Aaron Oakley

Polymorphism of glutathione transferase Omega 1 in a population exposed to a high environmental arsenic burden

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2008

DOI: 10.1097/FPC.0B013E3282F29663

Front Cover: Rational Design of a 310‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp (Chem. Eur. J. 44/2018)

Publisher: Wiley

Date: 19-07-2018

DOI: 10.1002/CHEM.201803279

Structural and Thermodynamic Dissection of Linear Motif Recognition by the E. coli Sliding Clamp

Publisher: American Chemical Society (ACS)

Date: 22-10-2013

DOI: 10.1021/JM401118F

Abstract: Protein-protein interactions based on linear motif (LM) recognition play roles in many cell regulatory processes. The E. coli sliding cl is a protein mediator of replisome formation, which uses a common surface pocket composed of two subsites (I and II) to interact with LMs in multiple binding partners. A structural and thermodynamic dissection of sliding cl -LM recognition has been performed, providing support for a sequential binding model. According to the model, a hydrophobic C-terminal LM dipeptide submotif acts as an anchor to establish initial contacts within subsite I, and this is followed by formation of a stabilizing hydrogen-bonding network between the flanking LM residues and subsite II. Differential solvation/desolvation during positioning of the submotifs is proposed as a driver for the sequential binding. Our model provides general insights into linear motif recognition and should guide the design of small-molecule inhibitors of the E. coli sliding cl , an emerging antibacterial target.

Three‐dimensional structure of (1,4)‐β‐d‐mannan mannanohydrolase from tomato fruit

Publisher: Wiley

Date: 05-2005

DOI: 10.1110/PS.041260905

Structure and function of the bacterial mechanosensitive channel of large conductance

Publisher: Wiley

Date: 1999

DOI: 10.1110/PS.8.10.1915

Abstract: Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.

Structural basis for cofactor-independent dioxygenation of N -heteroaromatic compounds at the α/β-hydrolase fold

Publisher: Proceedings of the National Academy of Sciences

Date: 22-12-2009

DOI: 10.1073/PNAS.0909033107

Abstract: Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1- H -3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Rü61a and 1- H -3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N -heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the α/β-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1- H -3-hydroxy-4-oxoquinaldine substrate, its N -acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical α/β-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The “oxyanion hole” of the α/β-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions.

Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp Using a Fragment-Based Approach

Publisher: American Chemical Society (ACS)

Date: 18-03-2014

DOI: 10.1021/JM500122R

Abstract: The bacterial sliding cl (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding cl inhibitors as lead compounds with broad-spectrum antibacterial activity.

Replisome speed determines the efficiency of the Tus-Ter replication termination barrier.

Publisher: Springer Science and Business Media LLC

Date: 31-08-2015

DOI: 10.1038/NATURE14866

Abstract: In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first ex le where intrinsic differences in rates of in idual replisomes have different biological outcomes.

The Identification and Structural Characterization of C7orf24 as γ-Glutamyl Cyclotransferase

Publisher: Elsevier BV

Date: 08-2008

DOI: 10.1074/JBC.M803623200

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

Publisher: Wiley

Date: 2005

DOI: 10.1002/RCM.2039

Abstract: The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare s les of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16- ion was the most abundant ion observed for the apo form and the 8- ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaMwith TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.

Structure and function of a spectrin-like regulator of bacterial cytokinesis

Publisher: Springer Science and Business Media LLC

Date: 18-11-2014

DOI: 10.1038/NCOMMS6421

Abstract: Bacterial cell ision is facilitated by a molecular machine—the isome—that assembles at mid-cell in iding cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the isome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound isome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The in idual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

Publisher: Portland Press Ltd.

Date: 24-02-2012

DOI: 10.1042/BJ20111166

Abstract: ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met49, Met93 and Met157), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoDM49-A and apoDM157-A all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met93 was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain s les. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippoc us of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met93, a process that may contribute to apoD antioxidant function.

Dynamics of Open DNA Sliding Clamps

Publisher: Public Library of Science (PLoS)

Date: 05-05-2016

DOI: 10.1371/JOURNAL.PONE.0154899

Targeting the JNK MAPK cascade for inhibition: basic science and therapeutic potential

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.BBAPAP.2003.11.016

Structural Flexibility Modulates the Activity of Human Glutathione Transferase P1-1

Publisher: Elsevier BV

Date: 07-1996

DOI: 10.1074/JBC.271.27.16193

The Anti-cancer Drug Chlorambucil as a Substrate for the Human Polymorphic Enzyme Glutathione Transferase P1-1: Kinetic Properties and Crystallographic Characterisation of Allelic Variants

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.JMB.2008.04.066

Abstract: The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 degrees C to 45 degrees C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.

