Jane F. Griffin

Molecular structure and mechanisms of action of cyclic and linear ion transport antibiotics

Ionophores are antibiotics that induce ion transport across natural and artificial membranes. The specific function of a given ionophore depends upon its selectivity and the kinetics of ion capture, transport, and release. Systematic studies of complexed and uncomplexed forms of linear and cyclic ionophores provide insight into molecular mechanisms of ion capture and release and the basis for ion selectivity. The cyclic dodecadepsipeptide valinomycin, cyclo[(-L-Val-D-Hyi-D-Val-L-Lac)3-], transports potassium ions across cellular membrane bilayers selectively. The x-ray crystallographic and nmr spectroscopic data concerning the structures of Na+, K+, and Ba+2 complexes are consistent and provide a rationale for the K+ selectivity of valinomycin. Three significantly different conformations of valinomycin are observed in anhydrous crystals, in hydrated crystals grown from dimethylsulfoxide, and in crystals grown from dioxane. Each of these conformations suggests a different mechanism of ion capture. One of the observed conformations has an elliptical structure stabilized by four 4<--1 intramolecular hydrogen bonds and two 5<--1 hydrogen bonds. Ion capture could be readily achieved by disruption of the 5<--1 hydrogen bonds to permit coordination to a potassium ion entering the cavity. The conformation found in crystals obtained from dimethyl sulfoxide is an open flower shape having three petals and three 4<--1 hydrogen bonds. Complexation could proceed by a closing up of the three petals of the flower around the desolvating ion. In the third form, water molecules reside in the central cavity of a bracelet structure having six 4<--1 hydrogen bonds. Two of these bracelets stack over one another with their valine-rich faces surrounding a dioxane molecule. The stacked molecules form a channel approximately 20 A in length, suggesting that under certain circumstances valinomycin might function as a channel. A series of analogues of valinomycin differing in ring composition and size have been synthesized and their transport properties tested. Peptide substitution and chiral variation in the dodecadepsipeptide can result in stabilization or modification of the different conformers. While contraction of the ring size results in loss of ion transport properties, expansion of the ring size permits complexation of larger ions and small positively charged molecules. Gramicidin A is a pentadecapeptide that functions as a transmembrane channel for transporting monovalent cations. Crystal structures of the cesium chloride complex and two uncomplexed forms of gramicidin A have been reported. In all three structures the gramicidin A molecule is a left-handed, antiparallel, double-stranded helical dimer. In the cesium complex the beta 7.2-helix has 6.4 residues per turn with an internal cavity large enough to accommodate cesium ions. In the uncomplexed structures the channel is 31 A long and has 5.6 amino acids per turn. Because the helix is too tightly wound to permit ion transport, ion transport would require breaking and reforming of hydrogen bonds.

Molecular conformation and protein binding affinity of progestins.

Analysis of X-ray data concerning 277 estranes, androstanes, and pregnanes and comparison with progesterone receptor binding data have prompted the following observations. In general: 1. The flexibility of natural steroid hormones permits them to take up conformations optimal for binding to sites on proteins that vary in individual structural requirements. 2. When substituents strain the fused ring system, the strain will be delocalized and often transmitted to the most flexible point of the molecule, thus giving rise to conformational transmission effects. Consequently, substituents will generally stabilize a specific conformation, limiting protein interaction and enhancing a specific hormone response. 3. Hydrogen bond patterns in crystals can be used to predict points of active site attachment. 4. Distortions resulting from crystal packing forms are insignificant. Progestin receptor binding affinity: 5. Complementarity of fit is not specific on the alpha and beta faces of the B, C, and D rings. 6. The delta4-3-one composition is the only consistently required element. 7. Five of the eight highest-affinity binders have inverted A rings. Others may be easily converted to it. 8. The inverted A ring is proposed as the optimal conformation and primary factor controlling binding. 9. An A ring binding pattern is apparent in other steroidal hormones. 10. The D-ring region is open to contribute to conformational change in the receptor or genome interaction.

