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Dominic J Campopiano

Publications and source records attributed to Dominic J Campopiano.

15 recordsLinked to original sources
PubMed2007

Histidine ligands in bacterial metallothionein enhance cluster stability.

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.

Amino Acid Sequence↗
PubMed2006

Discovery of glutathione S-transferase inhibitors using dynamic combinatorial chemistry.

Protein-directed dynamic combinatorial chemistry (DCC) relies on reversible chemical reactions that can function under the near-physiological conditions required by the biological target. Few classes of reaction have so far proven effective at generating dynamic combinatorial libraries (DCLs) under such constraints. In this study, we establish the conjugate addition of thiols to enones as a reaction well-suited for the synthesis of dynamic combinatorial libraries (DCLs) directed by the active site of the enzyme glutathione S-transferase (GST). The reaction is fast, freely reversible at basic pH, and easily interfaced with the protein, which is a target for the design of inhibitors in cancer therapy and the treatment of parasitic diseases such as schistosomiasis. We have synthesized DCLs based on glutathione (GSH, 1) and the enone ethacrynic acid, 2a. By varying either set of components, we can choose to probe either the GSH binding region ("G site") or the adjacent hydrophobic acceptor binding region ("H site") of the GST active site. In both cases the strongest binding DCL components are identified due to molecular amplification by GST which, in the latter system, leads to the identification of two new inhibitors for the GST enzyme.

Binding Sites↗
PubMed2006

Suicide inhibition of alpha-oxamine synthases: structures of the covalent adducts of 8-amino-7-oxononanoate synthase with trifluoroalanine.

The irreversible inhibition of 8-amino-7-oxononanoate synthase by trifluoroalanine involves decarboxylative defluorination of the inhibitor-PLP aldimine followed by attack of the conjugated imine by the amino group of the active site lysine to afford a covalently bound difluorinated intermediate which can subsequently undergo further HF losses and hydrolysis to afford a 2-(pyridoximine phosphate) acetoyl protein adduct.

Acyltransferases↗
PubMed2005

Mechanism of alpha-oxoamine synthases: identification of the intermediate Claisen product in the 8-amino-7-oxononanoate synthase reaction.

The reactive beta-ketoacid pyridoxal-5'-phosphate aldimine formed in the condensation step of the 8-amino-7-oxononanoate synthase reaction was 'trapped' in the enzyme-bound form by carrying out the reaction with l-alanine methyl ester and pimeloyl-CoA affording the more stable methyl ester of the putative intermediate, the characterisation of which provides the first definitive evidence for a beta-ketoacid intermediate in an alpha-oxamine synthase mechanism.

Acyltransferases↗
PubMed2004

Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full-length single-stranded DNA-binding protein from the hyperthermophilic bacterium Aquifex aeolicus.

Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)(68)]. All crystals diffract to around 2.9 A resolution and those without bound DNA (native) belong to space group P2(1), with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b = 73.40, c = 109.76 A, beta = 95.11 degrees . Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 A and could belong to three closely related space groups (I222, I2(1)2(1)2(1) or I4(1)) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way.

Amino Acid Sequence↗
PubMed2004

Structure-activity relationships in defensin dimers: a novel link between beta-defensin tertiary structure and antimicrobial activity.

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a beta-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mm NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have beta-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

Alleles↗
PubMed2003

Oxo-iron clusters in a bacterial iron-trafficking protein: new roles for a conserved motif.

We report a set of three 1.8-1.9 A resolution X-ray crystal structures of Neisseria gonorrhoeae Fbp (ferric-ion binding protein): (i) open-cleft apo-Fbp containing bound phosphate, (ii) open-cleft mono-Fe Fbp capped by nitrilotriacetate, and (iii) open-cleft trinuclear oxo-iron Fbp, the first structure of an iron-cluster adduct of a transferrin. The nine independent molecules in the unit cells provide 'snapshots' of the versatile dynamic structural roles of the conserved dityrosyl iron-binding motif (Tyr195-Tyr196) which control the capture and, possibly, processing of iron. These findings have implications for understanding bacterial iron acquisition and dissimilation, and organic/mineral interfaces.