Preliminary X-ray crystallographic studies of a newly defined human theta-class glutathione transferase

Publisher: International Union of Crystallography (IUCr)

Date: 1998

DOI: 10.1107/S0907444997008366

Abstract: Human theta-class glutathione S-transferases (GST's) appear to play a critical role in the metabolism of a variety of environmental pollutants but in some cases the products of the reaction are carcinogenic. Crystals of a human theta-class GST, namely hGSTT2-2, have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. The crystals belong to the trigonal space group P 3 1 21 with cell dimensions of a = b = 94.0 and c = 120.5 Å. They contain two monomers in the asymmetric unit and diffract to 3.0 Å resolution.

Crystallization of two glutathione S-transferases from an unusual gene family

Publisher: International Union of Crystallography (IUCr)

Date: 25-05-2001

DOI: 10.1107/S0907444901004929

Abstract: Two glutathione S-transferase isozymes from the mosquito Anopheles dirus (AdGST1-3 and AdGST1-4) from an alternately spliced gene family have been expressed, purified and crystallized. The isozymes share an N-terminal domain derived from a single exon and C-terminal domains from unique exons. Despite the high level of sequence identity (64% overall), the two isozymes crystallize in different space groups, the 1-3 isozyme in P3(1)21 or P3(2)21 (unit-cell parameters a = 49.9, c = 271.8 A at 100 K) and the 1-4 isozyme in P4(1) or P4(3) (unit-cell parameters a = 87.8, c = 166.1 at 100 K). Determination of these structures will advance our understanding of how these enzymes inactivate pesticides and the structural consequences of alternate splicing.

Identification and Characterization of γ-Glutamylamine Cyclotransferase, an Enzyme Responsible for γ-Glutamyl-ϵ-lysine Catabolism

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1074/JBC.M109.082099

Macromolecular Crystallography As A Tool For Investigating Drug, Enzyme And Receptor Interactions

Publisher: Wiley

Date: 11-03-2000

DOI: 10.1046/J.1440-1681.2000.03215.X

Abstract: 1. Protein crystallography is an essential tool for the discovery and investigation of pharmacological interactions at the molecular level. It allows investigators to directly visualize the three-dimensional structures of proteins, including enzymes, receptors and hormones. 2. Increasingly, knowledge of these interactions is being used in the drug-discovery process. This is popularly called structure-based drug design. The desired drug could be an enzyme inhibitor or an agonist that mimics endogenous transmitters or hormones. 3. Once the 3-D structure of a pharmacologically relevant target is known, computational processes can be used to search databases of compounds to identify ones that may interact strongly with the target. Lead compounds can be improved using the 3-D structure of the complex of the lead compound and its biological target. 4. The present review describes the processes involved in the determination of a structure by means of protein crystallography and the use of structures in the drug-discovery process. A number of successful ex les of structure-based drug design are described. The limitations of the techniques are discussed.

The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution

Publisher: Elsevier BV

Date: 11-1997

DOI: 10.1006/JMBI.1997.1364

Abstract: The human pi-class glutathione S-transferase (hGST P1-1) is a target for structure-based inhibitor design with the aim of developing drugs that could be used as adjuvants in chemotherapeutic treatment. Here we present seven crystal structures of the enzyme in complex with substrate (glutathione) and two inhibitors (S-hexyl glutathione and gamma-glutamyl- (S-benzyl)cysteinyl-D-phenylglycine). The binding of the modified glutathione inhibitor, gamma-glutamyl-(S-benzyl)cysteinyl-D-phenylglycine, has been characterized with the phenyl group stacking against the benzyl moiety of the inhibitor and making interactions with the active-site residues Phe8 and Trp38. The structure provides an explanation as to why this compound inhibits the pi-class GST much better than the other GST classes. The structure of the enzyme in complex with glutathione has been determined to high resolution (1.9 to 2.2 A) in three different crystal forms and at two different temperatures (100 and 288 K). In one crystal form, the direct hydrogen-bonding interaction between the hydroxyl group of Tyr7, a residue involved in catalysis, and the thiol group of the substrate, glutathione, is broken and replaced by a water molecule that mediates the interaction. The hydrogen-bonding partner of the hydroxyl group of Tyr108, another residue implicated in the catalysis, is space-group dependent. A high-resolution (2.0 A) structure of the enzyme in complex with S-hexyl glutathione in a new crystal form is presented. The enzyme-inhibitor complexes show that the binding of ligand into the electrophilic binding site does not lead to any conformational changes of the protein.

