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Biomedical subjects

C M Weeks

Publications and source records attributed to C M Weeks.

At least 19 recordsLinked to original sources
PubMed1994

The refined three-dimensional structure of 3 alpha,20 beta-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases.

BACKGROUND: Bacterial 3 alpha,20 beta-hydroxysteroid dehydrogenase reversibly oxidizes the 3 alpha and 20 beta hydroxyl groups of steroids derived from androstanes and pregnanes. It was the first short-chain dehydrogenase to be studied by X-ray crystallography. The previous description of the structure of this enzyme, at 2.6 A resolution, did not permit unambiguous assignment of several important groups. We have further refined the structure of the complex of the enzyme with its cofactor, nicotinamide adenine dinucleotide (NAD), and solvent molecules, at the same resolution. RESULTS: The asymmetric unit of the crystal contains four monomers, each with 253 amino acid residues, 38 water molecules, and 176 cofactor atoms belonging to four NAD molecules--one for each subunit. The positioning of the cofactor molecule has been modified from our previous model and is deeper in the catalytic cavity as observed for other members of both the long-chain and short-chain dehydrogenase families. The nicotinamide-ribose end of the cofactor has several possible conformations or is dynamically disordered. CONCLUSIONS: The catalytic site contains residues Tyr152 and Lys156. These two amino acids are strictly conserved in the short-chain dehydrogenase superfamily. Modeling studies with a cortisone molecule in the catalytic site suggest that the Tyr152, Lys156 and Ser139 side chains promote electrophilic attack on the (C20-O) carbonyl oxygen atom, thus enabling the carbon atom to accept a hydride from the reduced cofactor.

Amino Acid Sequence↗
PubMed1994

Structure solution by minimal-function phase refinement and Fourier filtering. I. Theoretical basis.

Eliminating the N atomic position vectors rj, j = 1, 2, ..., N, from the system of equations defining the normalized structure factors EH yields a system of identities that the EH's must satisfy, provided that the set of EH's is sufficiently large. Clearly, for fixed N and specified space group, this system of identities depends only on the set [H], consisting of n reciprocal-lattice vectors H, and is independent of the crystal structure, which is assumed for simplicity to consist of N identical atoms per unit cell. However, for a fixed crystal structure, the magnitudes magnitude of /EH/ are uniquely determined so that a system of identities is obtained among the corresponding phases psi H alone, which depends on the presumed known magnitudes magnitude of /EH/ and which must of necessity be satisfied. The known conditional probability distributions of triplets and quartets, given the values of certain magnitudes magnitude of /E/, lead to a function R(psi) of phases, uniquely determined by magnitudes magnitude of /E/ and having the property that RT < 1/2 < RR, where RT is the value of R(psi) when the phases are equal to their true values, no matter what the choice of origin and enantiomorph, and RR is the value of R(psi) when the phases are chosen at random. The following conjecture is therefore plausible: the global minimum of R(psi), where the phases are constrained to satisfy all identities among them that are known to exist, is attained when the phases are equal to their true values and is thus equal to RT.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallography, X-Ray↗
PubMed1994

Structure solution by minimal-function phase refinement and Fourier filtering. II. Implementation and applications.

The minimal function, R(psi), has been used to provide the basis for a new computer-intensive direct-methods procedure that shows potential for providing fully automatic routine solutions for structures in the 200-400 atom range. This procedure, which has been called shake-and-bake, is an iterative process in which real-space filtering is alternated with phase refinement using a technique that reduces the value of R(psi). It has been successfully tested using experimental data for a dozen known structures ranging in size from 25 to 317 atoms and crystallizing in a variety of space groups. The details of this procedure, the parameters used and the results of these applications are described.

Computers↗
PubMed1993

On the application of the minimal principle to solve unknown structures.