Steroid structure and function—II. Conformational transmission and receptor binding of medroxyprogesterone acetate

This is a discussion of the chemical structure of MPA (medroxyprogesterone acetate) and the ways in which the chemical structure of the substance influences its conformational transmission and receptor binding. The A-ring of MPA has been analyzed by single crystal X-ray analysis to be in the inverted lbeta. 2 x 1/2-chair conformation. This discussion is illustrated by tables of atomic coordinates and several diagrams of molecular conformations and bindings. The fact that MPA and progesterone have different conformations and electronic natures of their A-rings influences their susceptibility to metabolism. This fact may also account for the 30-fold higher affinity of MPA for the progestogen receptor in the rabbit uterus.

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

Crystallographic data on over 400 steroids collected in theAtlas of Steroid Structure provide information concerning preferred conformations, relative stabilities and substituent influence on 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° and −46°. The 4 torsion angles that lie outside this range do so because of a 16β-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° 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 8β,9α-half-chair conformation. However, in structures containing more than one molecule 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 distinct 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.

Molecular details of receptor binding and hormonal action of steroids derived from X-ray crystallographic investigations

Abstract Analysis of X-ray crystallographic data on steroids provides information concerning preferred conformations, relative stabilities, and substituent influence on the interactive potential of steroid hormones. Analysis of the data on the 4-ene-3-one ring indicates that it normally has a conformation midway between the 1α,2β-half chair and the 1α-sofa forms. Strain introduced into the molecule by substitution on the fused ring system shifts the A-ring conformation toward the more symmetric forms with an attendant change in the conjugation of the 4-ene-3-one system. Crystallographic data on 85 pregnane structures having a 20-one substituent provide information on 17β-side chain flexibility. In eighty-one structures the C(16)-C(17)-C(20)-O(20) torsion angle is between 0° and −46°. This is consistent with CD, i.r. and n.m.r. solution spectra and more precisely defines the side chain conformation in solution. The four structures whose side chains lie outside this range do so because of the presence of a 16β-substituent and not because of crystal packing forces. The steroid A ring appears to be primarily responsible for initiating and maintaining hormone binding to the estrogen and progestin receptors. When the structures of agonists and antagonists of specific steroid hormones are compared, they generally exhibit similarities in their A-ring region and dissimilarities in the D-ring region further suggesting that the steroid A-ring bears responsibility for receptor binding while the D-ring controls expression of activity. Antihormoncs that compete for the receptor site of a steroid hormone may be expected to have structural features appropriate for receptor binding (A-ring composition and conformation) and lack structural features that induce or stabilize subsequent receptor functions (D-ring conformatjonal features and functional groups).

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.

The fascinating complexities of steroid-binding enzymes.

Enzymes that modulate the level of circulating steroid hormone can be used to combat steroid-dependent disorders. Members of the NADPH-dependent short chain dehydrogenase/reductase (SDR) family control blood pressure, fertility, and natural and neoplastic growth. Despite the fact that only one amino acid residue is strictly conserved in the 60 known members of the family, all appear to have the dinucleotide-binding Rossmann fold and homologous catalytic residues containing the conserved tyrosine. Variation in the amino acid composition of the substrate binding pocket creates specificity of binding for steroids, prostaglandins, sugars and alcohols. Licorice induces high blood pressure by inhibiting an SDR in the kidney, and appears to combat ulcers by inhibiting another in the stomach. Detailed X-ray analyses of various members of the family should allow the design of potent, tissue-specific, highly selective inhibitors.