T Cell Determinants Incorporating β-Amino Acid Residues Are Protease Resistant and Remain Immunogenic In Vivo

Publisher: The American Association of Immunologists

Date: 15-09-2005

DOI: 10.4049/JIMMUNOL.175.6.3810

Abstract: A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of β-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope in idually with the corresponding β-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with β-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using β-amino acids. We conclude that the rational incorporation of β-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring α-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BBRC.2010.06.024

Abstract: Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme's preference for the 3'-phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced-fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability.

Rational Design of a 3₁₀‐Helical PIP‐Box Mimetic Targeting PCNA, the Human Sliding Clamp

Publisher: Wiley

Date: 19-07-2018

DOI: 10.1002/CHEM.201801734

Abstract: The human sliding cl (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 3

Identification, characterization and structure of a new Delta class glutathione transferase isoenzyme

Publisher: Portland Press Ltd.

Date: 07-06-2005

DOI: 10.1042/BJ20042015

Abstract: The insect GST (glutathione transferase) supergene family encodes a varied group of proteins belonging to at least six in idual classes. Interest in insect GSTs has focused on their role in conferring insecticide resistance. Previously from the mosquito malaria vector Anopheles dirus, two genes encoding five Delta class GSTs have been characterized for structural as well as enzyme activities. We have obtained a new Delta class GST gene and isoenzyme from A. dirus, which we name adGSTD5-5. The adGSTD5-5 isoenzyme was identified and was only detectably expressed in A. dirus adult females. A putative promoter analysis suggests that this GST has an involvement in oogenesis. The enzyme displayed little activity for classical GST substrates, although it possessed the greatest activity for DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] observed for Delta GSTs. However, GST activity was inhibited or enhanced in the presence of various fatty acids, suggesting that the enzyme may be modulated by fatty acids. We obtained a crystal structure for adGSTD5-5 and compared it with other Delta GSTs, which showed that adGSTD5-5 possesses an elongated and more polar active-site topology.

The ligandin (non-substrate) binding site of human pi class glutathione transferase is located in the electrophile binding site (H-site)

Publisher: Elsevier BV

Date: 08-1999

DOI: 10.1006/JMBI.1999.3029

Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability

Publisher: Elsevier BV

Date: 04-2025

DOI: 10.1074/JBC.M116.763896

Proposed mechanism for monomethylarsonate reductase activity of human omega-class glutathione transferase GSTO1-1

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.BBRC.2021.12.072

Abstract: Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26:  X-ray Crystallographic Studies of Dehalogenation of Brominated Substrates^,

Publisher: American Chemical Society (ACS)

Date: 09-08-2003

DOI: 10.1021/BI027280A

Abstract: The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.

Identification of a novel tetrameric structure for human apolipoprotein-D

Publisher: Cold Spring Harbor Laboratory

Date: 31-05-2018

DOI: 10.1101/335232

Abstract: Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer’s disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer’s disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∽95 to ∽100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

Glutathione transferases: a structural perspective

Publisher: Informa UK Limited

Date: 23-03-2011

DOI: 10.3109/03602532.2011.558093

Abstract: The glutathione transferases (GSTs) are one of the most important families of detoxifying enzymes in nature. The classic activity of the GSTs is conjugation of compounds with electrophilic centers to the tripeptide glutathione (GSH), but many other activities are now associated with GSTs, including steroid and leukotriene biosynthesis, peroxide degradation, double-bond cis-trans isomerization, dehydroascorbate reduction, Michael addition, and noncatalytic "ligandin" activity (ligand binding and transport). Since the first GST structure was determined in 1991, there has been an explosion in structural data across GSTs of all three families: the cytosolic GSTs, the mitochondrial GSTs, and the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG family). In this review, the major insights into GST structure and function will be discussed.

