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R N Perham

Publications and source records attributed to R N Perham.

At least 19 recordsLinked to original sources
PubMed1999

Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes.

The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands. BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn. The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant.

Acetyl-CoA Carboxylase↗
PubMed1999

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1999

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

Animals↗
PubMed1999

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

Capsid↗
PubMed1999

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes.

The pyruvate dehydrogenase multienzyme complex (Mr of 5-10 million) is assembled around a structural core formed of multiple copies of dihydrolipoyl acetyltransferase (E2p), which exhibits the shape of either a cube or a dodecahedron, depending on the source. The crystal structures of the 60-meric dihydrolipoyl acyltransferase cores of Bacillus stearothermophilus and Enterococcus faecalis pyruvate dehydrogenase complexes were determined and revealed a remarkably hollow dodecahedron with an outer diameter of approximately 237 A, 12 large openings of approximately 52 A diameter across the fivefold axes, and an inner cavity with a diameter of approximately 118 A. Comparison of cubic and dodecahedral E2p assemblies shows that combining the principles of quasi-equivalence formulated by Caspar and Klug [Caspar, D. L. & Klug, A. (1962) Cold Spring Harbor Symp. Quant. Biol. 27, 1-4] with strict Euclidean geometric considerations results in predictions of the major features of the E2p dodecahedron matching the observed features almost exactly.

Acetyltransferases↗
PubMed1999

Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

Binding Sites↗
PubMed1998

Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered.

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.

Capsid↗
PubMed1998

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Genes, Bacterial↗
PubMed1998

Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase.

Many small peptides can be displayed on every copy of the major coat protein in recombinant filamentous bacteriophages but larger peptides can only be accommodated in hybrid virions mixed with wild-type protein subunits. A peptide insert of 12 residues capable of display at high copy number in a hybrid virion was found to be incapable of supporting recombinant virion assembly, a defect that could not be overcome by over-expressing leader peptidase in the same Escherichia coli cell. In contrast, over-expressing leader peptidase did increase the copy number of two 9-residue peptides that were poorly incorporated into hybrid virions. The factors that limit peptide display are varied and not restricted to the early stages of viral assembly.

Amino Acid Sequence↗
PubMed1998

Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli.

Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain. A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.

Acetyl-CoA Carboxylase↗
PubMed1998

Structure of the capsid of Pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 A resolution.

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of alpha-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely alpha-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

Amino Acid Sequence↗
PubMed1998

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.

Binding Sites↗
PubMed1998

Three-dimensional structure of the major autoantigen in primary biliary cirrhosis.

BACKGROUND & AIMS: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in patients' serum. The major autoantigen, recognized by antibodies from > 95% of patients with PBC, has been identified as the E2 component (E2p) of the pyruvate dehydrogenase multienzyme complex. Immunodominant sites on E2p have been localized to the inner of the two lipoyl domains, where the essential cofactor lipoic acid is attached covalently. The aim of this study was to determine the three-dimensional structure of the inner lipoyl domain of human E2p. METHODS: The domain was expressed in Escherichia coli; after purification, its structure was analyzed using nuclear magnetic resonance spectroscopy. RESULTS: The structure of the lipoyl domain from human E2p was determined, and the implications of the structure for autoimmune recognition were assessed. CONCLUSIONS: Knowledge of the structure further defines the major epitope and may help in the design of antigen-specific immunotherapy for treatment of PBC.

Amino Acid Sequence↗
PubMed1997

The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Expression, purification and reversible denaturation.

A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The Km values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 microM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.

Acetyl Coenzyme A↗
PubMed1997

NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein.

An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1. This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd. NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography. This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived. This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

Capsid↗
PubMed1997

Simultaneous display of different peptides on the surface of filamentous bacteriophage.

We have developed a new system for producing hybrid virions of filamentous bacteriophage fd simultaneously displaying two different peptides by infecting cells harbouring a plasmid containing a modified gene VIII with an engineered bacteriophage carrying a second and different copy of a modified gene VIII. The simultaneous display of different peptides has many potential applications in exploring the immune response and studying protein-protein interaction.

Amino Acid Sequence↗
PubMed1997

Accessibility of peptides displayed on filamentous bacteriophage virions: susceptibility to proteinases.

The genome of the filamentous bacteriophage fd has been engineered so that small peptides can be inserted into the exposed N-terminal segment of pVIII, the major protein of the virus capsid. Most small peptides can be displayed on all 2700 copies of pVIII (a recombinant virion), but larger peptides can be displayed only in virions in which modified and wild-type proteins are intermingled (hybrid virions). Peptides displayed in this way are highly immunogenic and capable of interacting with receptors and other ligands. The physical accessibility of the displayed peptides was tested by examining their susceptibility to digestion with proteinases. Potential cleavage sites in peptides displayed on recombinant or hybrid virions were in general found to be accessible to trypsin and chymotrypsin; and the density of incorporation of peptides in the virion had no effect on the susceptibility to cleavage. However, peptide bonds towards the C-terminal end of an insert, located approximately 47 residues or fewer from the C-terminus of the coat protein, were protected from digestion, presumably because of their proximity to the bulk viral surface. These results have important implications for the design and optimization of peptide display systems using filamentous bacteriophages.

Amino Acid Sequence↗