Structural features which distinguish estrogen agonists and antagonists

The molecular structures of compounds that compete for the estrogen binding site suggest that when estradiol is bound to the receptor, there is a close fit only at the A-ring end of the steroid. The most potent antagonists have phenolic rings capable of mimicking the estradiol A-ring in promoting high affinity binding to the receptor. They fail to stabilize the conformational change or molecular interaction needed to achieve hormonal response because they either lack an essential functional group (hydrogen bond donor) or they present a steric block or topological feature incompatible with transformation or interaction subsequent to the initiation of binding by the A-ring or A-ring analogues. X-Ray analyses of 12 triphenylethylene derivatives demonstrate the stability of a specific conformation that is unaffected by crystal packing forces. This conformation has a pinwheel-like orientation of phenyl rings, the direction of which is correlated with a 10 degrees twist about the central double bond and appears to depend upon the orientation of the non-phenyl substituent relative to the double bond. Empirical energy calculations are insensitive to this intramolecular structural dependence and incorrectly predict that the pinwheel of the opposite direction is of lower energy.

On the structure, biosynthesis, function and phylogeny of isoarborinol and motiol

The solid-state conformations of the C-3 acetates of two isomeric hopanoids—1, isoarborinol (D∶C-friedo-B1∶A1-3β,5α,8α,10β,13β,14α,17β,18α,21β) and 2, motion (D∶C-friedo-B1∶A1-neogammacer-7(8)-en-3β-ol[3β,5α,9α,10β,13α,14β,17α,18β,21α])—have been determined by X-ray crystallography. The data show that whereas both molecules are planar, 1 orients into a chair-halfchair-chair-chair-halfchair conformation while 2 orients into a chair-sofa-twist-halfchair-halfchair conformation. To explain the biogenesis of 1 and 2 from squalene oxide, a step-wise mechanism is proposed which proceeds through the protosteroid cation (for 1) and dammarenyl cation (for 2). After ring enlargement from the corresponding 13(17)bond followed by concerted 1,2-migrations and loss of the 11β-H and 7β-H as protons, respectively, a 9,11-double bond (in 1) and a 7,8-double bond (in 2) is introduced into the nucleus. The mechanism is discussed in relation to the classical view of a non-stop cyclization process where, for example, squalene oxide folds in a chair-chair-chair-chair-boat conformation to give a cyclized product (motiol) presumably with the same conformational disposition as the cyclizing material. The three-dimensional geometry of 1 and 2 was found to be structurally dissimilar from sterols. For instance, 1 and 2 are shorter and volumetrically smaller molecules than cholesterol, and this may explain their diminished importance as membrane inserts compared with sterols in eukaryote evolution.

Steroid structure and function—IX. molecular conformation of catechol estrogens

Abstract The catechol estrogens have varying degrees of affinity for the estrogen receptor, catecholamine enzymes and the dopamine receptor. The X-ray crystal structures of 2-hydroxyestradiol, 2-hydroxyestrone and 4-hydroxyestrone are reported here and compared with those of estradiol, estrone, dopamine and apomorphine. The overall molecular shapes and hydrogen bonding patterns have been examined for possible relevance to protein binding and activity. The principal structural observations and their potential significance are the following: (1) The 4-hydroxyestrone results indicate that the 4-substituent does not constitute a steric impediment to estrogen receptor binding but does restrict the probable location of the hydrogen bond acceptor atom on the receptor. (2) The reduced affinity of 2-hydroxyestradiol for the estrogen receptor may be due to a combination of steric interaction and interference with hydrogen bond formation of O(3). (3) The reduced affinity of 17-one substituted steroids in this series for the estrogen receptor most strongly supports the probable importance of a hydrogen bond donor at that position interacting directly with the receptor. (4) In the catechol estrogens the formation of an intramolecular and two or more intermolecular hydrogen bonds together with the extension of the A rings planar surface to include the hydroxyl substituents and part of the B ring must account for its high binding affinity for catechol amine related enzymes, the dopamine receptor and a putative catechol estrogen receptor. (5) Two orientations of the catechol estrogens relative to that of dopamine when bound to its receptor are suggested by this study. One achieves maximum overlap of the hydrogen bond pattern of the dihydroxy benzene ring and the other maximizes similarity in overall shape. The results of this investigation are consistent with the hypothesis that the A-ring plays a primary role in initiating estrogen binding to its receptor and suggests an even more highly specific A-ring interaction with the dopamine receptor and catechol amine enzymes.