Engineering of microheterogeneity-resistant p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

AbstractBy site-directed mutagenesis, Cys-116 was converted to Ser-116 in p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens. In contrast to wild-type enzyme, the C116S mutant is no longer susceptible to oxidation by hydrogen peroxide and shows no reactivity towards 5,5'-dithiobis(2-nitrobenzoate). Crystals of the C116S mutant are isomorphous with the crystal form of wild-type enzyme. A difference electron density confirms the mutation made

Structure of Bovine Pancreatic Phospholipase A2 at 1.7Å Resolution

The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å. This model was adjusted to the multiple isomorphous replacement map with Diamond’s real space refinement program and subsequently refined using Agarwal’s least-squares method. The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH3+ group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.

Crystallographic Refinement by Incorporation of Molecular Dynamics: Thermostable Serine Protease Thermitase Complexed with Eglin c

In order to investigate the principles of protein thermostability, the crystal structure of thermitase from Thermoactinomyces vulgaris, a thermostable member of the subtilisin family of serine proteases, has been determined in a complex with eglin c. Eglin c is a serine protease inhibitor from the leech Hirudo medicinalis. After data collection with a television area-detector diffractometer and initial structure solution by molecular-replacement methods, crystallographic refinement proceeded with incorporation of molecular-dynamics techniques. It appeared that this refinement procedure has a large convergence radius with movements of more than 5 Å for many atoms. Two procedures for the crystallographic molecular-dynamics refinement have been tested. They differed mainly in time span and weight on the X-ray 'energy'. The best results were obtained with a procedure which allowed the molecular-dynamics technique to search a large area in conformational space by having less weight on the X-ray restraints and allowing more time. The use of molecular-dynamics refinement considerably simplified the laborious and difficult task of fitting the model in its electron density during the refinement process. The final crystallographic R factor is 17.9% at 2.2 Å resolution.

Structure of Porcine Pancreatic Phospholipase A2 at 2.6 Å Resolution and Comparison with Bovine Phospholipase A2

The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 Å resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 Å resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val → porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules

Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography

RECOGNITION of the oligosaccharide portion of ganglioside G(M1) in membranes of target cells by the heat-labile enterotoxin from Escherichia coli is the crucial first step in its pathogenesis, as it is for the closely related cholera toxin 1-3. These toxins have five B subunits, which are essential for G(M1) binding, and a single A subunit, which needs to be nicked by proteolysis and reduced, yielding an A1-'enzyme' and an A2-'linker' peptide. A1 is translocated across the membrane of intestinal epithelial cells, possibly after endocytosis 4,5, upon which it ADP-ribosylates the G protein G(s-alpha) (reviewed in refs 2, 3, 6). The mechanism of binding and translocation of these toxins has been extensively investigated 1,2,7-20, but how the protein is orientated on binding is still not clear 10-12,18. Knowing the precise arrangement of the ganglioside binding sites of the toxins will be useful for designing drugs against the diarrhoeal diseases caused by organisms secreting these toxins and in the development of oral vaccines against them 21,22. We present here the three-dimensional structure of the E. coli heat-labile enterotoxin complexed with lactose. This reveals the location of the binding site of the terminal galactose of G(M1), which is consistent with toxin binding to the target cell with its A1 fragment pointing away from the membrane. A small helix is identified at the carboxy terminus of A2 which emerges through the central pore of the B subunits and probably comes into contact with the membrane upon binding, whereas the A1 subunit is flexible with respect to the B pentamer

The Structure of Bovine Pancreatic Prophospholipase A2 at 3.0 Å Resolution

Bovine pancreatic prophospholipase A2, the precursor of the lipolytic enzyme phospholipase A2, crystallizes in space group P3121 with cell dimensions a = b = 46.95, c = 102.0 Å. The structure was determined at 3.0 Å resolution using rotation and translation functions with transaminated phospholipase A2 as the model structure. The rotation-function calculations appeared to be sensitive to the resolution range selected. The results of the translation function were very well defined. Positioning of the model molecule according to the rotation- and translation-function results yielded an R factor of 0.44 for 2697 reflections between 7.1 and 3.0 Å. This could be decreased to 0.40 by a rigid-body R-factor search. Subsequent restrained refinement gave R = 0.27. The position of the calcium ion, which was excluded from the structure factor calculations, shows up as one of the highest features in difference Fourier syntheses. From the difference maps it also appears that the ten N-terminal residues of prophospholipase A2 are disordered. Disorder is also observed in the loop consisting of residues 62 to 73. This is quite in contrast to the situation in phospholipase A2 where this loop is well defined. The observed disorder may account for the large difference in activity of phospholipase and prophospholipase with respect to aggregated substrates like micelles.

X-Ray studies reveal lanthanide binding sites at the A/B5 interface of E. coli heat labile enterotoxin

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.

X-Ray studies reveal lanthanide binding sites at the A/B5 interface of E. coli heat labile enterotoxin

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.