Vγ9Vδ2 T cells recognize butyrophilin 2A1 and 3A1 heteromers.

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.

Implementation of robotic pancreaticoduodenectomy at a community tertiary care hospital utilizing a comprehensive curriculum.

BACKGROUND: We evaluated the outcomes of a robotic pancreaticoduodenectomy (RPD) program implemented at a community tertiary care hospital. METHODS: A retrospective review of 65 RPD cases compared surgical outcomes and performance to benchmark data. RESULTS: Postoperative complications occurred in 31% (20) of patients vs. ≤73% (variance -42), with grade IV complications in 3% (2) vs. ≤5% (variance -2). Postoperative pancreatic fistula type B frequency was 12% (8) vs. ≤15% (variance -3). One 90-day mortality occurred (1.5% vs. 1.6%). Failure to rescue rate was 7% vs. ≤9% (variance -2), and R1 resection rate was 2% vs. ≤39% (variance -37). There was a downward trend of operative time (rho â€‹= â€‹-0.600, P â€‹< â€‹0.001), with a learning curve of 27 cases. Median hospital length of stay was 6 days vs. ≤15 days (variance -9). CONCLUSION: Our comprehensive RPD training program resulted in improved operative performance and outcomes commensurate with benchmark thresholds.

Inhibitors of the Oncogenic PA2G4-MYCN Protein-Protein Interface.

MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R1 position. Replacing the trifluoromethyl group on the phenyl ring at the R2 position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.

Bromodomain and extraterminal protein-targeted probe enables tumour visualisation in vivo using positron emission tomography.

Bromodomain and extraterminal (BET) proteins, a family of epigenetic regulators, have emerged as important oncology drug targets. BET proteins have not been targeted for molecular imaging of cancer. Here, we report the development of a novel molecule radiolabelled with positron emitting fluorine-18, [18F]BiPET-2, and its in vitro and preclinical evaluation in glioblastoma models.

A structural view of PA2G4 isoforms with opposing functions in cancer.

The role of proliferation-associated protein 2G4 (PA2G4), alternatively known as ErbB3-binding protein 1 (EBP1), in cancer has become apparent over the past 20 years. PA2G4 expression levels are correlated with prognosis in a range of human cancers, including neuroblastoma, cervical, brain, breast, prostate, pancreatic, hepatocellular, and other tumors. There are two PA2G4 isoforms, PA2G4-p42 and PA2G4-p48, and although both isoforms of PA2G4 regulate cellular growth and differentiation, these isoforms often have opposing roles depending on the context. Therefore, PA2G4 can function either as a contextual tumor suppressor or as an oncogene, depending on the tissue being studied. However, it is unclear how distinct structural features of the two PA2G4 isoforms translate into different functional outcomes. In this review, we examine published structures to identify important structural and functional components of PA2G4 and consider how they may explain its crucial role in the malignant phenotype. We will highlight the lysine-rich regions, protein-protein interaction sites, and post-translational modifications of the two PA2G4 isoforms and relate these to the functional cellular role of PA2G4. These data will enable a better understanding of the function and structure relationship of the two PA2G4 isoforms and highlight the care that will need to be undertaken for those who wish to conduct isoform-specific structure-based drug design campaigns.

Drugging MYCN Oncogenic Signaling through the MYCN-PA2G4 Binding Interface.

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN-PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN-PA2G4 protein-protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. SIGNIFICANCE: Competitive chemical inhibition of the PA2G4-MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

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.

Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations.

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd2+-loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target.

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.

QM/MM simulations provide insight into the mechanism of bioluminescence triggering in ctenophore photoproteins.

Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.

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.

Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 (Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS.

Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

The GM-CSF, IL-3, and IL-5 receptors constitute the ßc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while ßc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal.

Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding.

The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae.

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.

Crystal structure of the Leishmania major MIX protein: a scaffold protein that mediates protein-protein interactions.

Infection by Leishmania and Trypanosoma causes severe disease and can be fatal. The reduced effectiveness of current treatments is largely due to drug resistance, hence the urgent need to develop new drugs, preferably against novel targets. We have recently identified a mitochondrial membrane-anchored protein, designated MIX, which occurs exclusively in these parasites and is essential for virulence. We have determined the crystal structure of Leishmania major MIX to a resolution of 2.4 Å. MIX forms an all α-helical fold comprising seven α-helices that fold into a single domain. The distribution of helices is similar to a number of scaffold proteins, namely HEAT repeats, 14-3-3, and tetratricopeptide repeat proteins, suggesting that MIX mediates protein-protein interactions. Accordingly, using copurification and mass spectroscopy we were able to identify several proteins that may interact with MIX in vivo. Being parasite specific, MIX is a promising new drug target and, thus, the structure and potential interacting partners provide a basis for structure-guided drug discovery.

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase.

The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Šresolution. The crystals belonged to space group P2, with unit-cell parameters a=25.8, b=34.6, c=63.2 Å, ß=99.4°.