Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Proteins and Proteomics (v.1804, #7)
Molecular mechanism of Thioflavin-T binding to amyloid fibrils by Matthew Biancalana; Shohei Koide (pp. 1405-1412).
Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used “gold standards” for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT–fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
Keywords: Cross-beta; Amyloid dye; Protein design; Molecular dynamics simulation; Molecular recognition; Self-assembly
Functional expression of horsegram ( Dolichos biflorus) Bowman–Birk inhibitor and its self-association by Deepa G. Muricken; Lalitha R. Gowda (pp. 1413-1423).
Horsegram ( Dolichos biflorus), a protein-rich leguminous pulse, native to Southeast Asia and tropical Africa, contains multiple forms of Bowman–Birk inhibitors. The major Bowman–Birk inhibitor from horsegram (HGI-III) was cloned and functionally expressed in E scherichia coli (rHGI), which moved as a dimer in solution similar to the natural inhibitor. The biochemical characterization of rHGI also points to its close resemblance with HGI-III not only in its structure but also in its inhibitory characteristics. To explore the electrostatic interactions involved in the dimerization, a site-directed mutagenesis approach was used. The role of reactive site residue K24 and the C-terminal Asp in the structure and stability of the dimer was accomplished by mutating K24 and D75/76. The mutants produced in this study confirm that the self-association of HGI-III is indeed due to the electrostatic interaction between K24 of one monomer and D75/76 of the second monomer, in agreement with our previous data. The functional expression of a Bowman–Birk inhibitor minus a fusion tag serves as a platform to study the structural and functional effects of the special pattern of seven conserved disulphide bridges.
Keywords: Abbreviations; APNE; N; -acetyl-; dl; -phenylalanine-β-naphthyl ester; BAPNA; N; -α-, benzoyl-DL-arginine-; p; -nitroanilide; BTPNA; N; -benzoyl-L-tyrosine-; p; -nitroanilide; NAPNA; N; -succinyl-Ala-Ala-Ala-; p; -nitroanilide; BBI; Bowman–Birk inhibitor; CIU; chymotrypsin inhibitory unit; DMSO; dimethyl sulfoxide; HGI; horsegram inhibitor; rHGI; recombinant horsegram inhibitor; TPCK; l; -1-tosylamido-2-phenylalanine chloromethyl ketone; TIU; trypsin inhibitory unit; T; total acrylamide concentration; C; degree of cross-linkingRecombinant horsegram inhibitor; Cloning; Site-directed mutagenesis; Monomer–dimer interaction; C-terminal Asp
Residue N84 of Yeast Cystathionine β-Synthase is a Determinant of Reaction Specificity by Pratik H. Lodha; Emily M.S. Hopwood; Adrienne L. Manders; Susan M. Aitken (pp. 1424-1431).
Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent condensation ofl-serine andl-homocysteine to forml-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The k cat /K m l -Ser of the N84A, N84D and N84H mutants for the β-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal β-elimination activity was detected for N84D ( k cat /K m l -Ser=0.23±0.02M-1 s-1) and N84H ( k cat /K m l -Ser=0.34±0.06M-1 s-1), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation withl-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site.
Keywords: Abbreviations; ALAS; 5-aminolevulinate synthase; AATase; aspartate aminotransferase; l; -Cth; l; -cystathionine; CBL; cystathionine β-lyase; CBS; cystathionine β-synthase; hCBS; human CBS; yCBS; yeast CBS; ytCBS; truncated yCBS (residues 1 - 353); DTNB; 5,5′-dithio-bis-(2-nitrobenzoic acid); l; -Hcys; l; -homocysteine; LDH; l; -lactate dehydrogenase; OASS; O; -acetylserine sulfhydrylase; PLP; pyridoxal 5′-phosphate; TrpS; tryptophan synthasePyridoxal phosphate; Amino acid metabolism; Enzyme kinetics
Conservation and divergence on plant seed 11S globulins based on crystal structures by Mary Rose G. Tandang-Silvas; Takako Fukuda; Chisato Fukuda; Krisna Prak; Cerrone Cabanos; Aiko Kimura; Takafumi Itoh; Bunzo Mikami; Shigeru Utsumi; Nobuyuki Maruyama (pp. 1432-1442).
The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with Tm values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.
Keywords: 11S globulin; Seed; Storage protein; Vacuolar sorting; Thermal stability; Surface hydrophobicity
Structure of a premicellar complex of alkyl sulfates with the interfacial binding surfaces of four subunits of phospholipase A2 by Ying H. Pan; Brian J. Bahnson (pp. 1443-1448).
