the binding of deleted in azoospermia (DAZ)-like (DAZL) to the 3'-UTRs of mRNAs, via a highly conserved RNA recognition motif (RRM), has been shown to be essential in germ-cell development. Crystal structures of the RRM from murine DAZL (Dazl), both alone and in complex with RNA sequences from the 3'-UTRs of mRNAs regulated by Dazl, reveal high-affinity sequence-specific recognition of a GUU triplet involving an extended, kinked, pair of ß-strands. Recognition of the GUU triplet is maintained, whereas the identity and position of bases flanking this triplet varies. The Dazl RRM is thus able to recognize GUU triplets in different sequence contexts. Mutation of bases within the GUU triplet reduces the affinity of binding. Together with the demonstration that multiple Dazl RRMs can bind to a single RNA containing multiple GUU triplets, these structures suggest that the number of DAZL molecules bound to GUU triplets in the 3'-UTR provides a method for modulating the translation of a target RNA. The conservation of RNA binding and structurally important residues between members of the DAZ family, together with the demonstration that mutation of these residues severely impairs RNA binding, indicate that the mode of RNA binding revealed by these structures is conserved in proteins essential for gamete development from flies to humans.