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Nature Nov 2019Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the...
Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed vertebrates. Here we report one crystal structure and five cryo-electron microscopy structures of Transib, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase.
Topics: Amino Acid Sequence; Animals; Apoenzymes; Base Sequence; Biocatalysis; Cryoelectron Microscopy; Crystallography, X-Ray; DNA; DNA Cleavage; DNA-Binding Proteins; Evolution, Molecular; Homeodomain Proteins; Models, Molecular; Moths; Protein Domains; Transposases
PubMed: 31723264
DOI: 10.1038/s41586-019-1753-7 -
The Protein Journal Jun 2017Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with...
Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.
Topics: Amino Acid Substitution; Apoenzymes; Enzyme Stability; Holoenzymes; Hot Temperature; Humans; Mutation, Missense; Ornithine-Oxo-Acid Transaminase; Protein Multimerization; Protein Structure, Quaternary
PubMed: 28345116
DOI: 10.1007/s10930-017-9710-5 -
Nature Communications Aug 2021There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein...
There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Apoenzymes; Binding Sites; Crystallography, X-Ray; Drug Design; Enzyme Inhibitors; Methyltransferases; Models, Molecular; Phosphates; Protein Conformation; RNA Helicases; RNA, Viral; SARS-CoV-2; Structure-Activity Relationship; Viral Nonstructural Proteins
PubMed: 34381037
DOI: 10.1038/s41467-021-25166-6 -
Proceedings of the National Academy of... Mar 2013Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic...
Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several unique computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally designed SOD1 variants. Allosteric interaction-free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together, reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.
Topics: Allosteric Regulation; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Apoenzymes; Biomechanical Phenomena; Biophysical Phenomena; Enzyme Stability; Genetic Variation; Humans; Kinetics; Metals; Models, Molecular; Molecular Dynamics Simulation; Mutant Proteins; Mutation, Missense; Protein Conformation; Protein Processing, Post-Translational; Sequence Deletion; Superoxide Dismutase; Superoxide Dismutase-1
PubMed: 23431152
DOI: 10.1073/pnas.1216597110 -
Biochemistry May 2016The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for...
The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
Topics: Apoenzymes; Candida; Formate Dehydrogenases; Fungal Proteins; Kinetics; NAD; Sodium Azide
PubMed: 27100912
DOI: 10.1021/acs.biochem.6b00181 -
The Journal of Biological Chemistry Jun 1988During the interconversion of the various cluster types observed in aconitase ([4Fe-4S]1+,2+, [3Fe-4S]0,1+, cubane, or linear types) and production of apoenzyme, changes...
During the interconversion of the various cluster types observed in aconitase ([4Fe-4S]1+,2+, [3Fe-4S]0,1+, cubane, or linear types) and production of apoenzyme, changes int he state of the sulfur ligands (RSH and S2-) are bound to occur. We have attempted to obtain information on such changes by interception of SH groups and/or by analysis of the resulting cluster or apoprotein for various forms of sulfur and enzymatic activity. During cluster interconversions no evidence was obtained for changes in SH titer that could be associated with cluster ligands. We conclude from this that the ligand exchange at the cluster is too rapid to allow trapping even by SH reagents of low Mr. The possibility that released SH becomes buried in the protein structure and unreactive cannot be entirely discounted. On formation of the apoenzyme by oxidation with ferricyanide, four SH groups per molecule became unavailable for reaction with 5,5'-dithiobis(2-nitrobenzoic acid); instead, while the holoenzyme contains no disulfides or S(0), two di- or polysulfides were found in the apoenzyme indicating that, on an average, four SH groups and three of the original four S(2-) ligands are trapped as RS-Sx-SR. In agreement with this conclusion is the fact that the apoenzyme can be reconstituted without addition of S(2-). A convenient preparative procedure for reconstitutable apoenzyme in 75% yield is [3Fe-4S] and [4Fe-4S] clusters with a variety of combinations of iron and sulfur isotopes as required for Mössbauer, resonance Raman, and electron nuclear double resonance studies.
Topics: Aconitate Hydratase; Apoenzymes; Apoproteins; Chemical Phenomena; Chemistry, Physical; Dithionitrobenzoic Acid; Dithiothreitol; Electron Spin Resonance Spectroscopy; Iodoacetamide; Iron-Sulfur Proteins; Models, Molecular
PubMed: 2836417
DOI: No ID Found -
Proteins Sep 2021The main protease M , 3CL is an important target from coronaviruses. In spite of having 96% sequence identity among M from SARS-CoV-1 and SARS-CoV-2; the inhibitors used... (Comparative Study)
Comparative Study
The main protease M , 3CL is an important target from coronaviruses. In spite of having 96% sequence identity among M from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 M so far, were found to have differential inhibitory effect on M of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of M from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 M in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo M from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in M . Moreover, we have also investigated the network connectivity on the quaternary structure of M and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.
Topics: Apoenzymes; Binding Sites; Coronavirus 3C Proteases; Holoenzymes; Models, Molecular; Protease Inhibitors; Protein Multimerization; Protein Structure, Quaternary; Severe acute respiratory syndrome-related coronavirus; SARS-CoV-2
PubMed: 33983654
DOI: 10.1002/prot.26143 -
Infection and Immunity Jul 1992Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted... (Comparative Study)
Comparative Study
Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.
Topics: Apoenzymes; Base Sequence; Blotting, Southern; Blotting, Western; Cloning, Molecular; DNA; Enzyme-Linked Immunosorbent Assay; Helicobacter Infections; Helicobacter pylori; Molecular Sequence Data; Mutagenesis; Oligonucleotide Probes; Polymerase Chain Reaction; Recombinant Proteins; Restriction Mapping; Urease
PubMed: 1612735
DOI: 10.1128/iai.60.7.2657-2666.1992 -
The Biochemical Journal Jan 19751. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid...
1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.
Topics: Alcohol Oxidoreductases; Ammonium Sulfate; Apoenzymes; Apoproteins; Electrophoresis, Polyacrylamide Gel; Flavin Mononucleotide; Kinetics; Lactates; Protein Binding; Spectrophotometry, Atomic
PubMed: 1191251
DOI: 10.1042/bj1450037 -
International Journal of Molecular... Mar 2019Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and...
Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.
Topics: Adenosine Triphosphate; Apoenzymes; Binding Sites; Catalysis; Catalytic Domain; Dihydroxyacetone; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Glyceraldehyde; Humans; Molecular Dynamics Simulation; Phosphorus-Oxygen Lyases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Substrate Specificity
PubMed: 30836629
DOI: 10.3390/ijms20051099