The properties of three discrete premicellar complexes (E1#, E2#, E3#) of pig pancreatic group-IB secreted phospholipase A2 (sPLA2) with monodisperse alkyl sulfates have been characterized [Berg, O. G. et al., Biochemistry 43, 7999–8013, 2004]. Here we have solved the 2.7Å crystal structure of group-IB sPLA2 complexed with 12 molecules of octyl sulfate (C8S) in a form consistent with a tetrameric oligomeric that exists during the E1# phase of premicellar complexes. The alkyl tails of the C8S molecules are centered in the middle of the tetrameric cluster of sPLA2 subunits. Three of the four sPLA2 subunits also contain a C8S molecule in the active site pocket. The sulfate oxygen of a C8S ligand is complexed to the active site calcium in three of the four protein active sites. The interactions of the alkyl sulfate head group with Arg-6 and Lys-10, as well as the backbone amide of Met-20, are analogous to those observed in the previously solved sPLA2 crystal structures with bound phosphate and sulfate anions. The cluster of three anions found in the present structure is postulated to be the site for nucleating the binding of anionic amphiphiles to the interfacial surface of the protein, and therefore this binding interaction has implications for interfacial activation of the enzyme.
Keywords: Abbreviations; B-factor; temperature factor; E; i; #; premicellar complex of enzyme; E*; interfacial bound form of enzyme; i-face; interface binding surface of enzyme; MJ33; 1-hexadecyl-3-(trifluoroethyl)-; sn; -glycero-2-phosphomethanol; C; 8; S; octyl sulfate; PDB; Protein Data Bank; sPLA; 2; 14; kDa secreted phospholipase A2; RMSD; root mean squared deviation; R; free; free; R; -factor; R; working; working; R; -factorsPLA; 2; , alkyl sulfate; Premicellar complex; Interfacial enzyme; Membrane associated protein
S -sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril by Toyofumi Nakanishi; Masanori Yoshioka; Kazuyoshi Moriuchi; Daisuke Yamamoto; Motomu Tsuji; Takayuki Takubo (pp. 1449-1456).
Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluororescent microscopic properties of TTR incubated with and without cysteine- S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37°. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine- S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent KD of 7.75×10 −6M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its KD was 3.52×10 −6M while the Bmax of reduced TTR was 0.86, and its KD was 2.86×10 −6M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluororescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.
Keywords: S; -sulfonation; Transthyretin; Amyloid fibril; Oxidative deposition
Aquifex aeolicus FlgM protein exhibits a temperature-dependent disordered nature by Rhett G. Molloy; Wai Kit Ma; Andrew C. Allen; Kevin Greenwood; Lynn Bryan; Rebecca Sacora; LaBrittney Williams; Matthew J. Gage (pp. 1457-1466).
Studies on the nature and function of intrinsically disordered proteins (IDP) over the past 10years have demonstrated the importance of IDPs in normal cellular function. Although many proteins predicted to be IDPs have been experimentally characterized on an individual basis, the conservation of disorder between homologous proteins from different organisms has not been fully studied. We now demonstrate that the FlgM protein from the thermophile Aquifex aeolicus exhibits a more ordered conformation at 20°C than the previously characterized FlgM protein from Salmonella typhimurium. FlgM is an inhibitor of the RNA transcription factor σ28, which is involved in regulation of the late-stage genes involved in flagella synthesis. Previous work has shown that the S. typhimurium FlgM protein is an intrinsically disordered protein, though the C-terminus becomes ordered when bound to σ28 or under crowded solution conditions. In this work, we demonstrate that at 20°C the A. aeolicus FlgM protein exhibits alpha-helical character in circular dichroism (CD) experiments, though the percentage of alpha-helical content decreases with increased temperature, consistent with the FlgM assuming a less folded conformation. We also show that the A. aeolicus FlgM exhibits cooperativity in chemical denaturation experiments, consistent with a globular nature. Furthermore, we use the fluorescent probe FlAsH to show that the H2 helix is ordered, even in the unbound state and that the H1 and H2 helices appear to be associated with each other in the absence of the σ28 protein. Finally, we demonstrate that the H2 helix assumes an extended conformation at 85°C. Based on our results, we propose that at 20°C the A. aeolicus FlgM assumes a four-helix bundle-like conformation that becomes a more extended conformation at the A. aeolicus' physiological temperature of 85°C.
Keywords: Abbreviations; IDP; intrinsically disordered protein; FlAsH; fluorescein arsenical helix binder (4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein)FlAsH; FlgM; Intrinsically disordered protein; Protein–protein association; Thermophile
Tyrosinase inactivation in its action on dopa by Munoz-Munoz J.L. Muñoz-Muñoz; J.R. Acosta-Motos; F. Garcia-Molina; R. Varon; Garcia-Ruiz P.A. Garcia-Ruíz; J. Tudela; Garcia-Canovas F. Garcia-Cánovas; Rodriguez-Lopez J.N. Rodríguez-López (pp. 1467-1475).
Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substratel-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomerd-dopa than forl-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.
Keywords: o; -Diphenol; Stereospecificity; Inactivation; Suicide; Tyrosinase
SufS protein from Haloferax volcanii involved in Fe-S cluster assembly in haloarchaea by Basilio Zafrilla; Martinez-Espinosa Rosa María Martínez-Espinosa; Julia Esclapez; Perez-Pomares Francisco Pérez-Pomares; María José Bonete (pp. 1476-1482).
NifS-like proteins are pyridoxal 5′-phosphate (PLP)-dependent enzymes involved in sulphur transfer metabolism. These enzymes have been catalogued as cysteine desulphurases (CDs) which catalyse the conversion of L-cysteine into L-alanine and an enzyme-bound persulphide radical. This reaction, assisted by different scaffold protein machineries, seems to be the main source of sulphur for the synthesis of essential cofactors of the[Fe-S] cluster. CDs genes have been detected in the tree domains of life, but, up until now, there has been no biochemical characterisation or study into the physiological role of this enzyme in haloarchaea. In this study, we have cloned, expressed and characterised a cysteine desulphurase (SufS) from Haloferax volcanii and demonstrated that this protein is able to reconstitute the [Fe-S] cluster of halophilic ferredoxin.
Keywords: Cysteine desulphurases; Fe-S cluster; Archaea; Haloarchaea
The role of Cys108 in Trigonopsis variabilisd-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D by Mario Mueller; Regina Kratzer; Margaretha Schiller; Anita Slavica; Gerald Rechberger; Manfred Kollroser; Bernd Nidetzky (pp. 1483-1491).
Oxidative modification of Trigonopsis variabilisd-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change ind-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction withD-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50°C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability ofd-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.
Keywords: Abbreviations; DAO; d; -amino acid oxidase; DCIP; 2,6-dichloroindophenol; DTNB; 5,5′-dithio-bis(2-nitrobenzoic acid); DTT; dithiothreitol; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; Tv; DAO; Trigonopsis variabilis; d; -amino acid oxidased; -amino acid oxidase; Oxidative inactivation; Cysteine sulfinic and sulfonic acid; Site-directed mutagenesis; Mass-spectrometric characterization of chemical oxidation; Denaturation pathway
Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata by María Victoria Frassa; Ceolin Marcelo Ceolín; Marcos S. Dreon; Horacio Heras (pp. 1492-1499).
There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130×44Å. It is an octamer of four 98kDa heterodimers composed of 67 and 31kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (α+β) at 6M urea or 60–70°C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.
Keywords: Abbreviations; PV2; perivitellin-2; SAXS; small angle X ray scattering; Rg; gyration radius; TEM; transmission electron microscopy; CD; circular dichroismNeurotoxin; Perivitellin; 3D structure; Protein stability; Quaternary protein structure; Snail; Mollusc
Polyglutamine tract-binding protein-1 binds to U5-15kD via a continuous 23-residue segment of the C-terminal domain by Masaki Takahashi; Mineyuki Mizuguchi; Hiroyuki Shinoda; Tomoyasu Aizawa; Makoto Demura; Hitoshi Okazawa; Keiichi Kawano (pp. 1500-1507).
Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts with various proteins, including RNA polymerase II and the spliceosomal protein U5-15kD. PQBP-1 is known to be associated with X-linked mental retardation in which a frameshift mutation in the PQBP-1 gene occurs. In the present study, we demonstrate that PQBP-1 binds to U5-15kD via a continuous 23-residue segment within its C-terminal domain. Intriguingly, this segment is lost in the frameshift mutants of PQBP-1 associated with X-linked mental retardation. These findings suggest that the frameshift mutations in the PQBP-1 gene lead to expression of mutants lacking the ability to interact with U5-15kD.
Keywords: Abbreviations; hDim2; human Dim2; HSQC; heteronuclear single-quantum correlation; PQBP-1; polyglutamine tract-binding protein-1; PQBP-1-CT43; PQBP-1(223–265); snRNP; small nuclear ribonucleoprotein particle; XLMR; X-linked mental retardationUnstructured protein; Protein structure; PQBP-1; Polyglutamine; U5-15kD
Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl, urea, and pH by Bogumil Zelent; Kim A. Sharp; Jane M. Vanderkooi (pp. 1508-1515).
The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2°C in the presence of 0.5, 1, and 2M guanidinium–HCl, respectively. The extrapolated value for Tm in the absence of guanidinium–HCl is 73.7°C, compared with 70.2°C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35°C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.
