ABSTRACT
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.
Subject(s)
Butyrophilins , Receptors, Antigen, T-Cell, gamma-delta , Butyrophilins/metabolism , Butyrophilins/immunology , Butyrophilins/chemistry , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Humans , Protein Binding , Protein Multimerization , Antigens, CD/metabolism , Antigens, CD/immunology , Antigens, CD/chemistry , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Crystallography, X-Ray , Lymphocyte Activation/immunology , Models, Molecular , Intraepithelial Lymphocytes/immunology , Intraepithelial Lymphocytes/metabolismABSTRACT
Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
Subject(s)
Histone Acetyltransferases/metabolism , Leukemia, Myeloid, Acute/metabolism , Neoplastic Stem Cells/metabolism , Animals , Cell Line, Tumor , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Structure, TertiaryABSTRACT
Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.
Subject(s)
Protein Engineering , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Enzyme Stability , BurkholderialesABSTRACT
Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.
Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Calcitonin Receptor-Like Protein/ultrastructure , Cryoelectron Microscopy , GTP-Binding Protein alpha Subunits, Gs/metabolism , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , Receptor Activity-Modifying Protein 1/ultrastructure , Receptors, Calcitonin Gene-Related Peptide/metabolism , Receptors, Calcitonin Gene-Related Peptide/ultrastructure , Binding Sites , Calcitonin Gene-Related Peptide/chemistry , Calcitonin Receptor-Like Protein/chemistry , Calcitonin Receptor-Like Protein/metabolism , GTP-Binding Protein alpha Subunits, Gs/chemistry , Humans , Molecular Dynamics Simulation , Protein Domains , Protein Stability , Receptor Activity-Modifying Protein 1/chemistry , Receptor Activity-Modifying Protein 1/metabolism , Receptors, Calcitonin Gene-Related Peptide/chemistry , ras Proteins/chemistry , ras Proteins/metabolismABSTRACT
Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
Subject(s)
Benzenesulfonates/pharmacology , Cellular Senescence/drug effects , Histone Acetyltransferases/antagonists & inhibitors , Hydrazines/pharmacology , Lymphoma/drug therapy , Lymphoma/pathology , Sulfonamides/pharmacology , Acetylation/drug effects , Animals , Benzenesulfonates/therapeutic use , Cell Proliferation/drug effects , Cells, Cultured , Drug Development , Fibroblasts , Gene Expression Regulation, Neoplastic/drug effects , Histone Acetyltransferases/deficiency , Histone Acetyltransferases/genetics , Histones/chemistry , Histones/metabolism , Hydrazines/therapeutic use , Lymphoma/enzymology , Lymphoma/genetics , Lysine/chemistry , Lysine/metabolism , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Sulfonamides/therapeutic useABSTRACT
We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human (h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII) and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a, 8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of the nearly matching KI values of 7a for both the hCA II and IX (i.e. 26.4 and 23.0 nM respectively) we explored its binding mode within the CA IX mimic isoform by means of X-ray crystal experiments on the corresponding adduct.
Subject(s)
Carbonic Anhydrase Inhibitors/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistry , Sulfonamides/chemistry , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/metabolism , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Catalytic Domain , Crystallography, X-Ray , Humans , Molecular Structure , Protein Binding , Pyrazoles/chemical synthesis , Pyrazoles/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/metabolismABSTRACT
The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen-deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.
Subject(s)
Carbonic Anhydrase Inhibitors/analysis , Carbonic Anhydrase Inhibitors/pharmacology , Drug Discovery , Drug Evaluation, Preclinical , Thiazolidinediones/analysis , Thiazolidinediones/pharmacology , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/chemistry , Crystallography, X-Ray , Humans , Hydrogen Deuterium Exchange-Mass Spectrometry , Models, Molecular , Thiazolidinediones/chemistryABSTRACT
Epacadostat (EPA), a new and promising anti-cancer small molecule is firmly established as selective inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). The X-Ray structure of the human CA IX mimic in complex with EPA is investigated here for the first time and compared to previously reported EPA-CA II adduct. The structural information obtained are all in agreement with the in vitro kinetic data which accounted for a selective inhibition of the CA IX over the CA II isoform.
