Biomedical subjects

Anthony J Wilkinson

Publications and source records attributed to Anthony J Wilkinson.

At least 37 records · Page 2Linked to original sources

PubMed2005

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein.

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain-Barré syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K(d) of approximately 10(-7) M consistent with a role as a receptor for a high-affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC-type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni.

ATP-Binding Cassette Transporters↗

PubMed2004

Crystallization and preliminary crystallographic studies of the cofactor-binding domain of the LysR-type transcriptional regulator Cbl from Escherichia coli.

Cbl (CysB-like protein) is a member of the family of LysR-type transcriptional regulators (LTTRs) and controls genes engaged in sulfur assimilation in Escherichia coli. It has been postulated that adenosine 5-phosphosulfate (APS) is responsible for abolishing Cbl-activated transcription from the ssu promoter (Bykowski et al., 2002). To elucidate the structural basis of Cbl function and to confirm the role of APS as an anti-inducer, the cofactor-binding domain of Cbl (c-Cbl, MW = 26 kDa) was cloned, purified and crystallized in the presence of APS. The crystals belong to space group C222(1), but show substantial variation of the unit-cell parameters and diffraction anisotropy. Despite this, X-ray data extending to 3.0 A resolution have been collected and solution of the structure by molecular replacement is in progress.

Adenosine Phosphosulfate↗

PubMed2004

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability.

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.

Amino Acid Motifs↗

PubMed2004

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins.

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.

Binding Sites↗

PubMed2004

Crystallization of YloQ, a GTPase of unknown function essential for Bacillus subtilis viability.

YloQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 A spacing.

Adenosine Triphosphate↗

PubMed2003

Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilis.

AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholinoethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3 A spacing has been collected.

Bacillus subtilis↗

PubMed2003

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps.

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.

Amino Acid Sequence↗

PubMed2003

The crystal structure of gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53.

Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.

Amino Acid Motifs↗

PubMed2003

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket.

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.

Amino Acid Substitution↗

PubMed2003

The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.

The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.

Amino Acid Sequence↗

PubMed2003

Crystallization of gankyrin, an oncoprotein that interacts with CDK4 and the S6b (rpt3) ATPase of the 19S regulator of the 26S proteasome.

Gankyrin is an oncoprotein overexpressed in hepatocarcinoma cells that binds to the cell-cycle regulator CDK4 and the S6b ATPase subunit of the regulatory component of the proteasome. It belongs to the family of ankyrin-repeat proteins that appear to mediate protein-protein interactions in diverse biochemical processes. Gankyrin has been crystallized from polyethylene glycol solutions and diffraction data have been obtained from these crystals that extend to 2.1 A spacing.

Crystallization↗

PubMed2002

Crystallization of the GTP-dependent transcriptional regulator CodY from Bacillus subtilis.

CodY is a GTP sensor that represses transcription of early stationary phase and sporulation genes in Bacillus subtilis. As nutrients become limiting, GTP levels fall and CodY-mediated repression is relieved. Crystals of CodY have been grown in the presence and absence of GTP from sodium citrate buffered solutions containing lithium sulfate and diffraction data have been collected extending to 3.5 A spacing.

Bacillus subtilis↗

PubMed2002

Bacillus subtilis division protein DivIVA - screen for stable oligomer state conditions.

The cell division protein DivIVA is predicted to be a coiled-coil, tropomyosin-like protein, that self-associates both in vivo and in vitro into oligomers of up to 10-12 monomers. A simple and quick screen for conditions supporting the stable oligomer structure has been developed revealing that DivIVA forms a homogeneous oligomer in the presence of PEGs (PEG 4 K or PEG 8K and PEG 1K).

Bacillus subtilis↗

PubMed2002

Crystallization of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli.

FtsH is a membrane-anchored ATP-dependent protease that degrades misfolded or misassembled membrane proteins as well as a subset of cytoplasmic regulatory proteins. It belongs to the family of AAA(+) ATPases with roles in diverse cellular processes. The ATPase domain of FtsH from Escherichia coli has been crystallized from ammonium sulfate solutions and crystals diffracting to 1.5 A resolution have been obtained.

ATP-Dependent Proteases↗

PubMed2002

Dimer formation and transcription activation in the sporulation response regulator Spo0A.

The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A.

Bacillus subtilis↗

PubMed2002

The crystal structure of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli at 1.5 A resolution.

Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including sigma(32), LpxC, and lambda CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an alpha/beta nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the beta-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis.

ATP-Dependent Proteases↗

PubMed2002

Protein engineering 20 years on.

It is 20 years since site-directed mutagenesis was first used to modify the active site of an enzyme of known structure and mechanism. Since then, this method has contributed far-reaching insights into catalysis, specificity, stability and folding of proteins. Engineered proteins are now being used in industry and for the improved treatment of human disease.

Antibodies↗

PubMed2002

Oligomerization of the Bacillus subtilis division protein DivIVA.

DivIVA appears to be a mediator of inhibition by MinCD of division at the cell poles in Bacillus subtilis. Gel permeation and ultracentrifugation techniques were used to show self-association of DivIVA into a form consisting of 10-12 monomers in vitro. Western blot analysis of non-denaturating polyacrylamide gels confirms the presence of similar oligomers in B. subtilis cell lysates. These oligomers persist in a B. subtilis strain containing the divIVA1 mutation, in which proper vegetative septum positioning is abolished. In contrast, the divIVA2 mutation, which has a similar biological impact, appears to produce a protein with different oligomerization properties. The results of the present study suggest that oligomerization of DivIVA is important, but not sufficient for its function in the cell division process.

Bacillus subtilis↗