Keywords: Abbreviations; DSC; differential scanning calorimetry; LPO; lactoperoxidase; Gdn–HCl; guanidinium hydrochlorideProtein stability; Denaturation
Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae by Guidi Francesca; Magherini Francesca; Gamberi Tania; Borro Marina; Simmaco Maurizio; Modesti Alessandra (pp. 1516-1525).
We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift.
Keywords: Yeast fermentation; Proteomics; Diauxic shift; Glucose metabolism
Specificity and mutational analysis of the metal-dependent 3-deoxy-d- manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans by Timothy M. Allison; Jeffrey A. Yeoman; Richard D. Hutton; Fiona C. Cochrane; Geoffrey B. Jameson; Emily J. Parker (pp. 1526-1536).
3-Deoxy-d- manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate andd-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d- manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn2+, Co2+ and Cd2+ (in decreasing order) being able to restore activity to metal-free enzyme. Cd2+ significantly enhanced the stability of the enzyme, raising the Tm by 14°C.d-Glucose 6-phosphate andd-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity withd-ribose 5-phosphate. The243AspGlyPro245 motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d- arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.
Keywords: Abbreviations; A5P; d-; arabinose 5-phosphate; Aae; Aquifex aeolicus; BTP; 1,3-bis[tris(hydroxymethyl)methylamino]propane; Apy; Aquifex pyrophilus; DAH7P; 3-deoxy-; d-; arabino; -heptulosonate 7-phosphate; DSF; Differential scanning fluorimetry; DTT; dithiothreitol; E4P; d-; erythrose 4-phosphate; Eco; Escherichia coli; EDTA; ethylenediaminetetraacetic acid; IPTG; isopropyl β-; d-; thiogalactopyranoside; ITC; isothermal titration calorimetry; KDO; 3-deoxy-; d-; manno; -octulosonate; KDO8P; 3-deoxy-; d-; manno; -octulosonate 8-phosphate; KDO8PS; 3-deoxy-; d-; manno; -octulosonate 8-phosphate synthase; LPS; lipopolysaccharide; MWCO; molecular weight cut-off; Nme; Neisseria meningitidis; PEP; phosphoenol pyruvate; SEC; size-exclusion chromatographyKDO8P; KDO8PS; DAH7P; Metal-dependent enzyme
Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase by Lynn McNaughton; Zhong Li; Patrick Van Roey; Steven D. Hanes; David M. LeMaster (pp. 1537-1541).
Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn α-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and15N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.
Keywords: Abbreviation; PPIase; peptidyl proline isomeraseProlyl isomerase; NMR; Flexibility; Domain; Chemical shift; 15; N relaxation
Structural basis for the different activities of yeast Grx1 and Grx2 by Wei-Fang Li; Jiang Yu; Xiao-Xiao Ma; Yan-Bin Teng; Ming Luo; Ya-Jun Tang; Cong-Zhao Zhou (pp. 1542-1547).
Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10Å and 1.50Å, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.
Keywords: Abbreviations; Grx; glutaredoxin; ROS; reactive oxygen species; GSH; reduced glutathione; GR; glutathione reductase; HED; β-hydroxyethyl disulfide; GSSG; oxidized glutathione, glutathione disulfide; β-ME-SG; glutathionylated β-mercaptoethanol; HEDS assay; glutathione:bis-(2-hydroxyethyl)-disulfide transhydrogenase assayGlutaredoxin; Glutathione; Crystal structure; Redox state; Conformational rearrangement; Glutathione-disulfide reductase activity
Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase by Franck E. Dayan; Pankaj R. Daga; Stephen O. Duke; Ryan M. Lee; Patrick J. Tranel; Robert J. Doerksen (pp. 1548-1556).
A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.
Keywords: Abbreviations; PPO; protoporphyrinogen oxidase; Protogen; protoporphyrinogen IX; Proto; protoporphyrin IX; S-PPO; wild-type PPO; R-PPO; herbicide-resistant mutant lacking Gly210 PPO; MD simulation; molecular dynamics simulationCodon deletion; Mechanism of resistance; Evolution of resistance; Helix capping; Herbicide resistance; Binding kinetics; Molecular dynamics simulation
Identification and analysis of dominant negative mutants of RAIDD and PIDD by Tae-Ho Jang; Ju Young Bae; Ok Kyoung Park; Ji Hoe Kim; Kyung-Hyun Cho; Ju-Hong Jeon; Hyun Ho Park (pp. 1557-1563).
Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.
Keywords: Apoptosis; Inflammation; Caspase-2; Apoptosome; DISC; RAIDD; PIDD PIDDosome; Dominant negative mutant