Vivian Cody

Membrane receptors mediating thyroid hormone action

A recently identified thyroid hormone cell surface receptor on the extracellular domain of integrin alphaVbeta3 leads in human cell lines to activation of the mitogen-activated protein kinase (MAPK) signal transduction cascade. Examples of MAPK-dependent thyroid hormone actions are plasma membrane ion pump stimulation and specific nuclear events. These events include serine phosphorylation of the nuclear thyroid hormone receptor, leading to coactivator protein recruitment and complex tissue responses, such as thyroid hormone-induced angiogenesis or tumor cell growth. The existence of this cell surface receptor means that the activity of administered hormone could be limited through structural modification of the molecule to reproduce or inhibit only those hormone actions initiated at the cell surface. Examples of such modifications are provided.

Molecular modeling of the thyroid hormone interactions with αvβ3 integrin

Abstract A cell surface receptor for thyroid hormone has recently been identified on the extracellular domain of integrin αvβ3. In a variety of human and animal cell lines this hormone receptor mediates activation by thyroid hormone of the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. An arginine-glycine-aspartate (RGD) recognition site on the heterodimeric integrin is essential to the binding of a variety of extracellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding by the integrin and consequent MAPK activation, suggesting that the hormone interaction site is located at or near the RGD recognition site on integrin αvβ3. A deaminated thyroid hormone ( l -thyroxine, T4) analogue, tetraiodothyroacetic acid (tetrac, T4ac), inhibits binding of T4 and 3,5,3′-triiodo- l -thyronine (T3) to αvβ3, but does not activate MAPK. Structural data show that the RGD cyclic peptide binds at the interface of the propeller of the αv and the B domains on the integrin head [Xiong JP, Stehle T, Zhang R, Joachimiack A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin αvβ3 in complexing with an Arg-Gly-Asp ligand. Science 2002;296:151–5]. To model potential interactions of thyroid hormone analogues with integrin, we mapped T4 and T4ac to the binding site of the RGD peptide. Modeling studies indicate that there is sufficient space in the cavity for the thyroid hormone to bind. Since the hormone is smaller in overall length than the RGD peptide, the hormone does not interact with the Arg recognition site in the propeller domain from αv. In this model, most of the hormone interactions are with βA domain of the integrin. Mutagenic studies can be carried out to validate the role of these residues in directing hormone interactions.

Understanding the role of Leu22 variants in methotrexate resistance: comparison of wild-type and Leu22Arg variant mouse and human dihydrofolate reductase ternary crystal complexes with methotrexate and NADPH.

Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-resistant Leu22Arg (L22R) variant of mouse dihydrofolate reductase (mDHFR) crystallized as a ternary complex with methotrexate (MTX) and the cofactor NADPH. These results are compared with the MTX and NADPH ternary complexes of L22R human DHFR (hDHFR) and those of mouse and human wild-type DHFR enzymes. The conformation of mDHFR Arg22 is such that it makes hydrogen-bonding contacts with Asp21, Trp24 and a structural water molecule, observations which were not made in the L22R hDHFR ternary complex. These data show that there is little difference between the structures of the wild type and L22R variant for either mouse or human DHFR; however, there are significant differences between the species. Comparison of these structures reveals that the active site of mDHFR is larger than that in the hDHFR structure. In mDHFR, the position of MTX is shifted 0.6 A toward helix C (residues 59-65), which in turn is shifted 1.2 A away from the active site relative to that observed in the hDHFR ternary complexes. In the L22R variant mDHFR structure, MTX makes shorter contacts to the conserved residues Ile7, Val115 and Tyr121 than in the L22R variant human DHFR structure. These contacts are comparable in both wild-type enzymes. The unexpected results from this comparison of the mouse and human DHFR complexes bound with the same ligand and cofactor illustrate the importance of detailed study of several species of enzyme, even when there is a high sequence homology between them. These data suggest that the differences in binding interactions of the L22R variant are in agreement with the weaker binding affinity for MTX in the variant enzymes; the larger size of the binding site in mDHFR supports the observation that the binding affinity of MTX for L22R mDHFR is significantly weaker than that of the L22R hDHFR enzyme.

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.

Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 Å resolution: first non‐biased insight into thyroxine binding reveals different hormone orientation in two binding sites

The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P4(3)2(1)2 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 A resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 A. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains.

