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A J Wilkinson

Publications and source records attributed to A J Wilkinson.

At least 19 recordsLinked to original sources
PubMed2000

A new mutation in spo0A with intragenic suppressors in the effector domain.

Spo0A is a two domain response regulator, a key protein in the initiation of sporulation of Bacillus subtilis. This protein controls a number of changes in gene expression that occur during the transition from stationary phase to the onset of sporulation. The phosphorylated form of Spo0A influences the transcription of a specific set of genes. In addition to others, it represses abrB and activates spoIIA and spoIIE transcription. Although the N-terminal phosphoacceptor domain is well characterised, there is limited information on the C-terminal, DNA-binding domain. Comparisons of Spo0A homologues from a number of Bacillus and Clostridium species show that the C-terminal domain contains three highly conserved regions. In this study, we have investigated the influence of spo0A mutations mapping within the C-terminal domain on transcription from the abrB, spoIIA and spoIIE promoters using lacZ fusions. Our results indicate that described mutations can be part of signalling between N- and C-terminal domains of the protein. Also, the increased expression observed from the spoIIE promoter in some Spo0A mutants might result from a stabilising function of these mutations on the transcriptional apparatus utilising sigma(A).

Amino Acid Sequence↗
PubMed2000

Domain swapping in the sporulation response regulator Spo0A.

Adaptive responses of micro-organisms, such as chemotaxis and sporulation, are governed by two-component systems consisting of sensor kinases, that interpret environmental signals, and response regulators which activate the appropriate physiological responses. Signal transduction via response regulator proteins is mediated through transient phosphorylation of aspartic acid residues. In Spo0A, the key regulator of development (sporulation) in Bacillus, phosphorylation of the N-terminal receiver domain (N-Spo0A) at aspartate-55 switches on the transcription activation functions residing in the C-terminal effector domain. Here we report the crystal structure of N-Spo0A from Bacillus stearothermophilus at 1.6 A spacing, revealing a dimer formed by an alpha-helix swap. Comparison of this structure with the recently described structure of phosphorylated N-Spo0A shows that dimer formation results from a cis-trans isomerization of the Lys106--Pro107 peptide bond. The quaternary reorganization is associated with alterations in the active site stereochemistry which may have implications for signalling. Remarkably, this 3-D domain swapped N-Spo0A dimer has an identical topology to a hypothetical CheY-like dimer, recently proposed as an intermediate in the evolution of the family of periplasmic substrate binding proteins.

Amino Acid Sequence↗
PubMed1999

Phosphorylated aspartate in the structure of a response regulator protein.

Phosphorylation of aspartic acid residues is the hallmark of two- component signal transduction systems that orchestrate the adaptive responses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmental signals and a response regulator that activates the appropriate physiological response. Although structures of response regulators are known, little is understood about their activated phosphorylated forms, due to the intrinsic instability of the acid phosphate linkage. Here, we report the phosphorylated structure of the receiver/phosphoacceptor domain of Spo0A, the master regulator of sporulation, from Bacillus stearothermophilus. The phosphoryl group is covalently bonded to the invariant aspartate 55, and co-ordinated to a nearby divalent metal cation, with both species fulfilling their electrostatic potential through interactions with solvent water molecules, the protein main chain, and with side-chains of amino acid residues strongly conserved across the response regulator family. This is the first direct visualisation of a phosphoryl group covalently linked to an aspartic acid residue in any protein, with implications for signalling within the response regulator family.

Adaptation, Biological↗
PubMed1999

Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.

The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity.

Amino Acid Sequence↗
PubMed1999

Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases. Site-directed mutagenesis of the ATP-dependent protease FtsH.

Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.

ATP-Dependent Proteases↗
PubMed1999

Crystallographic and calorimetric analysis of peptide binding to OppA protein.

Isothermal titration calorimetry has been used to study the binding of 20 different peptides to the peptide binding protein OppA, and the crystal structures of the ligand complexes have been refined. This periplasmic binding protein, part of the oligopeptide permease system of Gram negative bacteria, has evolved to bind and enclose small peptides of widely varying sequences. The peptides used in this study have the sequence Lys-X-Lys, where X is any of the 20 commonly occurring amino acids. The various side-chains found at position 2 on the ligand fit into a hydrated pocket. The majority of side-chains are restrained to particular conformations within the pocket. Water molecules act as flexible adapters, matching the hydrogen-bonding requirements of the protein and ligand and shielding charges on the buried ligand. This use of water by OppA to broaden the repertoire of its binding site is not unique, but contrasts sharply with other proteins which use water to help bind ligands highly selectively. Predicting the thermodynamics of binding from the structure of the complexes is highly complicated by the influence of water on the system.

Amino Acid Sequence↗
PubMed1999

Solution of the structure of the cofactor-binding fragment of CysB: a struggle against non-isomorphism.