Hidden Glutathione Transferases in the Human Genome

Publisher: MDPI AG

Date: 12-08-2023

DOI: 10.3390/BIOM13081240

Abstract: With the development of accurate protein structure prediction algorithms, artificial intelligence (AI) has emerged as a powerful tool in the field of structural biology. AI-based algorithms have been used to analyze large amounts of protein sequence data including the human proteome, complementing experimental structure data found in resources such as the Protein Data Bank. The EBI AlphaFold Protein Structure Database (for ex le) contains over 230 million structures. In this study, these data have been analyzed to find all human proteins containing (or predicted to contain) the cytosolic glutathione transferase (cGST) fold. A total of 39 proteins were found, including the alpha-, mu-, pi-, sigma-, zeta- and omega-class GSTs, intracellular chloride channels, metaxins, multisynthetase complex components, elongation factor 1 complex components and others. Three broad themes emerge: cGST domains as enzymes, as chloride ion channels and as protein–protein interaction mediators. As the majority of cGSTs are dimers, the AI-based structure prediction algorithm AlphaFold-multimer was used to predict structures of all pairwise combinations of these cGST domains. Potential homo- and heterodimers are described. Experimental biochemical and structure data is used to highlight the strengths and limitations of AI-predicted structures.

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

Publisher: Informa UK Limited

Date: 2009

DOI: 10.1080/09687680903150027

Abstract: The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.

Novel Folding and Stability Defects Cause a Deficiency of Human Glutathione Transferase Omega 1

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1074/JBC.M110.197822

Identification of a novel tetrameric structure for human apolipoprotein-D

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.JSB.2018.05.012

Abstract: Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

Characterization of a family 11 xylanase fromBacillus subtillisB230 used for paper bleaching

Publisher: International Union of Crystallography (IUCr)

Date: 25-03-2003

DOI: 10.1107/S0907444903001227

Abstract: Enzymes such as family 11 xylanases are increasingly being used for industrial applications. Here, the cloning, structure determination and temperature-stability data of a family 11 xylanase, Xyn11X, from the alkali-tolerant Bacillus subtilis subspecies B230 are reported. This enzyme, which degrades xylan polymers, is being produced on an industrial scale for use in the paper-bleaching industry. Xyn11X adopts the canonical family 11 xylanase fold. It has a greater abundance of side chain to side chain hydrogen bonds compared with all other family 11 xylanase crystal structures. Means by which the thermostability of Xyn11X might be improved are suggested.

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site.

Publisher: Elsevier BV

Date: 03-1998

DOI: 10.1016/S0969-2126(98)00034-3

Abstract: Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be ided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes. Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group. hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Cold Spring Harbor Laboratory

Date: 19-08-2021

DOI: 10.1101/2021.08.18.456904

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Wiley

Date: 12-2021

DOI: 10.1002/MBO3.1251

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long‐lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP‐dependent motor proteins distantly related to helicases. It was first identified in the model Gram‐positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram‐positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram‐negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Hotspots of age-related protein degradation: the importance of neighboring residues for the formation of non-disulfide crosslinks derived from cysteine

Publisher: Portland Press Ltd.

Date: 11-07-2017

DOI: 10.1042/BCJ20170268

Abstract: Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating ‘hotpsots’ for the spontaneous processes responsible for protein degradation in aged tissues.

Multifunctional Role of Tyr 108 in the Catalytic Mechanism of Human Glutathione Transferase P1-1. Crystallographic and Kinetic Studies on the Y108F Mutant Enzyme^,

Publisher: American Chemical Society (ACS)

Date: 05-1997

DOI: 10.1021/BI962813Z

Crystallization and diffraction data of 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase: a cofactor-free oxygenase of the α/β-hydrolase family

Publisher: International Union of Crystallography (IUCr)

Date: 06-04-2007

DOI: 10.1107/S1744309107013760

Bacterial Sliding Clamp Inhibitors that Mimic the Sequential Binding Mechanism of Endogenous Linear Motifs

Publisher: American Chemical Society (ACS)

Date: 28-05-2015

DOI: 10.1021/ACS.JMEDCHEM.5B00232

Abstract: The bacterial DNA replication machinery presents new targets for the development of antibiotics acting via novel mechanisms. One such target is the protein-protein interaction between the DNA sliding cl and the conserved peptide linear motifs in DNA polymerases. We previously established that binding of linear motifs to the Escherichia coli sliding cl occurs via a sequential mechanism that involves two subsites (I and II). Here, we report the development of small-molecule inhibitors that mimic this mechanism. The compounds contain tetrahydrocarbazole moieties as "anchors" to occupy subsite I. Functional groups appended at the tetrahydrocarbazole nitrogen bind to a channel gated by the side chain of M362 and lie at the edge of subsite II. One derivative induced the formation of a new binding pocket, termed subsite III, by rearrangement of a loop adjacent to subsite I. Discovery of the extended binding area will guide further inhibitor development.