Subject(s)
Antigens, Neoplasm/metabolism , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/pharmacology , Oximes/chemistry , Oximes/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Antigens, Neoplasm/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carbonic Anhydrase IX/antagonists & inhibitors , Carbonic Anhydrase IX/chemistry , Crystallography, X-Ray , Humans , Molecular Docking SimulationABSTRACT
Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP, and ATP with micromolar-range affinities. X-ray crystal structures of apo and nucleotide-bound SbtB7001 revealed that while AMP, ADP, and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop, and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased 10-fold in the presence of Ca2+, and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bicarbonates/metabolism , Cyanobacteria , Adenine Nucleotides/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Calcium/metabolism , Models, Molecular , Protein ConformationABSTRACT
Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2ß2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction.
Subject(s)
Amidohydrolases/chemistry , Bacterial Proteins/chemistry , Herbicides/chemistry , Pseudomonas/enzymology , Triazines/chemistry , Amidohydrolases/genetics , Amidohydrolases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Evolution, Molecular , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Herbicides/metabolism , Hydrolysis , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Models, Molecular , Operon , Organophosphates/chemistry , Organophosphates/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Pseudomonas/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Triazines/metabolismABSTRACT
The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.
Subject(s)
Antibodies, Protozoan/immunology , B-Lymphocytes/immunology , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , T-Lymphocytes/immunology , Animals , Antibody Affinity , Crystallography, X-Ray , Female , Humans , Malaria Vaccines/administration & dosage , Malaria Vaccines/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Protozoan Proteins/administration & dosage , Protozoan Proteins/chemistry , Protozoan Proteins/geneticsABSTRACT
Dihydropteroate synthase (DHPS) is an enzyme of the folate biosynthesis pathway, which catalyzes the formation of 7,8-dihydropteroate (DHPt) from 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP) and para-aminobenzoic acid (pABA). DHPS is the long-standing target of the sulfonamide class of antibiotics that compete with pABA. In the wake of sulfa drug resistance, targeting the structurally rigid (and more conserved) pterin site has been proposed as an alternate strategy to inhibit DHPS in wild-type and sulfa drug resistant strains. Following the work on developing pterin-site inhibitors of the adjacent enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), we now present derivatives of 8-mercaptoguanine, a fragment that binds weakly within both enzymes, and quantify sub-µm binding using surface plasmon resonance (SPR) to Escherichia coli DHPS (EcDHPS). Eleven ligand-bound EcDHPS crystal structures delineate the structure-activity relationship observed providing a structural framework for the rational development of novel, substrate-envelope-compliant DHPS inhibitors.
Subject(s)
Dihydropteroate Synthase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Guanine/analogs & derivatives , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Dihydropteroate Synthase/metabolism , Enzyme Inhibitors/metabolism , Escherichia coli/enzymology , Guanine/metabolism , Hydrogen Bonding , Ligands , Protein Binding , Protein Structure, Tertiary , Structure-Activity Relationship , Substrate Specificity , Sulfonamides/chemistry , Surface Plasmon ResonanceABSTRACT
Here we report the synthesis of natural products (NPs) 5'-O-sulfamoyl adenosine 1 and 5'-O-sulfamoyl-2-chloroadenosine 2. As primary sulfamates these compounds represent an uncommon class of NPs, furthermore there are few NPs known that contain a NS bond. Compounds 1 and 2 were evaluated for inhibition of carbonic anhydrases (CA), a metalloenzyme family where the primary sulfamate is known to coordinate to the active site zinc and form key hydrogen bonds with adjacent CA active site residues. Both NPs were good to moderate CA inhibitors, with compound 2 a 20-50-fold stronger CA inhibitor (Ki values 65-234â¯nM) than compound 1. The protein X-ray crystal structures of 1 and 2 in complex with CA II show that it is not the halogen-hydrophobic interactions that give compound 2 a greater binding energy but a slight movement in orientation of the ribose ring that allows better hydrogen bonds to CA residues. Compounds 1 and 2 were further investigated for antimicrobial activity against a panel of microbes relevant to human health, including Gram-negative bacteria (4 strains), Gram-positive bacteria (1 strain) and yeast (2 strains). Antimicrobial activity and selectivity was observed. The minimum inhibitory concentration (MIC) of NP 1 was 10⯵M against Gram-positive Staphylococcus aureus and NP 2 was 5⯵M against Gram-negative Escherichia coli. This is the first time that NP primary sulfamates have been assessed for inhibition and binding to CAs, with systematic antimicrobial activity studies also reported.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Biological Products/pharmacology , Carbonic Anhydrase Inhibitors/pharmacology , Sulfonic Acids/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Candida albicans/drug effects , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/metabolism , Cell Survival/drug effects , Cryptococcus neoformans/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , HEK293 Cells , Humans , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/chemistryABSTRACT
The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.
