Conformational differences in liganded and unliganded states of Galectin-3

The conformation of the carbohydrate recognition domain of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems, has been investigated in the absence of ligand and in the presence of N-acetylactosamine. A new methodology based on the measurement of residual dipolar couplings from NMR spectra has been used to characterize differences in protein structure along the backbone in the presence and absence of ligand, as well as the binding geometry of the ligand itself. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. The implications for ligand specificity and protein functionality are discussed.     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.     

Modulation of transcriptional sensitivity of mineralocorticoid and estrogen receptors

Recent reports describe the ability of factors to modulate the position of the dose–response curve of receptor–agonist complexes, and the amount of partial agonist activity of receptor–antagonist complexes, of androgen, glucocorticoid (GRs), and progesterone receptors (PRs). We now ask whether this modulation extends to the two remaining steroid receptors: mineralocorticoid (MRs) and estrogen receptors (ERs). These studies of MR were facilitated by our discovery that the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) is a new antimineralocorticoid with significant amounts of partial agonist activity. Elevated levels of MR, the co-activators TIF2 and SRC-1, and the co-repressor SMRT do modulate the dose–response curve and partial agonist activity of MR complexes. Interestingly, the precise responses are indistinguishable from those seen with GRs in the same cells. Thus, the unequal transactivation of common genes by MRs versus GRs probably cannot be explained by differential responses to changing cellular concentrations of homologous receptor, co-activators, or co-repressors. We also find that the dose–response curve of ER–estradiol complexes is left-shifted to lower steroid concentrations by higher amounts of exogenous ER. Therefore, the modulation of either the dose–response curve of agonists or the partial agonist activity of antisteroid, and in many cases the modulation of both properties, is a common phenomenon for all of the classical steroid receptors.     

Crystal structures of the liganded and unliganded nickel-binding protein NikA from Escherichia coli

Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be approximately 10 microm and that for cobalt was approximately 20 times higher.     

Functional roles of plasma membrane localized estrogen receptors

A series of emerging data supports the existence and importance of plasma membrane localized estrogen receptors in a variety of cells that are targets for the steroid hormone action. When estradiol (E2) binds to the cell surface protein, the ensuing signal transduction event triggers downstream signaling cascades that contribute to important biological functions. Aside from the classical signaling through nuclear estrogen receptors, we have provided evidence for the functional roles of an estrogen receptor localized in the plasma membrane. This review highlights some of the recent advances made in the understanding of the genomic/non-genomic actions of plasma membrane localized estrogen receptors.     

Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase.

The refined crystal structures of chicken, yeast and trypanosomal triosephosphate isomerase (TIM) have been compared. TIM is known to exist in an "open" (unliganded) and "closed" (liganded) conformation. For chicken TIM only the refined open structure is available, whereas for yeast TIM and trypanosomal TIM refined structures of both the open and the closed structure have been used for this study. Comparison of these structures shows that the open structures of chicken TIM, yeast TIM and trypanosomal TIM are essentially identical. Also it is shown that the closed structures of yeast TIM and trypanosomal TIM are essentially identical. The conformational difference between the open and closed structures concerns a major shift (7 A) in loop-6. Minor shifts are observed in the two adjacent loops, loop-5 (1 A) and loop-7 (1 A). The pairwise comparison of the three different TIM barrels shows that the 105C alpha atoms of the core superimpose within 0.9 A. The sequences of these three TIMs have a pairwise sequence identity of approximately 50%. The residues that line the active site are 100% conserved. The residues interacting with each other across the dimer interface show extensive variability, but the direct hydrogen bonds between the two subunits are well conserved. The orientation of the two monomers with respect to each other is almost identical in the three different TIM structures. There are 56 (22%) conserved residues out of approximately 250 residues in 13 sequences. The functions of most of these conserved residues can be understood from the available open and closed structures of the three different TIMs. Some of these residues are quite far from the active site. For example, at a distance of 19 A from the active site there is a conserved saltbridge interaction between residues at the C-terminal ends of alpha-helix-6 and alpha-helix-7. This anchoring contrasts with the large conformational flexibility of loop-6 and loop-7 near the N termini of these helices. The flexibility of loop-6 is facilitated by a conserved large empty cavity near the N terminus of alpha-helix-6, which exists only in the open conformation.     

Distinct roles for retinoic acid receptors alpha and beta in early lung morphogenesis

Retinoic acid (RA) signaling is required for normal development of multiple organs. However, little is known about how RA influences the initial stages of lung development. Here, we used a combination of genetic, pharmacological and explant culture approaches to address this issue, and to investigate how signaling by different RA receptors (RAR) mediates the RA effects. We analyzed initiation of lung development in retinaldehyde dehydrogenase-2 (Raldh2) null mice, a model in which RA signaling is absent from the foregut from its earliest developmental stages. We provide evidence that RA is dispensable for specification of lung cell fate in the endoderm. By using synthetic retinoids to selectively activate RAR alpha or beta signaling in this model, we demonstrate novel and unique functions of these receptors in the early lung. We show that activation of RAR beta, but not alpha, induces expression of the fibroblast growth factor Fgf10 and bud morphogenesis in the lung field. Similar analysis of wild type foregut shows that endogenous RAR alpha activity is required to maintain overall RA signaling, and to refine the RAR beta effects in the lung field. Our data support the idea that balanced activation of RAR alpha and beta is critical for proper lung bud initiation and endodermal differentiation.