Molecular Basis for RNA Polymerase-Dependent Transcription Complex Recycling By The Helicase-like Motor Protein HelD.

Publisher: Research Square Platform LLC

Date: 16-07-2020

DOI: 10.21203/RS.3.RS-40738/V1

Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using cryo-electron microscopy and single-particle analysis, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the extraordinary conformational changes that occur in RNAP driven by HelD interaction. HelD has a unique 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a dramatic conformational change in the primary channel leading to removal and recycling of the stalled polymerase.

Molecular dynamics analysis of apolipoprotein-D - lipid hydroperoxide interactions: Mechanism for selective oxidation of Met-93

Publisher: Public Library of Science (PLoS)

Date: 30-03-2012

DOI: 10.1371/JOURNAL.PONE.0034057

Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

Publisher: Elsevier BV

Date: 02-2004

DOI: 10.1016/J.PEP.2003.10.001

Abstract: In Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E. coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage lambda-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5mM s le in 10mM phosphate, pH 6.05, 0.1M NaCl, recorded at 36 degrees C, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a=b=142.2A, c=192.1A, and diffracted beyond 2.7A resolution with synchrotron radiation.

Structural Insights into Omega-Class Glutathione Transferases: A Snapshot of Enzyme Reduction and Identification of a Non-Catalytic Ligandin Site

Publisher: Public Library of Science (PLoS)

Date: 09-04-2013

DOI: 10.1371/JOURNAL.PONE.0060324

Intramolecular binding mode of the C-terminus ofEscherichia colisingle-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

Publisher: Oxford University Press (OUP)

Date: 27-11-2013

DOI: 10.1093/NAR/GKT1238

Abstract: Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB–ssDNA complex. 15N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

Structural and Functional Basis of Resistance to Neuraminidase Inhibitors of Influenza B Viruses

Publisher: American Chemical Society (ACS)

Date: 09-08-2010

DOI: 10.1021/JM100621S

Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example

Publisher: American Chemical Society (ACS)

Date: 13-04-2018

DOI: 10.1021/ACS.JMEDCHEM.8B00318

Abstract: Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an ex le of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

Efficient synthesis and antioxidant activity of coelenterazine analogues

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.TETLET.2014.09.065

Atomistic Insights into Photoprotein Formation: Computational Prediction of the Properties of Coelenterazine and Oxygen Binding in Obelin

Publisher: Wiley

Date: 16-12-2020

DOI: 10.1002/JCC.26125

Abstract: Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca

The glutathione conjugate of ethacrynic acid can bind to human pi class glutathione transferase P1‐1 in two different modes

Publisher: Wiley

Date: 08-12-1997

DOI: 10.1016/S0014-5793(97)01424-5

Abstract: The diuretic drug ethacrynic acid, an inhibitor of pi class glutathione S-transferase, has been tested in clinical trials as an adjuvant in chemotherapy. We recently solved the crystal structure of this enzyme in complex with ethacrynic acid and its glutathione conjugate. Here we present a new structure of the ethacrynic-glutathione conjugate complex. In this structure the ethacrynic moiety of the complex is shown to bind in a completely different orientation to that previously observed. Thus there are at least two binding modes possible, an observation of great importance to the design of second generation inhibitors of the enzyme.

Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.JSB.2018.10.008

Abstract: Bacterial sliding cl s bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding cl s via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of β sliding cl s from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli cl s and similar patterns of binding to linear cl -binding motif peptides. The results suggest that linear motif-sliding cl interactions are well conserved and an antibiotic targeting the sliding cl should have broad-spectrum activity against Gram-negative pathogens.

A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.CELL.2006.04.040

Abstract: During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

Anisotropic atomic motions in high-resolution protein crystallography molecular dynamics simulations

Publisher: IOP Publishing

Date: 11-06-2007

DOI: 10.1088/1478-3975/4/2/002

Abstract: Molecular dynamics (MD) simulations using empirical force fields are popular for the study of proteins. In this work, we compare anisotropic atomic fluctuations in nanosecond-timescale MD simulations with those observed in an ultra-high-resolution crystal structure of crambin. In order to make our comparisons, we have developed a compact graphical technique for assessing agreement between spatial atomic distributions determined by MD simulations and observed anisotropic temperature factors.

Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe2+ metal-ion preference

Publisher: International Union of Crystallography (IUCr)

Date: 05-07-2008

DOI: 10.1107/S1744309108017600

A structural view of bacterial DNA replication

Publisher: Wiley

Date: 17-04-2019

DOI: 10.1002/PRO.3615

Identification and characterisation of new inhibitors for the human hematopoietic prostaglandin D 2 synthase

Publisher: Elsevier BV

Date: 02-2010

DOI: 10.1016/J.EJMECH.2009.10.025

Abstract: Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.

Evidence for an Induced-Fit Mechanism Operating in Pi Class Glutathione Transferases^,

Publisher: American Chemical Society (ACS)

Date: 23-06-1998

DOI: 10.1021/BI980323W

Purification, crystallization and preliminary crystallographic analysis of DehIVa, a dehalogenase fromBurkholderia cepaciaMBA4

Publisher: International Union of Crystallography (IUCr)

Date: 08-02-2005

DOI: 10.1107/S1744309105002472

DNA Replication Is the Target for the Antibacterial Effects of Nonsteroidal Anti-Inflammatory Drugs

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.CHEMBIOL.2014.02.009

Abstract: Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding cl at a common binding site required for partner binding. Inhibition of interaction of the cl loader and/or the replicative polymerase α subunit with the sliding cl is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding cl .

Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1.

Publisher: American Chemical Society (ACS)

Date: 12-2000

DOI: 10.1021/BI0007122

Abstract: We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.

Fragment-based discovery of inhibitors of the bacterial DnaG-SSB interaction

Publisher: MDPI AG

Date: 22-02-2018

DOI: 10.3390/ANTIBIOTICS7010014

Crystal Structure of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å Resolution:  Dynamics of Catalytic Residues^,

Publisher: American Chemical Society (ACS)

Date: 07-01-2004

DOI: 10.1021/BI034748G

Abstract: We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C(alpha)-C(beta)-C(gamma) angle (126 degrees ) that deviated by 13.4 degrees from the "ideal" bond angle of 112.6 degrees. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C(alpha)-C(beta)-C(gamma) angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N(delta)(1) and Asp108 in conformation A gave the most exothermic reaction (DeltaH = -22 kcal/mol). With His272 doubly protonated at N(delta)(1) and N(epsilon)(2), the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C(alpha)-C(beta)-C(gamma) angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.

The 2.2 Å Crystal Structure of a Pocilloporin Pigment Reveals a Nonplanar Chromophore Conformation

Publisher: Elsevier BV

Date: 03-2003

DOI: 10.1016/S0969-2126(03)00028-5

Abstract: Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, nonfluorescent pocilloporin to 2.2 A resolution and a genetically engineered fluorescent variant to 2.4 A resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gln-Tyr-Gly) being identical the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment.

Solution Structure of Glutathione Bound to Human Glutathione Transferase P1-1:  Comparison of NMR Measurements with the Crystal Structure

Publisher: American Chemical Society (ACS)

Date: 12-02-1998

DOI: 10.1021/BI971902O

The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor

Publisher: Elsevier BV

Date: 11-2003

DOI: 10.1074/JBC.M307896200

Structural insights into the dehydroascorbate reductase activity of human omega-class glutathione transferases

Publisher: Elsevier BV

Date: 07-2012

DOI: 10.1016/J.JMB.2012.04.014

Abstract: The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.

RNA polymerases from Low G+C Gram Positive Bacteria

Publisher: Cold Spring Harbor Laboratory

Date: 07-06-2021

DOI: 10.1101/2021.06.06.447298

Abstract: The low G+C Gram positive bacteria represent some of the most medically and industrially important microorganisms. They are relied on for the production of food and dietary supplements, enzymes and antibiotics, as well as being responsible for the majority of nosocomial infections and serving as a reservoir for antibiotic resistance. Control of gene expression in this group is more highly studied than in any bacteria other than the Gram negative model Escherichia coli , yet until recently no structural information on RNA polymerase (RNAP) from this group was available. This review will summarise recent reports on the high resolution structure of RNAP from the model low G+C representative Bacillus subtilis , including the role of auxiliary subunits δ and ε, and outline approaches for the development of antimicrobials to target RNAP from this group.

