Biomedical subjects

R J Read

Publications and source records attributed to R J Read.

5 recordsLinked to original sources

PubMed1992

Fitting electron density by systematic search.

A systematic search approach to the automatic refinement of protein structures could reduce the need for manual intervention. In this approach, possible conformations for a segment of the polypeptide chain are generated systematically and the trial segments are scored for their agreement with the observed diffraction data. The sampling of conformational space is sufficiently exhaustive that reasonable conformations should be included. A number of score functions have been tested, including local electron-density correlations and global structure-factor agreements. The score functions vary in their predictive power as well as in their bias toward the conformation found in the current refined model, but the best score functions have reasonable predictive power. Related functions can be used to indicate which regions of the model fit poorly, reducing the need for manual inspection of models in electron density.

Algorithms↗

PubMed1992

Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli.

The Shiga toxin family, a group of cytotoxins associated with diarrhoeal diseases and the haemolytic uraemic syndrome, includes Shiga toxin from Shigella dysenteriae type 1 and verotoxins produced by enteropathogenic Escherichia coli. The family belongs to the A-B class of bacterial toxins, which includes the cholera toxin family, pertussis and diphtheria toxins. These toxins all have bipartite structures consisting of an enzymatic A subunit associated with a B oligomer which binds to specific cell-surface receptors, but their amino-acid sequences and pathogenic mechanisms differ. We have determined the crystal structure of the B oligomer of verotoxin-1 from E. coli. The structure unexpectedly resembles that of the B oligomer of the cholera toxin-like heat-labile enterotoxin from E. coli, despite the absence of detectable sequence similarity between these two proteins. This result implies a distant evolutionary relationship between the Shiga toxin and cholera toxin families. We suggest that the cell surface receptor-binding site lies in a cleft between adjacent subunits of the B pentamer, providing a potential target for drugs and vaccines to prevent toxin binding and effect.

Bacterial Toxins↗

PubMed1992

A multiple-start Monte Carlo docking method.

We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure. Our method is based on a Monte Carlo (MC) algorithm applied to the translational and rotational degrees of freedom of the probe fragment. Starting from a randomly generated initial configuration, favorable binding modes are generated using a two-step process. An MC run is first performed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the target surface. This has the effect of making buried probes move toward the target surface and also allows enhanced sampling of deep pockets. In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing). We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favorable docking modes in a specified region around the target. We test this method using two inhibitor-receptor systems: Streptomyces griseus proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E. coli in complex with methotrexate. The method could consistently reproduce the complex found in the crystal structure searching from random initial positions in cubes ranging from 25 A to 50 A about the binding site. In the case of SGPB, we were also successful in docking to the native structure. In addition, we were successful in docking small probes in a search that included the entire protein surface.

Algorithms↗

PubMed1991

Crystallization and preliminary X-ray crystallographic analysis of verotoxin-1 B-subunit.

The B-subunit of verotoxin-1, which is believed to form a pentamer (monomer Mr = 7691), has been crystallized by vapor diffusion over a wide range of conditions. The best crystals, obtained with polyethylene glycol 8000 as the precipitant, belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 59.2 A, b = 102.7 A, c = 56.3 A. The cell dimensions are consistent with one B-subunit pentamer per asymmetric unit, and the crystals diffract to at least 2.0 A resolution. Data collected using synchrotron radiation at a wavelength of 2.070 A may allow the structure to be solved using the anomalous signal from three sulfur atoms in the monomer, combined with averaging over the non-crystallographic symmetry.

Bacterial Toxins↗

PubMed1991

Refined structure of porcine pepsinogen at 1.8 A resolution.

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.

Amino Acid Sequence↗