Transitional changes in the structure of C-reactive protein create highly pro-inflammatory molecules: Therapeutic implications for cardiovascular diseases.

C-reactive protein (CRP) is the prototypic acute-phase reactant that has long been recognized almost exclusively as a marker of inflammation and predictor of cardiovascular risk. However, accumulating evidence indicates that CRP is also a direct pathogenic pro-inflammatory mediator in atherosclerosis and cardiovascular diseases. The 'CRP system' consists of at least two protein conformations with distinct pathophysiological functions. The binding of the native, pentameric CRP (pCRP) to activated cell membranes leads to a conformational change resulting in two highly pro-inflammatory isoforms, pCRP* and monomeric CRP (mCRP). The deposition of these pro-inflammatory isoforms has been shown to aggravate the localized tissue injury in a broad range of pathological conditions including atherosclerosis and thrombosis, myocardial infarction, and stroke. Here, we review recent findings on how these structural changes contribute to the inflammatory response and discuss the transitional changes in the structure of CRP as a novel therapeutic target in cardiovascular diseases and overshooting inflammation.

Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins.

The recently determined structures of three different protein toxins by X-ray crystallography has unexpectedly revealed a common membrane-insertion domain. This domain consists of an alpha-helical bundle of between seven and ten helices, some of which are hydrophobic. The three toxins, colicin, insecticidal delta-endotoxin and diphtheria toxin are directed towards different hosts, have different killing mechanisms and bear no sequence homology. The observation of a common membrane-insertion domain has implications for the design of therapeutic agents in combating disease.

Insights into membrane insertion based on studies of colicins.

The recently determined three-dimensional structure of the pore-forming domain of colicin A has led to a hypothetical model for membrane insertion and channel formation. Certain features of this model have implications for understanding the mechanism of membrane insertion by other toxins and may have a broader relevance to protein transport in general.

Substrate and pseudosubstrate interactions with protein kinases: determinants of specificity.

Protein crystallography has revealed that protein kinases have extended protein-substrate-binding grooves associated with their active sites. Some protein kinases are autoinhibited by a mechanism in which part of their structure, termed a pseudosubstrate, occupies the active site. Substrates and pseudosubstrates occupy overlapping regions within the extended substrate-binding groove, making multiple specific electrostatic and non-polar contacts. With masterly economy, Nature has exploited the active site in many protein kinases to both recognize substrates with great specificity and autoregulate by remaining inactive until the appropriate activation signal is received.

An updated unified pharmacophore model of the benzodiazepine binding site on gamma-aminobutyric acid(a) receptors: correlation with comparative models.

A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Crystal structure of a colicin N fragment suggests a model for toxicity.

BACKGROUND: Pore-forming colicins are water-soluble bacteriocins capable of binding to and translocating through the Escherichia coli cell envelope. They then undergo a transition to a transmembrane ion channel in the cytoplasmic membrane leading to bacterial death. Colicin N is the smallest pore-forming colicin known to date (40 kDa instead of the more usual 60 kDa) and the crystal structure of its membrane receptor, the porin OmpF, is already known. Structural knowledge of colicin N is therefore important for a molecular understanding of colicin toxicity and is relevant to toxic mechanisms in general. RESULTS: The crystal structure of colicin N reveals a novel receptor-binding domain containing a six-stranded antiparallel beta sheet wrapped around the 63 A long N-terminal alpha helix of the pore-forming domain. The pore-forming domain adopts a ten alpha-helix bundle that has been observed previously in the pore-forming domains of colicin A, la and E1. The translocation domain, however, does not appear to adopt any regular structure. Models for receptor binding and translocation through the outer membrane are proposed based on the structure and biochemical data. CONCLUSIONS: The colicin N-ompF system is now the structurally best-defined translocation pathway. Knowledge of the colicin N structure, coupled with the structure of its receptor, OmpF, and previously published biochemical data, limits the numerous possibilities of translocation and leads to a model in which the translocation domain inserts itself through the porin pore, the receptor-binding domain stays outside and the pore-forming domain translocates along the outer wall of the trimeric porin channel.

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

BACKGROUND: 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 divided 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. RESULTS: 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. CONCLUSIONS: 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.