S-(4-Nitrophenacyl)glutathione is a specific substrate for glutathione transferase omega 1-1

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1016/J.AB.2007.09.029

Abstract: Glutathione transferase omega 1-1 (GSTO1-1) catalyzes the biotransformation of arsenic and is implicated as a factor influencing the age-at-onset of Alzheimer's disease and the posttranslational activation of interleukin 1beta (IL-1beta). Investigation of the biological role of GSTO1-1 variants has been h ered by the lack of a specific assay for GSTO1-1 activity in tissue s les that contain other GSTs and other enzymes with similar catalytic specificities. Previous studies (P. G. Board and M. W. Anders, Chem. Res. Toxicol. 20 (2007) 149-154) have shown that GSTO1-1 catalyzes the reduction of S-(phenacyl)glutathiones to acetophenones. A new substrate, S-(4-nitrophenacyl)glutathione (4NPG), has been prepared and found to have a high turnover with GSTO1-1 but negligible activity with GSTO2-2 and other members of the glutathione transferase superfamily. A spectrophotometric assay with 4NPG as a substrate has been used to determine GSTO1-1 activity in several human breast cancer cell lines and in mouse liver and brain tissues.

Mechanism and putative structure of B0-like neutral amino acid transporters

Publisher: Springer Science and Business Media LLC

Date: 09-2006

DOI: 10.1007/S00232-006-0879-3

Abstract: The Na(+)-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by B(0)-type neutral amino acid transporters. Two B(0)-type amino acid transporters have been identified in the neurotransmitter transporter family SLC6, namely B(0)AT1 (SLC6A19) and B(0)AT2 (SLC6A15). In contrast to other members of this family, B(0)-like transporters are chloride-independent. B(0)AT1 and B(0)AT2 preferentially bind the substrate prior to the Na(+)-ion. The Na(+)-concentration affects the K ( m ) of the substrate and vice versa. A kinetic scheme is proposed that is consistent with the experimental data. An overlapping binding site of substrate and cosubstrate has been demonstrated in the bacterial orthologue LeuT( Aa ) from Aquifex aeolicus, which elegantly explains the mutual effect of substrate and cosubstrate on each other's K ( m )-value. LeuT( Aa ) is sequence-related to transporters of the SLC6 family, allowing homology modeling of B(0)-like transporters along its structure.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation‐contraction coupling

Publisher: Wiley

Date: 20-04-2022

DOI: 10.1002/PRO.4311

Abstract: Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca 2+ channels, dihydropyridine receptor (DHPR voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α 1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

AIG2A and AIG2B limit the activation of salicylic acid-regulated defenses by tryptophan-derived secondary metabolism in Arabidopsis

Publisher: Oxford University Press (OUP)

Date: 16-08-2022

DOI: 10.1093/PLCELL/KOAC255

Abstract: Chemical defense systems involving tryptophan-derived secondary metabolites (TDSMs) and salicylic acid (SA) are induced by general nonself signals and pathogen signals, respectively, in Arabidopsis thaliana. Whether and how these chemical defense systems are connected and balanced is largely unknown. In this study, we identified the AVRRPT2-INDUCED GENE2A (AIG2A) and AIG2B genes as gatekeepers that prevent activation of SA defense systems by TDSMs. These genes also were identified as important contributors to natural variation in disease resistance among A. thaliana natural accessions. The loss of AIG2A and AIG2B function leads to upregulation of both SA and TDSM defense systems. Suppressor screens and genetic analysis revealed that a functional TDSM system is required for the upregulation of the SA pathway in the absence of AIG2A and AIG2B, but not vice versa. Furthermore, the AIG2A and AIG2B genes are co-induced with TDSM biosynthesis genes by general pathogen elicitors and nonself signals, thereby functioning as a feedback control of the TDSM defense system, as well as limiting activation of the SA defense system by TDSMs. Thus, this study uncovers an AIG2A- and AIG2B-mediated mechanism that fine-tunes and balances SA and TDSM chemical defense systems in response to nonpathogenic and pathogenic microbes.

Effects of i‐propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence

Publisher: Wiley

Date: 02-2001

DOI: 10.1110/PS.21201

Abstract: Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady-state, time-resolved, and stopped-flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild-type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so-called "lid," an alpha-helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild-type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild-type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (tau(1) = 0.5--1.2 ns) for the wild-type TLL nor in the longer fluorescence lifetime component (tau(2) = 4.8--6.0 ns) in the wild-type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for tau(1) increased significantly, from 0.43 to 1.5 ns. The shorter correlation time phi(1) of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r(infinity) the litude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped-flow fluorescence spectroscopy measurements suggested the lid to open within approximately 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.

Time to face the fats: What can mass spectrometry reveal about the structure of lipids and their interactions with proteins?