Amino Acid Motifs↗
PubMed2003

A novel protein-mineral interface.

Transferrins transport Fe3+ and other metal ions in mononuclear-binding sites. We present the first evidence that a member of the transferrin superfamily is able to recognize multi-nuclear oxo-metal clusters, small mineral fragments that are the most abundant forms of many metals in the environment. We show that the ferric ion-binding protein from Neisseria gonorrhoeae (nFbp) readily binds clusters of Fe3+, Ti4+, Zr4+ or Hf4+ in solution. The 1.7 A resolution crystal structure of Hf-nFbp reveals three distinct types of clusters in an open, positively charged cleft between two hinged protein domains. A di-tyrosyl cluster nucleation motif (Tyr195-Tyr196) is situated at the bottom of this cleft and binds either a trinuclear oxo-Hf cluster, which is capped by phosphate, or a pentanuclear cluster, which in turn can be capped with phosphate. This first high-resolution structure of a protein-mineral interface suggests a novel metal-uptake mechanism and provides a model for protein-mediated mineralization/dissimilation, which plays a critical role in geochemical processes.

Animals↗
PubMed2003

Biotinylation in the hyperthermophile Aquifex aeolicus.

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein-protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP Delta 67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP Delta 67 at temperatures of up to 70 degrees C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP Delta 67 complex. Furthermore, this complex was also formed between BPL and a BCCP Delta 67 mutant lacking the lysine residue (BCCP Delta 67 K117L) however, complex formation was considerably reduced using holo-BCCP Delta 67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP Delta 67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition.

Acetyl-CoA Carboxylase↗
PubMed2002

Synergistic anion and metal binding to the ferric ion-binding protein from Neisseria gonorrhoeae.

The 34-kDa periplasmic iron-transport protein (FBP) from Neisseria gonorrhoeae (nFBP) contains Fe(III) and (hydrogen)phosphate (synergistic anion). It has a characteristic ligand-to-metal charge-transfer absorption band at 481 nm. Phosphate can be displaced by (bi)carbonate to give Fe.CO(3).nFBP (lambda(max) 459 nm). The local structures of native Fe-PO(4)-nFBP and Fe.CO(3).nFBP were determined by EXAFS at the FeK edge using full multiple scattering analysis. The EXAFS analysis reveals that both phosphate and carbonate ligands bind to FBP in monodentate mode in contrast to transferrins, which bind carbonate in bidentate mode. The EXAFS analysis also suggests an alternative to the crystallographically determined position of the Glu ligand, and this in turn suggests that an H-bonding network may help to stabilize monodentate binding of the synergistic anion. The anions oxalate, pyrophosphate, and nitrilotriacetate also appear to serve as synergistic anions but not sulfate or perchlorate. The oxidation of Fe(II) in the presence of nFBP led to a weak Fe(III).nFBP complex (lambda(max) 471 nm). Iron and phosphate can be removed from FBP at low pH (pH 4.5) in the presence of a large excess of citrate. Apo-FBP is less soluble and less stable than Fe.nFBP and binds relatively weakly to Ga(III) and Bi(III) but not to Co(III) ions, all of which bind strongly to apo-human serum transferrin.

Anions↗
PubMed2002

Purification and characterisation of the BIOH protein from the biotin biosynthetic pathway.

Conversion of pimeloyl-coenzyme A (CoA) to biotin in Escherichia coli requires at least four enzymes encoded by genes in the bio operon. One gene, bioH, which is not present in the bioABFCD operon, is required for the synthesis of pimeloyl-CoA but its exact role in formation of this intermediate is unknown. To investigate this further, we have overexpressed and purified the bioH gene products from both E. coli (BIOH EC) and Neisseria meningitis (BIOH NM) in E. coli. When purified BIOH was incubated with excess CoA and analysed by electrospray mass spectrometry a species of mass corresponding to a BIOH:CoA complex was observed. Mutation of a conserved serine residue to alanine (BIOH EC S82A) did not prevent CoA binding. This is the first report of the purification of BIOH and the observation of a small molecule bound to the protein provides clues to its role in pimeloyl-CoA synthesis.

Amino Acid Sequence↗