Structure-function relationships of the raloxifene-estrogen receptor-alpha complex for regulating transforming growth factor-alpha expression in breast cancer cells.

Amino acid Asp-351 in the ligand binding domain of estrogen receptor alpha (ERalpha) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERalpha complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor alpha target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERalpha. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERalpha allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ERalpha complex with the complete loss of estrogen-like activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERalpha expression vectors. Profound differences in the agonist and antagonist actions of Ralcenter dotERalpha complexes were noted only in stable transfectants. The agonist activity of the Ralcenter dotERalpha complex was enhanced with D351E and D351Y ERalpha, but raloxifene lost its agonist activity with D351F ERalpha. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERalpha. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERalpha complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERalpha complex.     

Targeted mutation of key residues at the start of helix 12 in the hERalpha ligand-binding domain identifies the role of hydrogen-bonding and hydrophobic interactions in the activity of the protein

Estradiol (E(2)) and tamoxifen exert their effects through two members of the nuclear receptor superfamily, estrogen receptor (ER)-alpha and -beta. We want to identify the key interactions linking ligand-binding and activity of the ERalpha. Asp-351 and Leu-536 participate in hydrogen bond (Asp-351) and hydrophobic (Leu-536) interactions at the start of helix 12 in the ligand-binding domain (LBD) of the ERalpha. Mutations at each position alter ER activity, but we do not know which is more important. We mutated these residues in combination and individually and assessed the activity of the mutated ERs in the absence and presence of E(2) and 4-OHT on an ERE-driven and an AP-1-driven promoter, as well as their ability to interact with coregulators. On an ERE-driven promoter, the residue at position 351 determined whether E(2) stimulated or reduced the activity of the ER, as well as the level of activity in the presence of 4-OHT. Surprisingly, mutation of both residues generally did not produce cumulative deleterious effects, and they exerted counterbalancing effects on the basal activity on both promoters. Our results identify the contributions of specific interactions to the activity of the hERalpha, and support the concept that this region couples ligand-binding with ER activity.     

Control of the estrogen-like actions of the tamoxifen-estrogen receptor complex by the surface amino acid at position 351

Tamoxifen is a valuable therapeutic agent with applications in the treatment and prevention of breast cancer. However, the development of drug resistance limits the usefulness of tamoxifen therapy. One form of drug resistance in breast cancer is tamoxifen-stimulated growth. We have addressed a mechanism how the tamoxifen–estrogen receptor (ER) complex can convert from being a blocking to stimulatory signal in breast cancer. We have described an effective assay system to study the action of antiestrogen–ER complex through the activation of transforming growth factor alpha gene in situ. The MDA-MB-231 breast cancer cells were stably transfected with cDNAs for wtER (D351), mutant Asp351Tyr ER (D351Y) and mutant Asp351Gly ER (D351G). The D351Y ER can enhance the estrogenic properties of 4OHT and change the pharmacology of raloxifene by converting it from antiestrogen to estrogen. We hypothesized that alterations in the charge of amino acid (aa) 351, and changes in the interaction with the side chain of an antiestrogen, are critical for the subsequent estrogenicity of the complex. Our goal was (1) to modulate the estrogenicity of the antiestrogen–ER complex by different aa substitutions at position 351 and (2) to examine the role of alterations in the side chain of antiestrogens on the estrogenicity of the complex. Substitution of tyrosine for aspartate at aa351 results in increased estrogenicity for a series of tamoxifen derivatives–ER complexes and the conversion of EM 652-ER and GW 7604-ER complexes from antiestrogenic to estrogen-like. Substitution of glycine for aspartate at aa 351 results in the conversion of 4OHT-ER complex from estrogen-like to antiestrogenic. We propose that the side chain of antiestrogens either neutralizes or displaces the charge at aspartate 351 thereby removing a charged site for the opportunistic binding of a novel coactivator. If no charge is present (D351G) then no coactivator can bind and the complex with any antiestrogen is not estrogen-like. However, if the charge is extended beyond the reach of an antiestrogen side chain (D351Y), then the coactivators bind and compounds are estrogen-like. The establishment of a relationship between the structure of the antiestrogen–ER complex and its function will enhance the development of novel compounds with unique biological activities and potentially avoid premature drug resistance.     

Site-directed mutagenesis in haemoglobin. Functional role of tyrosine-42(C7) alpha at the alpha 1-beta 2 interface

To clarify the functional role of Tyr-42(C7) alpha, which forms a hydrogen bond with Asp-99(G1) beta at the alpha 1-beta 2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42 alpha was replaced by Phe (Hb Phe-42 alpha) or His (Hb His-42 alpha), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99 beta mutants, Hb Phe-42 alpha showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42 alpha, the deoxy T quaternary structure is highly destabilized and the strain imposed on the Fe-N epsilon (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42 alpha, Hb His-42 alpha showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42 alpha and Asp-99 beta. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.     

Long-range side-chain-main-chain interactions play crucial roles in stabilizing the (betaalpha)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.     

The crystal structure of staphylococcal nuclease refined at 1.7 A resolution

The crystal structure of staphylococcal nuclease has been determined to 1.7 A resolution with a final R-factor of 16.2% using stereochemically restrained Hendrickson-Konnert least-squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease 1,2 bound with deoxythymidine-3',5'-diphosphate and Ca2+. Tyr-113 and Tyr-115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp-21 and Asp-40 shift as does Glu-43, the proposed general base in the hydrolysis of the 5'-phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys-70 side chain which hydrogen bonds to both Asp-21 and Glu-43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg-35, Lys-84, Tyr-85, and Arg-87, which hydrogen bond to the 3'- and 5'-phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure at 1.65 A, provides a wealth of structural information for the increasing number of studies using staphylococcal nuclease as a model system of protein structure and function.     

Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1. 9-A resolution. In the native structure, His-255 Nepsilon2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.     

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.     

A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride.

A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of Escherichia coli CheY refined at 1.7-A resolution

The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57.     

2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.