The elucidation of the structure of CysB(88-324) by multiple isomorphous replacement (MIR) techniques was seriously delayed by problems encountered at every stage of the analysis. There was extensive non-isomorphism both between different native crystals and between native and heavy-atom-soaked crystals. The heavy-atom substitution was invariably weak and different soaking experiments frequently led to substitution at common sites. These correlated heavy-atom binding sites resulted in an overestimation of the phase information. Missing low-resolution reflections in the native data set, constituting only 2% of the total observations, reduced the power of density modification and phase refinement. Finally, the extensive dimer interface made it difficult to isolate a single molecule in the course of model building into the MIR maps. The power of maximum likelihood refinement (REFMAC) was exploited in solving the structure by means of iterative cycles of refinement of a partial model, initially comprising only 30% of the protein atoms in the final coordinate set. This technique, which uses experimental phases, can automatically discriminate the correct and incorrect parts of electron-density maps and give properly weighted combined phases which are better than the experimental or calculated ones. This allowed the model to be gradually extended by manual building into improved electron-density maps. A model generated in this way, containing just 50% of the protein atoms, proved good enough to find the transformations needed for multi-crystal averaging between different crystal forms. The averaging regime im-proved the phasing dramatically such that the complete model could be built. The problems, final solutions and some possible causes for the observed lack of isomorphism are discussed.

Bacterial Proteins↗
PubMed1999

Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A.

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2. 5 A spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 A spacing using synchrotron radiation.

Amino Acid Sequence↗
PubMed1998

An evolutionary link between sporulation and prophage induction in the structure of a repressor:anti-repressor complex.

Spore formation is an extreme response of some bacteria to adversity. In Bacillus subtilis the proteins of the sin, sporulation inhibition, region form a component of an elaborate molecular circuitry that regulates the commitment to sporulation. SinR is a tetrameric repressor protein that binds to the promoters of genes essential for entry into sporulation and prevents their transcription. This repression is overcome through the activity of SinI, which disrupts the SinR tetramer through the formation of a SinI-SinR heterodimer. The interactions governing this curious quaternary transition are revealed in the crystal structure of the SinI-SinR complex. The most striking, and unexpected, finding is that the tertiary structure of the DNA-binding domain of SinR is identical with that of the corresponding domains of the repressor proteins, CI and Cro, of bacteriophage 434 that regulate lysis/lysogeny. This structural similarity greatly exceeds that between SinR and any bacterial protein or between the 434 repressor proteins and their homologues in the closely related bacteriophage lambda. The close evolutionary relationship implied by the structures of SinR and the 434 repressors provokes both comparison of their functions and a speculative consideration of the intriguing possibility of an evolutionary link between the two adaptive responses, sporulation and prophage induction.

Amino Acid Sequence↗
PubMed1998

Stabilizing bound O2 in myoglobin by valine68 (E11) to asparagine substitution.

The isopropyl side chain of valine68 in myoglobin has been replaced by the acetamide side chain of asparagine in an attempt to engineer higher oxygen affinity. The asparagine replacement introduces a second hydrogen bond donor group into the distal heme pocket which could further stabilize bound oxygen. The Val68 to Asn substitution leads to approximately 3-fold increases in oxygen affinity and 4-6-fold decreases in CO affinity. As a result, the M-value (KCO/KO2) is lowered 15-20-fold to a value close to unity. An even larger enhancement of O2 affinity is seen when asparagine68 is inserted into H64L sperm whale myoglobin which lacks a distal histidine. The overall rate constants for oxygen and carbon monoxide binding to the single V68N myoglobin mutants are uniformly lower than those for the wild-type protein. In contrast, the overall rate constant for NO association is unchanged. Analyses of time courses monitoring the geminate recombination of ligands following nanosecond and picosecond flash photolysis of MbNO and MbO2 indicate that the barrier to ligand binding from within the heme pocket has been raised with little effect on the barrier to diffusion of the ligand into the pocket from the solvent. The crystal structures of the aquomet, deoxy, oxy, and carbon monoxy forms of the V68N mutant have been determined to resolutions ranging from 1.75 to 2.2 A at 150 K. The overall structures are very similar to those of the wild-type protein with the principal alterations taking place within and around the distal heme pocket. In all four structures the asparagine68 side chain lies almost parallel to the plane of the heme with its amide group directed toward the back of the distal heme pocket. The coordinated water molecule in the aquomet form and the bound oxygen in the oxy form can form hydrogen-bonding interactions with both the Asn68 amide group and the imidazole side chain of His64. Surprisingly, in the carbon monoxy form of the V68N mutant, the histidine64 side chain has swung completely out the distal pocket, its place being taken by two ordered water molecules. Overall, these functional and structural results show that the asparagine68 side chain (i) forms a strong hydrogen bond with bound oxygen through its -NH2 group but (ii) sterically hinders the approach of ligands to the iron from within the distal heme pocket.

Amino Acid Substitution↗
PubMed1998

Bacillus subtilis regulatory protein GerE.

GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus. The gene encoding GerE has been cloned from B. subtilis and overexpressed in Escherichia coli. Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 A resolution with a synchrotron radiation X-ray source.

Bacillus subtilis↗
PubMed1998

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

Algorithms↗
PubMed1998

The stereochemical mechanism of the cooperative effects in hemoglobin revisited.