The Shake-and-Bake method of structure determination is a new direct methods phasing algorithm based on a minimum-variance, phase invariant residual, which is referred to as the minimal principle. Previously, the algorithm had been applied only to known structures. This algorithm has now been applied to two previously unknown structures that contain 105 and 110 non-hydrogen atoms, respectively. This report focuses on (i) algorithmic and parametric optimizations of Shake-and-Bake and (ii) the determination of two previously unknown structures. Traditional tangent formula phasing techniques were unable to unravel these two new structures.

Algorithms↗
PubMed1991

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site.

Amino Acid Sequence↗
PubMed1988

The mechanism of action of steroid antagonists: insights from crystallographic studies.

Examination of the structures of compounds having high affinity for estrogen, progestin, mineralocorticoid and glucocorticoid receptors strongly suggests that receptor binding is primarily the result of a tight association between the receptor and the steroidal A-ring. High affinity binding to the estrogen receptor appears to be dependent upon the presence of a phenolic ring in the substrate. An inverted 1 beta, 2 alpha conformation of the 4-ene-3-one A-ring appears to be most conductive to high affinity binding to the progesterone receptor. Binding to the mineralocorticoid receptor appears to be correlated to a complementary fit between amino acids of the receptor site and a flat 4-en-3-one A-ring similar to that imposed upon aldosterone by the 11,18-epoxide formation. The glucocorticoid receptor appears to prefer a 4-en-3-one A-ring that is bowed toward the alpha-face as is the case in structures having a 9 alpha-fluoro substituent or additional unsaturation at C(1)-C(2). The binding of androgens to their receptor differs in appearing to have an essential dependence upon functional groups at the A- and D-ring end of the steroid. With the exception of the androgens, the data suggest that specific interactions between the steroid B-, C- and D-rings and the receptor play at best a minor role in receptor binding but are the most important factor in determining agonist versus antagonist behavior subsequent to binding. Antagonists that compete for a steroid receptor site may be expected to have the A-ring composition and conformation necessary for receptor binding but lack the 11 beta-OH and the D-ring conformational features and functional groups that induce or stabilize subsequent receptor functions. Antagonists might also be compounds with A-ring conformations appropriate for binding but other structural features that interfere with subsequent receptor functions essential to activity.

Androgen Antagonists↗
PubMed1988

Crystallization of prostatic binding protein.

Prostatic binding protein is a dimeric glycoprotein capable of binding a variety of steroids. This protein is a major component of rat prostate cytosol making it possible to purify milligram quantities. Hexagonal crystals of X-ray diffraction quality have been grown from phosphate buffered ammonium sulfate solution by vapor diffusion methods. These crystals which are reasonably stable to X-rays, show diffraction to 6.3 A and belong to space group P6(1) or P6(1)22 or the enantiomorphs. The unit cell has dimensions a = 88.7(5) A, c = 405(2) A, contains 24 molecules and has a specific volume of 2.8 A3/Dalton.

Ammonium Sulfate↗
PubMed1985

Molecular conformation, receptor binding, and hormone action of natural and synthetic estrogens and antiestrogens.

The X-ray crystallographic structural determinations of synthetic estrogens and antiestrogens provide reliable information on the global minimum energy conformation of these molecules or a local minimum energy conformation that is within 1 or 2 kcal/mole of the global minimum. In favorable cases, state-of-the-art molecular mechanics calculations provide quantitative agreement with X-ray results and information on the relative energy of other local minimum energy conformations not observed crystallographically. Because the conformation of diethylstilbestrol (DES) observed in solvated crystals has an overall conformation and dipole moment more similar to estradiol it is the form more likely to bind to the receptor and produce hormone activity. Either phenol ring of DES can successfully mimic the estradiol A-ring in binding to the receptor. Indenestrol A (INDA) and indenestrol B (INDB) have nearly identical fully extended planar conformations. Either the alpha or gamma rings of these compounds may mimic the A ring of estradiol and compete for the estrogen receptor. Although there are eight distinct ways in which molecules of a racemic mixture of INDA or INDB can bind to the receptor, not all of them may be able to elicit a hormonal response. This may account for the reduced biological activity of the compounds despite their successful competition for receptor binding. The minimum energy conformations of Z-pseudodiethylstilbestrol (ZPD) and E-pseudodiethylstilbestrol (EPD) are bent in a fashion similar to that of indanestrol (INDC). These molecules have good binding affinity suggesting that the receptor does not require a flat molecule. Therefore these conformations would appear to be compatible with receptor binding, but only the Z isomer has an energetically allowed extended conformation that accounts for its observed biological activity relative to DES.