Publisher: American Chemical Society (ACS)

Date: 06-06-2012

DOI: 10.1007/S13361-012-0410-2

Abstract: Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally erse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.

Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

Publisher: Cold Spring Harbor Laboratory

Date: 08-11-2021

DOI: 10.1101/2021.11.07.467205

Abstract: Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

The Three-Dimensional Structure of the Human Pi Class Glutathione Transferase P1-1 in Complex with the Inhibitor Ethacrynic Acid and Its Glutathione Conjugate^,

Publisher: American Chemical Society (ACS)

Date: 1997

DOI: 10.1021/BI962316I

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

Publisher: Oxford University Press (OUP)

Date: 10-04-2013

DOI: 10.1093/NAR/GKT162

Rational Design of a 310 -Helical PIP-Box Mimetic Targeting PCNA, the Human Sliding Clamp

Publisher: Wiley

Date: 23-07-2018

DOI: 10.1002/CHEM.201803280

Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

Publisher: MDPI AG

Date: 08-10-2012

DOI: 10.3390/IJMS131012857

Glutathione transferases: new functions

Publisher: Elsevier BV

Date: 12-2005

DOI: 10.1016/J.SBI.2005.10.005

Abstract: Well known as detoxification enzymes, the glutathione transferases also function in prostaglandin and steroid hormone synthesis. New uses for the canonical glutathione transferase fold are becoming apparent the bacterial stringent starvation protein SspA and the yeast prion protein Ure2p (both transcription factors) were found to adopt this fold, but their roles remain unclear. The intracellular chloride ion channel CLIC1 adopts the canonical glutathione transferase fold in its soluble form and appears to undergo radical structural modification as part of its membrane insertion process. The structures of rat and human mitochondrial glutathione transferases have been solved: they adopt a topology similar to that of bacterial disulfide bond isomerases, leading to the suggestion that they have evolved independently of the canonical enzymes. Recent structural studies of integral membrane glutathione S-transferases from microsomes have revealed common patterns of tertiary and quaternary structure.

Mutations of gly to ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione 1 1Edited by R. Huber

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1006/JMBI.1998.2270

Abstract: Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.

Flexibility Revealed by the 1.85 Angstrom Crystal Structure of the Beta Sliding-Clamp Subunit of Escherichia Coli Dna Polymerase Iii

Publisher: International Union of Crystallography (IUCr)

Date: 27-06-2003

DOI: 10.1107/S0907444903009958

Abstract: The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding cl that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity

Publisher: Springer Science and Business Media LLC

Date: 19-12-2017

DOI: 10.1038/S41598-017-17861-6

Abstract: Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo . In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

Crystal and Solution Structures of the Helicase-binding Domain of Escherichia coli Primase

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1074/JBC.M412645200

Exploring the Structure and Activity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26: Evidence for Product- and Water-Mediated Inhibition^,

Publisher: American Chemical Society (ACS)

Date: 22-03-2002

DOI: 10.1021/BI015734I

Abstract: The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for ex le, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Publisher: Springer Science and Business Media LLC

Date: 18-12-2020

DOI: 10.1038/S41467-020-20157-5

Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Mechanism of transcription modulation by the transcription-repair coupling factor

Publisher: Oxford University Press (OUP)

Date: 31-05-2022

DOI: 10.1093/NAR/GKAC449

Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

Structures of thermolabile mutants of human glutathione transferase P1-1.

Publisher: Elsevier BV

Date: 09-2000

DOI: 10.1006/JMBI.2000.4054

University Of Melbourne

Location: Australia

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University Of Tasmania

Location: Australia

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ARC Future Fellowships - Grant ID: FT0990287

Start Date: 01-2010

End Date: 01-2014

Amount: $686,400.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0877658

Start Date: 05-2008

End Date: 12-2013

Amount: $1,098,934.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180100805

Start Date: 2018

End Date: 02-2021

Amount: $534,573.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100660

Start Date: 2011

End Date: 12-2013

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100047

Start Date: 2016

End Date: 09-2016

Amount: $380,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210100365

Start Date: 02-2021

End Date: 02-2024

Amount: $692,195.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100027

Start Date: 04-2012

End Date: 12-2013

Amount: $320,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100085

Start Date: 2011

End Date: 04-2012

Amount: $450,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE190100165

Start Date: 2019

End Date: 12-2019

Amount: $407,609.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100078

Start Date: 02-2011

End Date: 12-2011

Amount: $500,000.00

Funder: Australian Research Council