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R Pickersgill

Publications and source records attributed to R Pickersgill.

At least 19 recordsLinked to original sources
PubMed2001

The architecture of parallel beta-helices and related folds.

Three-dimensional structures have been determined of a large number of proteins characterized by a repetitive fold where each of the repeats (coils) supplies a strand to one or more parallel beta-sheets. Some of these proteins form superfamilies of proteins, which have probably arisen by divergent evolution from a common ancestor. The classical example is the family including four families of pectinases without obviously related primary sequences, the phage P22 tailspike endorhamnosidase, chrondroitinase B and possibly pertactin from Bordetella pertusis. These show extensive stacking of similar residues to give aliphatic, aromatic and polar stacks such as the asparagine ladder. This suggests that coils can be added or removed by duplication or deletion of the DNA corresponding to one or more coils and explains how homologous proteins can have different numbers of coils. This process can also account for the evolution of other families of proteins such as the beta-rolls, the leucine-rich repeat proteins, the hexapeptide repeat family, two separate families of beta-helical antifreeze proteins and the spiral folds. These families need not be related to each other but will share features such as relative untwisted beta-sheets, stacking of similar residues and turns between beta-strands of approximately 90 degrees often stabilized by hydrogen bonding along the direction of the parallel beta-helix.Repetitive folds present special problems in the comparison of structures but offer attractive targets for structure prediction. The stacking of similar residues on a flat parallel beta-sheet may account for the formation of amyloid with beta-strands at right-angles to the fibril axis from many unrelated peptides.

Amino Acid Sequence↗
PubMed2001

Anisotropic refinement of the structure of Thermoascus aurantiacus xylanase I.

The isotropic crystallographic model of the structure of xylanase I from Thermoascus aurantiacus (TAXI) has now been refined anisotropically at 1.14 A resolution to a standard residual of R = 11.1% for all data. TAXI is amongst the five largest proteins deposited in the Protein Data Bank to have been refined with anisotropic displacement parameters (ADPs) at this level of resolution. The anisotropy analysis revealed a more isotropic distribution of anisotropy than usually observed previously. Adding ADPs resulted in high-quality electron-density maps which revealed discrepancies from the previously suggested primary sequences for this enzyme. Side-chain conformational disorder was modelled for 16 residues, including Trp275, a bulky residue at the active site. An unrestrained refinement was consistent with the protonation of the catalytic acid/base glutamate and the deprotonation of the nucleophile glutamate, as required for catalysis. The thermal stability of TAXI is reinterpreted in the light of the new refined model.

Amino Acid Sequence↗
PubMed2000

Crystallization and preliminary X-ray analysis of the auxin receptor ABP1.

Auxin-binding protein (ABP1) is an important receptor for the plant hormone auxin that is involved with many growth and developmental responses in plants. The maize ABP1 gene has been expressed in insect cells, purified and crystallized. Type II crystals are monoclinic, with two glycosylated homodimers in the asymmetric unit, and diffract to 1.9 A using synchrotron radiation.

Crystallization↗
PubMed1999

Crystallization and preliminary crystallographic analysis of the endo-polygalacturonase from Erwinia carotovora ssp. carotovora.

Crystals of endo-polygalacturonase from Erwinia carotovora ssp. carotovora have been grown from polyethylene glycol 6000 by the hanging-drop method. Polygalacturonase is important in the virulence of this plant pathogen. The protein crystallizes in space group C2 with unit-cell parameters a = 81.3, b = 53.0, c = 103.1 A, beta = 112.6 degrees and with a single molecule in the asymmetric unit. The crystals diffract to 1.9 A.

Bacillus subtilis↗
PubMed1998

Crystal structure of polygalacturonase from Erwinia carotovora ssp. carotovora.

The crystal structure of the 40-kDa endo-polygalacturonase from Erwinia carotovora ssp. carotovora was solved by multiple isomorphous replacement and refined at 1.9 A to a conventional crystallographic R-factor of 0.198 and Rfree of 0.239. This is the first structure of a polygalacturonase and comprises a 10 turn right-handed parallel beta-helix domain with two loop regions forming a "tunnel like" substrate-binding cleft. Sequence conservation indicates that the active site of polygalacturonase is between these two loop regions, and comparison of the structure of polygalacturonase with that of rhamnogalacturonase A from Aspergillus aculeatus enables two conserved aspartates, presumed to be catalytic residues, to be identified. An adjacent histidine, in accord with biochemical results, is also seen. A similarity in overall electrostatic properties of the substrate-binding clefts of polygalacturonase and pectate lyase, which bind and cleave the same substrate, polygalacturonic acid, is also revealed.

Amino Acid Sequence↗
PubMed1998

Structure and evolution of parallel beta-helix proteins.

Three bacterial pectate lyases, a pectin lyase from Aspergillus niger, the structures of rhamnogalacturonase A from Aspergillus aculeatus, RGase A, and the P22-phage tailspike protein, TSP, display the right-handed parallel beta-helix architecture first seen in pectate lyase. The lyases have 7 complete coils while RGase A and TSP have 11 and 12, respectively. Each coil contains three beta-strands and three turn regions named PB1, T1, PB2, T2, PB3, and T3 in their order of occurrence. The lyases have homologous sequences but RGase A and TSP do not show obvious sequence homology either to the lyases or to each other. However, the structural similarities between all these molecules are so extensive that divergence from a common ancestor is much more probable than convergence to the same fold. The region PB2-T2-PB3 is the best conserved region in the lyases and shows the clearest structural similarity. Not only is the pleating and the direction of the hydrogen bonding in the sheets conserved, but so is the unusual alphaL-conformation turn between the two sheets. However, the overall shape, the position of long loops, a conserved alpha-helix that covers the amino-terminal end of the parallel beta-helix and stacks of residues in alphaR-conformation at the start of PB1 all suggest a common ancestor. The functional similarity, that the enzymes all bind alpha-galactose containing polymers at an equivalent site involving PB1 and its two flanking turn regions, further supports divergent evolution. We suggest that the stacking of the coils and the unusual near perpendicular junction of PB2 and PB3 make the parallel beta-helix fold especially likely to maintain similar main chain conformations during divergent evolution even after all vestige of similarity in primary structure has vanished.

