ABSTRACT
Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
Subject(s)
Antibodies , Proto-Oncogene Proteins p21(ras) , Proto-Oncogene Proteins p21(ras)/genetics , Recognition, Psychology , Hydrophobic and Hydrophilic Interactions , HLA-A Antigens/geneticsABSTRACT
Replication of the coronavirus genome starts with the formation of viral RNA-containing double-membrane vesicles (DMV) following viral entry into the host cell. The multi-domain nonstructural protein 3 (nsp3) is the largest protein encoded by the known coronavirus genome and serves as a central component of the viral replication and transcription machinery. Previous studies demonstrated that the highly-conserved C-terminal region of nsp3 is essential for subcellular membrane rearrangement, yet the underlying mechanisms remain elusive. Here we report the crystal structure of the CoV-Y domain, the most C-terminal domain of the SARS-CoV-2 nsp3, at 2.4 Å-resolution. CoV-Y adopts a previously uncharacterized V-shaped fold featuring three distinct subdomains. Sequence alignment and structure prediction suggest that this fold is likely shared by the CoV-Y domains from closely related nsp3 homologs. NMR-based fragment screening combined with molecular docking identifies surface cavities in CoV-Y for interaction with potential ligands and other nsps. These studies provide the first structural view on a complete nsp3 CoV-Y domain, and the molecular framework for understanding the architecture, assembly and function of the nsp3 C-terminal domains in coronavirus replication. Our work illuminates nsp3 as a potential target for therapeutic interventions to aid in the on-going battle against the COVID-19 pandemic and diseases caused by other coronaviruses.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Molecular Docking Simulation , Pandemics , Protein Domains , Viral Nonstructural Proteins/geneticsABSTRACT
De novo structure determination from single-wavelength anomalous diffraction using native sulfur or phospho-rus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production of heavy-atom derivatives and seleno-methio-nyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phospho-rus. However, at lower energies, the scattering and absorption of air contribute to the background noise, reduce the signals and are thus adverse to native-SAD phasing. We have previously demonstrated native-SAD phasing at an energy of 5â keV in air at the NSLS-II FMX beamline. Here, the use of a helium path developed to reduce both the noise from background scattering and the air absorption of the diffracted X-ray beam are described. The helium path was used for collection of anomalous diffraction data at 5â keV for two proteins: thaumatin and the membrane protein TehA. Although anomalous signals from each individual crystal are very weak, robust anomalous signals are obtained from data assembled from micrometre-sized crystals. The thaumatin structure was determined from 15 microcrystals and the TehA structure from 18 microcrystals. These results demonstrate the usefulness of a helium environment in support of native-SAD phasing at 5â keV.
ABSTRACT
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.
Subject(s)
COVID-19 Drug Treatment , Antiviral Agents/therapeutic use , Coronavirus 3C Proteases , Cysteine Endopeptidases/metabolism , Hepacivirus/metabolism , Humans , Molecular Docking Simulation , Protease Inhibitors/chemistry , SARS-CoV-2 , Viral Nonstructural Proteins/geneticsABSTRACT
KAMO and BLEND provide particularly effective tools to automatically manage the merging of large numbers of data sets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending BLEND to support additional clustering options such as the use of more accurate cell distance metrics and the use of reflection-intensity correlation coefficients to infer `distances' among sets of reflections. This increases the sensitivity to differences in unit-cell parameters and allows clustering to assemble nearly complete data sets on the basis of intensity or amplitude differences. If the data sets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. When starting from incomplete data sets, one applies KAMO twice, first using unit-cell parameters. In this step, either the simple cell vector distance of the original BLEND or the more sensitive NCDist is used. This step tends to find clusters of sufficient size such that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between reflections with a common hkl to merge clusters in a way that is sensitive to structural differences that may not have perturbed the unit-cell parameters sufficiently to make meaningful clusters. Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into categories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent of one another, it should be possible to greatly improve the efficacy of data-clustering software by using a multi-stage partitioning strategy. Here, one possible approach to multi-stage data clustering is demonstrated. The strategy is to use unit-cell clustering until the merged data are sufficiently complete and then to use intensity-based clustering. Using this strategy, it is demonstrated that it is possible to accurately cluster data sets from crystals that have subtle differences.
Subject(s)
Algorithms , Software , Cluster Analysis , Crystallography, X-Ray , Proteins/chemistryABSTRACT
BACKGROUND AND AIMS: Radiolabeled short peptide ligands targeting prostate-specific membrane antigen (PSMA) were developed initially for imaging and treatment of prostate cancers. While many nonprostate solid tumors including hepatocellular carcinoma (HCC) express little PSMA, their neovasculature expresses a high level of PSMA, which is avid for Gallium-68-labeled PSMA-targeting radio-ligand (68Ga-PSMA-11) for positron emission tomography (PET). However, the lack of a spontaneous animal model of tumor-associated vascular PSMA overexpression has hindered the development and assessment of PSMA-targeting radioligands for imaging and therapy of the nonprostatic cancers. We identified detectable indigenous PSMA expression on tumor neovascular endothelia in a naturally occurring woodchuck model of HCC. METHODS: Molecular docking was performed with 3 bait PSMA ligands and compared between human and woodchuck PSMA. Initially, PET images were acquired dynamically after intravenously injecting 37 MBq (1.0 mCi) of 68Ga-PSMA-11 into woodchuck models of HCC. Subsequently, 10-minute static PET scans were conducted for other animals 1-hour after injection due to HCC and liver background uptake stabilization at 30-45 minutes after injection. Liver tissue samples were harvested after imaging, fresh-frozen for quantitative reverse transcription polymerase chain reaction and western blot for validation, or fixed for histology for correlation. RESULTS: Our preclinical studies confirmed the initial clinical findings of 68Ga-PSMA-11 uptake in HCC. The agents (ligands and antibodies) developed against human PSMA were found to be reactive against the woodchuck PSMA. CONCLUSION: This animal model offers a unique opportunity for investigating the biogenesis of tumor-associated vascular PSMA, its functional role(s), and potentials for future treatment strategies targeting tumor vascular PSMA using already developed PSMA-targeting agents.