Diethylstilbestrol↗
PubMed1982

The molecular structure of (20R)-20-phenyl-5-pregnene-3 beta, 20-diol, an inhibitor of cholesterol conversion to pregnenolone.

The crystal and molecular structure of (20R)-20-phenyl-5-pregnene-3 beta, 20-diol hemihydrate has been determined by X-ray analysis in order to establish the configuration and conformation at C(20). Interest in this compound was stimulated by its high affinity inhibitory binding to cytochrome P-450SCC, the enzyme which catalyzes the biosynthesis of pregnenolone (3 beta-hydroxy-5-pregnen-20-one) from cholesterol. The results of the analysis suggest a possible conformation for the cholesterol side chain in the enzyme complex.

Cholesterol↗
PubMed1980

Conformational analysis of sterols: comparison of X-ray crystallographic observations with data from other sources.

Cystallographic data on over 400 steroids collected in the Atlas of Steroid Structure provide information concerning preferred conformations, relative stabilities and substituent influence of the interactive potential of steroid hormones. Analysis of these data indicates that observed conformational details are intramolecularly controlled and that the influence of crystal packing forces is negligible. Crystallographic data on the orientation of the progesterone side chain contradict published force-field calculations. In 84 of 88 structures having a 20-one substituent, the C(16)-C(17)-C(20)-O(20) torsion angle is between 0 degrees and -46 degrees. The 4 torsion angles that lie outside this range do so because of a 16 beta-substituent and not because of crystal packing forces. Not one of the 88 structures is found to have a conformation in which the C(16)-C(17)-C(20)-O(20) torsion angle is within +/- 15 degrees of the most commonly calculated minimum energy value. The narrow range of side chain conformations seen in very different crystalline environments in the 88 crystal structure determinations and the predictable substituent influence apparent in the data strongly suggest that crystallographically observed conformers seldom deviate from minimum energy positions, regardless of hypothetical broad energy minima, metastable states and small barriers to rotation. The 96 crystallographically independent determinations of the cholestane 17-side chain show that the chain has 4 principal conformations (A:B:C:D), occurring in the ratio 69:8:8:11. Although the fully extended side chain is clearly the energetically most favored one, in 16 observations of cholesterol itself only 6 are in the extended conformation. Some of the correlated conformational changes in the chains can be rationalized on the basis of model studies, but others apparently result from subtle intramolecular forces. The unsaturated B ring provides another element of flexibility in the structure of cholesterol. The 5-ene B ring is normally observed in an 8 beta, 9 alpha-half-chair conformation. However, in structures containing more than one molecular in the crystallographic asymmetric unit, at least one of the 2 molecules is found to differ significantly from this form. It may be that this inherent flexibility is responsible for the presence of conformationally distnct molecules in the same crystal. The intermolecular interaction observed in the crystal structure of cholesterol and its fatty acid derivatives illustrate the type of interaction between the steroid ring system and hydrocarbon chains that can be expected in membrane bilayers.

Cholestanes↗
PubMed1977

A steroid structure which refutes the isolation of C(17)-C(20) rotational isomers.

Isolation of two C(17)-C(20) rotamers of 20-methyl-20-(2-hydroxyethoxy)-5-pregnene-3beta, 17alpha-diol has been reported. X-Ray analysis of a diacetate derivative of one of the "rotamers" shows that the actual structure is 3beta-acetoxy-17aalpha-(2-acetoxyethoxy)-17alpha,17abeta-dimethyl-D-homo-5-androsten-17beta-ol (C28H44O6). Thus, although this investigation refutes the existence of C(17)-C(20) rotamers, it suggests a possible new pathway for D-homo steroid synthesis.

Homosteroids↗