Amino Acid Sequence↗
PubMed1997

Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases.

BACKGROUND: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. RESULTS: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization. CONCLUSIONS: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.

Aspergillus niger↗
PubMed1997

Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan.

In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-beta1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-beta-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of pectin lyase A from Aspergillus niger.

The major secreted pectin lyase (E.C. 4.2.2.10) from Aspergillus niger, strain 4M-147, has been purified and crystallized by the hanging-drop method using polyethylene glycol as precipitant. The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.2, b = 83.2, c = 93.1 A (1 A = 0.1 nm) and a single molecule in the asymmetric unit. The crystals diffract to at least 2.0 A resolution and are suitable for structure determination.

Journal Article↗
PubMed1995

Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven.

Comparison of the recently determined crystal structures Pseudomonas fluorescens subsp. cellulosa family F xylanase, (1-3)-beta-glucanase and (1-3,1-4)-beta-glucanase and the catalytic domain of E. coli beta-galactosidase reveals that they belong to a superfamily of 8-fold beta/alpha-barrels with similar amino acid residues at their active sites. In the three families that these enzymes represent, the nucleophile is a glutamate, which is located close to the carboxy-terminus of beta-strand seven. In addition all three enzymes have the sequence asparagine-glutamate close to the carboxy-terminus of beta-strand four. This glutamate has been identified as the acid/base in the family F xylanases and is essential for catalysis in beta-galactosidase. We suggest that the equivalent residue in the barley glucanases is the acid/base. Analysis of the sequences of family 1 beta-glucosidases and family 5 cellulases shows that these enzymes also belong to this superfamily which we call the 4/7 superfamily.

Amino Acid Sequence↗
PubMed1994

Making a small enzyme smaller; removing the conserved loop structure of hen lysozyme.

Engineering a smaller lysozyme is a challenge for both random and site-directed mutagenesis. This paper illustrates the power of knowledge-based protein engineering in the design of a smaller lysozyme that folds correctly and has activity against bacterial cell walls. In this smaller lysozyme the conserved disulphide bridged loop is replaced by a short loop. The long loop was selected because it buries a predominantly hydrophilic surface. The short loop was discovered by searching for appropriate fragments in the protein databank. This approach is important in the design of small enzymes useful to the food industry.

Animals↗
PubMed1994

Parallel beta-domains: a new fold in protein structures.

A new type of structural domain, composed of parallel beta-strands folded into a coiled structure, has been observed in several protein structures within the past year. An analysis of the basic motif indicates that there are two distinct types, with variations likely to be discovered in the future.

Amino Acid Sequence↗
PubMed1994

The structure of Bacillus subtilis pectate lyase in complex with calcium.

We have solved the structure of the Bacillus subtilis pectate lyase (BsPel) in complex with calcium. The structure consists of a parallel beta-helix domain and a loop region. The alpha L-bounded beta-strand seen in BsPel is a new element of protein structure and its frequent occurrence suggests it is an important characteristic of the parallel beta-helix. A pronounced cleft is formed between the loops and the parallel beta-helix domain and we propose that this is the active site cleft. Calcium, essential for the activity of the enzyme, binds at the bottom of this cleft and an arginine residue close to the calcium, which is conserved across all pectin and pectate lyases, may be involved in catalysis.

Amino Acid Sequence↗
PubMed1993

Crystal structure determination and refinement at 2.3-A resolution of the lentil lectin.

We report on the X-ray structure determination of the orthorhombic crystal form of lentil lectin by molecular replacement using the pea lectin coordinates as a starting model. The structure was refined at 2.3-A resolution with a combination of molecular dynamics refinement and classical restrained least-squares refinement. The final R value for all data Fo > 1 sigma (Fo) between 7.0- and 2.3-A resolution is 0.164%, and deviations from ideal bond distances are 0.014 A. The C-terminus of the beta-chain proved to be 23 amino acids longer than found in previous studies. This together with several inconsistencies between the previously determined amino acid sequence and the observed electron density forced a redetermination of the amino acid sequence of the protein. The overall structure is very similar to that of pea lectin and isolectin I of Lathyrus ochrus, the most prominent deviations being confined to loop regions and the regions of intermolecular contact. The largest difference between the pea and lentil lectin monomers is situated in the loop region of amino acids 73-79 of the beta chain. There are no significant differences between the two crystallographic independent lentil lectin monomers in the asymmetric unit. The model includes 104 well-defined water molecules, of which a significant number have a counterpart in the pea lectin structure. As for the other legume lectins, each lentil lectin monomer contains one calcium ion in a highly conserved environment. On the contrary, the manganese binding sites are distorted with respect to the pea lectin and concanavalin A structures. The Asp beta 121 side chain apparently does not ligate the Mn2+ ion. This difference is consistent in both lentil lectin monomers and agrees with earlier solution studies. Possible implications for oligosaccharide binding are discussed.

Amino Acid Sequence↗
PubMed1992

Crystallization and preliminary X-ray studies of the pectate lyase from Bacillus subtilis.

The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized. Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution. They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit. A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees. These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit. Both crystal forms are suitable for the determination of high-resolution structures.

Bacillus subtilis↗
PubMed1992

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.

Crystallography↗