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 flavonoids: crystal and molecular structures of morin hydrate and myricetin (1:2) triphenylphosphine oxide complex

Abstract The crystal and molecular structures of morin (2′,3,4′,5,7-pentahydroxyflavone) hydrate ( I ), and myricetin (3′,4′,5′,3,5,7-hexahydroxyflavone) triphenylphosphine oxide (TPPO) (1:2) co-crystal complex ( II ) have been studied by X-ray analysis and AM1 molecular orbital methods. The molecular conformation of the two flavones described by the torsion angle θ[C(3)-C(2)-C(1t)-C(2′)] between the benzopyrone and phenyl ring is −43.3° and 51.0° for molecules A and B of morin, respectively, and −37.0° for myricetin. Minimum energy conformations from AM1 molecular orbital calculations have θ values of −38.2° for morin and −27.0° for myricetin. The energy profile for rotation about θ for morin has a 28 kcal mol −1 barrier at 0° due to steric interactions between the 2′-hydroxy and the 3-hydroxy group. There are two local minima near 30 and 140°, in good agreement with structural results. The profile for myricetin has two equivalent minima near 30 and 150° with a barrier of less than 2 kcal mol −1 . In the crystal both flavones form extensive networks of intra- and intermolecular hydrogen bonds. In ( I ), each morin conformer packs in alternating layers linked by water molecules, while in ( II ), TPPO stabilizes the crystal by formation of short hydrogen bonds (2.58–2.65 A) of the phosphoryl oxygen to the flavone. Myricetin also forms a two dimensional sheet-like packing in which myricetin molecules hydrogen bond to each other, as well as to TPPO. These conformational and hydrogen bonding patterns provide insight into specific types of ligand-receptor interactions and support structure activity data which suggest the importance of electronic and hydrogen bonding properties in the bioactivity of flavones.

Conformational Analysis of Methylphenidate and Its Structural Relationship to Other Dopamine Reuptake Blockers Such as CFT

AbstractPurpose. This work was performed 1) to determine the conformational preferences of the threo and erythro isomers of the dopamine reuptake blocker methylphenidate, 2) to determine the crystal conformation of the threo isomer, 3) to confirm the absolute configuration of the more active threo enantiomer, and 4) to incorporate the compound into a previously determined pharmacophore for dopamine reuptake blockers. Methods. A conformational analysis was performed with the MM2-87 program, a crystal of the (– )-threo HC1 salt was analyzed by x-ray crystallography, and the global minima of the (+ }-threo isomer and the potent dopamine reuptake blocker CFT were superimposed. Results. In the global minimum of the threo isomer, the carbonyl oxygen of the ester group is oriented toward the ammonium group as was also found in the crystal state. In the erythro isomer, the ester group prefers an extended conformation relative to the piperidine group. The absolute configuration of the biologically active ( + )-threo enantiomer was confirmed to be R,R. The atomic sequence from the amine group through the ester group is identical in the active enantiomers of methylphenidate and CFT. Conclusions. The dopamine reuptake protein requires a precise orientation of the ammonium and ester groups but allows considerable leeway in the position of the phenyl ring. The pKa of the threo isomer is predicted to be higher than that of the erythro isomer.

Structural characteristics of antifolate dihydrofolate reductase enzyme interactions

The ubiquitous enzyme dihydrofolate reductase (DHFR) is responsible for the reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate in an NADPH-dependent manner. It is also a key pharmacological target for the treatment of cancer, as well as bacterial and opportunistic pathogenic infections. Interest in the design of potent and selective antifolate inhibitors has made DHFR one of the most studied enzymes, in particular its structural and biochemical properties. This review surveys more than 129 DHFR solution and crystal structures currently (02/07) reported in the Protein Data Bank representing 15 species of enzyme. Comparison of these DHFR sequences shows that while there is a high sequence homology among vertebrate species (75–95%), there is only about 30% homology between vertebrate and bacterial species. Despite the highly conserved nature of the ligand and cofactor binding sites, DHFR can bind a wide range of compounds that can have a high degree of flexibility. The enzyme itself can also undergo ligand-induced conformational changes that reflect its catalytic mechanism of action. Mechanistic questions can now be addressed with the structural data available for atomic resolution enzyme complexes as well as from neutron diffraction data that have recently become available. These data provide new insight into the design of novel inhibitors that can target specific species with high selectivity of binding.

New insights into DHFR interactions: analysis of Pneumocystis carinii and mouse DHFR complexes with NADPH and two highly potent 5-(omega-carboxy(alkyloxy) trimethoprim derivatives reveals conformational correlations with activity and novel parallel ring stacking interactions.

Structural data are reported for two highly potent antifolates, 2,4‐diamino‐5‐[3′,4′‐dimethoxy‐5′‐(5‐carboxy‐1‐pentynyl)]benzylpyrimidine (PY1011), with 5000‐fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4‐diamino‐5‐[2‐methoxy‐5‐(4‐carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80‐fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 Å resolution; with PY1011 and pcDHFR, refined to 2.0 Å resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 Å resolution. These results reveal that the carboxylate of the ω‐carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1‐pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C‐terminal β sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174–His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability. Proteins 2006.