ABSTRACT
Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30â keV photon energy range and delivers a flux of 4.0â ×â 1012â photonsâ s-1 at 1â Å into a 1â µmâ ×â 1.5â µm to 10â µmâ ×â 10â µm (Vâ ×â H) variable focus, expected to reach 5â ×â 1012â photonsâ s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.
Subject(s)
Synchrotrons , Crystallography , Crystallography, X-Ray , Data Collection , Macromolecular Substances , ViscosityABSTRACT
Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.
Subject(s)
3-Mercaptopropionic Acid/metabolism , Azotobacter vinelandii/enzymology , Dioxygenases/chemistry , Dioxygenases/metabolism , Iron/chemistry , Iron/metabolism , 3-Mercaptopropionic Acid/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray/methods , Kinetics , Models, Molecular , Substrate SpecificityABSTRACT
Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ß-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ß-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.
Subject(s)
Archaea/enzymology , Archaeal Proteins/chemistry , Carotenoids/metabolism , Dioxygenases/chemistry , Archaea/chemistry , Archaea/classification , Archaea/genetics , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carotenoids/chemistry , Catalysis , Catalytic Domain , Dioxygenases/genetics , Dioxygenases/metabolism , Substrate Specificity , Synechocystis/chemistry , Synechocystis/enzymology , Synechocystis/geneticsABSTRACT
The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.
Subject(s)
Metals/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 1/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Catalytic Domain , Metals/chemistry , Models, Molecular , Nuclear Proteins/chemistry , Phosphoprotein Phosphatases/chemistry , Phosphorylation , Protein Conformation , Protein Phosphatase 1/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7â Å) were obtained and diffracted to 3.0â Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an Rwork of 23.0% and an Rfree of 27.2% at 3.0â Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form.
Subject(s)
Acyltransferases/chemistry , Escherichia coli/enzymology , Amidohydrolases/chemistry , Arabidopsis/enzymology , Catalytic Domain , Crystallography, X-Ray , Models, MolecularABSTRACT
De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements - primarily sulfur in proteins and phospho-rus in nucleic acids - increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10â µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5â keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.
ABSTRACT
Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD â¼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD â¼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.
Subject(s)
Alkenes/metabolism , Oxygenases/metabolism , Binding Sites , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Deuterium Exchange Measurement , Hydrogen-Ion Concentration , Iron/chemistry , Iron/metabolism , Kinetics , Mutagenesis, Site-Directed , Neurospora crassa/enzymology , Oxygenases/chemistry , Oxygenases/genetics , Solvents/chemistry , Solvents/metabolism , Sphingomonadaceae/enzymology , Substrate SpecificityABSTRACT
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).
Subject(s)
Crystallography, X-Ray , Models, Molecular , Proteins/chemistry , Crystallography, X-Ray/methods , Phosphatidylinositol 3-Kinases/chemistry , Protein Conformation , Pyrophosphatases/chemistryABSTRACT
The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins, mediates the receptor's transactivation function. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. Here, we show that the NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion on chemical denaturation. By combining small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map revealing metastable long-range contacts, e.g., between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) nuclear magnetic resonance that mutations near I33 alter 19F chemical shifts at S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of how specific contact metastability mediates critical functions of disordered proteins.
Subject(s)
Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Mutation , Estrogen Receptor alpha/genetics , Fluorine-19 Magnetic Resonance Imaging , Humans , Isoleucine/genetics , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Scattering, Small Angle , Serine/genetics , Transcriptional Activation , X-Ray DiffractionABSTRACT
The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1â µm beam size and photon flux of 3 × 1012 photonsâ s-1 at a photon energy of 12.66â keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100â nm raster-scanning precision at over 10 grid-linepairsâ s-1 frequency for fly scans of a 200â µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750â Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.
ABSTRACT
Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs.
Subject(s)
Cobalt/metabolism , Oxygenases/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Synechocystis/enzymology , X-Ray Absorption SpectroscopyABSTRACT
The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1ß release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Caspase Inhibitors/chemistry , Neoplasm Proteins/chemistry , Peptides/chemistry , Animals , Apoptosis Regulatory Proteins/metabolism , Caspase 3/chemistry , Caspase 3/metabolism , Caspase Inhibitors/metabolism , Catalytic Domain , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins , Jurkat Cells , Mice , Neoplasm Proteins/metabolism , Peptides/metabolism , Phosphate-Binding Proteins , Protein Structure, Secondary , RAW 264.7 Cells , THP-1 CellsABSTRACT
With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallo-graphy with crystal sizes of less than 10â µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. The method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data sets from synchrotron microdiffraction beamlines.
ABSTRACT
The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.