In 1970, Perutz tried to put the allosteric mechanism of hemoglobin, proposed by Monod, Wyman and Changeux in 1965, on a stereochemical basis. He interpreted their two-state model in terms of an equilibrium between two alternative structures, a tense one (T) with low oxygen affinity, constrained by salt-bridges between the C-termini of the four subunits, and a relaxed one (R) lacking these bridges. The equilibrium was thought to be governed primarily by the positions of the iron atoms relative to the porphyrin: out-of-plane in five-coordinated, high-spin deoxyhemoglobin, and in-plane in six-coordinated, low-spin oxyhemoglobin. The tension exercised by the salt-bridges in the T-structure was to be transmitted to the heme-linked histidines and to restrain the movement of the iron atoms into the porphyrin plane that is necessary for oxygen binding. At the beta-hemes, the distal valine and histidine block the oxygen-combining site in the T-structure; its tension was thought to strengthen that blockage. Finally, Perutz attributed the linearity of proton release with early oxygen uptake to the sequential rupture of salt-bridges in the T-structure and to the accompanying drop in pKa of the weak bases that form part of them. Almost every feature of this mechanism has been disputed, but evidence that has come to light more than 25 years later now shows it to have been substantially correct. That new evidence is reviewed below.

Allosteric Regulation↗
PubMed1997

Tension in haemoglobin revealed by Fe-His(F8) bond rupture in the fully liganded T-state.

In 1972, Perutz proposed that the low affinity of T-state haemoglobin is caused by tension in the bond between the iron and the proximal histidine, restraining the Fe from moving into the porphyrin plane on binding oxygen. This proposal has often been disputed. If such tension does exist, it will be manifest in the liganded T-state. Here we describe the structure of the fully liganded T-state cyanide complex of haemoglobin, in which the Fe-proximal histidine bond in the alpha-subunits, but not in the beta-subunits, is ruptured. This rupture uncouples the structural changes at the alpha-haem from those in the globin and the beta-haem, and demonstrates unequivocally the existence of tension and its transmission through this bond.

Binding Sites↗
PubMed1997

The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement.

BACKGROUND: CysB is a tetrameric protein of identical subunits (M(r) = 36,000) which controls the expression of genes associated with the biosynthesis of cysteine in bacteria. CysB is both an activator and a repressor of transcription whose activity is responsive to the inducer N-acetylserine; thiosulphate and sulphide act as anti-inducers. CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over approximately 280 residues including a putative helix-turn-helix DNA-binding motif at their N terminus. The aims of the present study were to explore further the complex molecular biology and curious ligand binding properties of CysB and to provide structural insights into the LysR family of proteins. RESULTS: The crystal structure of a dimeric chymotryptic fragment of Klebsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refined against data extending to 1.8 A resolution. The protein comprises two alpha/beta domains (I and II) connected by two short segments of polypeptide. The two domains enclose a cavity lined by polar sidechains, including those of two residues whose mutation is associated with constitutive expression of the cysteine regulon. A sulphate anion and a number of well ordered water molecules have been modelled into discrete electron-density peaks within this cavity. In the dimer, strands beta B from domain I and strands beta G from domain II come together so that a pair of antiparallel symmetry-related 11-stranded twisted beta-pleated sheets is formed. CONCLUSIONS: The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins. A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins. In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.

Amino Acid Sequence↗
PubMed1997

Peptide binding in OppA, the crystal structures of the periplasmic oligopeptide binding protein in the unliganded form and in complex with lysyllysine.

The periplasmic oligopeptide binding protein, OppA, acts as the initial receptor for the uptake of peptides by the oligopeptide permease (Opp) in Gram-negative bacteria. Opp will handle peptides between two and five amino acid residues regardless of their sequence. The crystal structures of a series of OppA-peptide complexes have revealed an enclosed but versatile peptide binding pocket and have illustrated how tri- and tetrapeptide ligands are accommodated. Here, the crystal structures of (i) OppA complexed with a dipeptide (lysyllysine) and (ii) unliganded OppA have been solved using X-ray data extending to 1.8 and 2.4 A spacing, respectively. In the dipeptide complex, the alpha-amino group of the ligand is anchored through an ion pair interaction with Asp419, as observed in complexes with longer peptides. However, its alpha-carboxylate group forms water-mediated interactions with the guanidinium groups of Arg404 and Arg413 rather than the direct salt bridges to Arg413 and His371 observed in the tripeptide and tetrapeptide complexes, respectively. Isothermal titration calorimetric measurements of the binding of lysine-containing peptides of different lengths to OppA show that the dipeptide, KK, is bound with approximately 60-fold lower affinity than related tri- and tetrapeptides (KKK and KKKA, respectively). These data are discussed with reference to the calculated enthalpic and entropic contributions to ligand binding and the structures of the OppA peptide complexes. In the unliganded molecule, domain III has rotated as a rigid body through 26 degrees away from domains I and II, exposing the ligand binding site. The water structure in the binding cleft shows similarities to that in the various OppA-peptide complexes.

Bacterial Proteins↗