Subject(s)
Amidohydrolases/chemistry , Frankia/enzymology , Rhodococcus/enzymology , Amidohydrolases/isolation & purification , Catalytic Domain , Cluster Analysis , Computational Biology , Crystallography, X-Ray , Metals/analysis , Models, Molecular , Phylogeny , Protein Conformation , Sequence HomologyABSTRACT
The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34-50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
Subject(s)
Computational Biology/statistics & numerical data , Models, Molecular , Models, Statistical , Proteins/chemistry , Software , Bacteria/chemistry , Computational Biology/methods , Computer Graphics , Crystallography, X-Ray , Databases, Protein , Humans , International Cooperation , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Sequence Homology, Amino Acid , Viruses/chemistryABSTRACT
UNLABELLED: We previously isolated the transaminase KES23458 from Pseudomonas sp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, ß-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from ß-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a ß-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions for Pseudomonas sp. strain AAC. IMPORTANCE: We describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics simulations were carried out to compare the conformational changes in the transaminase protein to better understand the determinants of specificity in the protein. This study makes a substantial contribution that is of interest to the broad biotechnology and enzymology communities, providing insights into the catalytic activity of an industrially relevant biocatalyst as well as the biological function of this operon.
Subject(s)
Metabolic Networks and Pathways/genetics , Pseudomonas/enzymology , Pseudomonas/metabolism , Transaminases/metabolism , beta-Alanine/metabolism , Acetates/metabolism , Acetyl Coenzyme A/metabolism , Catalytic Domain , Crystallography, X-Ray , Metabolism , Models, Molecular , Protein Conformation , Protein Multimerization , Pseudomonas/genetics , Substrate Specificity , Transaminases/chemistry , Transaminases/geneticsABSTRACT
The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99â Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded ß-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316â K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.
Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Clostridium perfringens/enzymology , Cysteine Endopeptidases/chemistry , Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Clostridium perfringens/chemistry , Clostridium perfringens/metabolism , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Dynamic Light Scattering , Metals/metabolism , Models, Molecular , Protein ConformationABSTRACT
The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.
Subject(s)
Allophanate Hydrolase/chemistry , Allophanate Hydrolase/metabolism , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Triazines/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Protein Conformation , Pseudomonas/enzymologyABSTRACT
Auxins are important for plant growth and development, including the control of fruit ripening. Conjugation to amino acids by indole-3-acetic acid (IAA)-amido synthetases is an important part of auxin homeostasis. The structure of the auxin-conjugating Gretchen Hagen3-1 (GH3-1) enzyme from grapevine (Vitis vinifera), in complex with an inhibitor (adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate), is presented. Comparison with a previously published benzoate-conjugating enzyme from Arabidopsis thaliana indicates that grapevine GH3-1 has a highly similar domain structure and also undergoes a large conformational change during catalysis. Mutational analyses and structural comparisons with other proteins have identified residues likely to be involved in acyl group, amino acid, and ATP substrate binding. Vv GH3-1 is a monomer in solution and requires magnesium ions solely for the adenlyation reaction. Modeling of IAA and two synthetic auxins, benzothiazole-2-oxyacetic acid (BTOA) and 1-naphthaleneacetic acid (NAA), into the active site indicates that NAA and BTOA are likely to be poor substrates for this enzyme, confirming previous enzyme kinetic studies. This suggests a reason for the increased effectiveness of NAA and BTOA as auxins in planta and provides a tool for designing new and effective auxins.
Subject(s)
Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/chemistry , Vitis/enzymology , Amino Acids/metabolism , Arabidopsis/chemistry , Arabidopsis/enzymology , Arabidopsis/genetics , Crystallography, X-Ray , Enzyme Inhibitors , Fruit/chemistry , Fruit/enzymology , Fruit/genetics , Gene Expression Regulation, Plant , Homeostasis , Kinetics , Ligases/antagonists & inhibitors , Ligases/chemistry , Ligases/genetics , Models, Molecular , Mutagenesis, Site-Directed , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Substrate Specificity , Vitis/chemistry , Vitis/geneticsABSTRACT
The X-ray crystal structure of the complex of protein tyrosine phosphatase 1B with nitrate anion has been determined and modelled quantum-mechanically. Two protomers were present in the structure, one with the mechanistically important WPD loop closed and the other with this loop open. Nitrate was observed bound to each protomer, making close contacts with the S atom of the catalytic cysteine and a tyrosine residue from a crystallographically related protomer.