Crystal structure of manganese superoxide dismutase from Bacillus stearothermophilus at 2.4 A resolution.

The crystal structure of manganese superoxide dismutase (MnSOD) from Bacillus stearothermophilus has been solved at 2.4 A resolution by a combination of multiple isomorphous replacement and molecular replacement (1 A = 0.1 nm). The structure has been refined to a conventional R-factor for all 16,560 unique reflections at 2.4 A of 0.26, and the 2Fo-Fc density maps show features more consistent with the known amino acid sequence of MnSOD from B. stearothermophilus than with the starting model, the MnSOD from Thermus thermophilus. The molecule is a dimer of identical subunits, each with 203 amino acid residues. The polypeptide chain of the monomer is organized into two domains, one of which has an "all-alpha" structure and the other an "alpha/beta" structure, with the manganese ion bound between them. The ion is co-ordinated by three histidine residues, 26, 81 and 167, and one aspartic acid residue, 173, in a tetrahedral arrangement strongly distorted towards trigonal pyramidal. We anticipate that Tyr34, whose hydroxyl group is only 5 A from the metal, is involved in the catalytic reaction. The active site is particularly rich in aromatic amino acid residues. As in the Cu/ZnSOD there are indications that MnSOD provides electrostatic guidance to the substrate entering the active site.

Crystallization of glutathione S-transferase from human placenta.

Crystals of an acidic pi class glutathione S-transferase from human placenta have been obtained by the hanging drop method using ammonium sulphate as a precipitant. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 60.1 A, c = 244.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.7 A.

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.

Crystallization of a proform of aerolysin, a hole-forming toxin from Aeromonas hydrophila.

Crystals of proaerolysin, the precursor of the hole-forming toxin from Aeromonas hydrophila, have been obtained. The mature form of this protein binds to a receptor on mammalian cells, aggregates and forms 30 A holes in the membrane. The crystals are tetragonal, space group P4(3)2(1)2, a = b = 104.00 A, c = 222.0 A. They contain a dimer in the asymmetric unit and diffract to a resolution of 2.6 A.

Crystallization and preliminary X-ray diffraction studies of a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

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

Glutathione S -transferases (GSTs) play a pivotal role in the detoxification of foreign chemicals and toxic metabolites. They were originally termed ligandins because of their ability to bind large molecules (molecular masses >400 Da), possibly for storage and transport roles. The location of the ligandin site in mammalian GSTs is still uncertain despite numerous studies in recent years. Here we show by X-ray crystallography that the ligandin binding site in human pi class GST P1-1 occupies part of one of the substrate binding sites. This work has been extended to the determination of a number of enzyme complex crystal structures which show that very large ligands are readily accommodated into this substrate binding site and in all, but one case, causes no significant movement of protein side-chains. Some of these molecules make use of a hitherto undescribed binding site located in a surface pocket of the enzyme. This site is conserved in most, but not all, classes of GSTs suggesting it may play an important functional role.

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Refined structure of the pore-forming domain of colicin A at 2.4 A resolution.

The E1 subgroup (E1, A, B, IA, IB, K and N) of anti-bacterial toxins called colicins is known to form voltage-dependent channels in lipid bilayers. The crystal structure of the pore-forming domain of colicin A from Escherichia coli has been refined to the diffraction limit of the crystals at 2.4 A resolution by means of molecular dynamics and restrained least-squares methods to a conventional R-factor of 0.18 for all data between 6.0 and 2.4 A resolution. The polypeptide chain of 204 amino acid residues consists of ten alpha-helices organized in a three-layer structure. The helices range in length from 9 to 23 residues with an average length of 125 residues. The packing arrangement of the helices has been analysed; the packing is different from that observed in four-helix bundle proteins. The sites of 83 water molecules have been located and refined. Analysis of the structure provides insights into the mechanism of formation of a voltage-gated channel by the protein. Although it is proposed that substantial tertiary structural changes occur during membrane insertion, the secondary structural elements remain conserved. This idea has been proposed recently for a number of other protein-membrane events and thus may have more general applicability.

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase.

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

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

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.

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

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.