Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor

Recently, many lines of evidence have been accumulated indicating that thyroid hormone receptor (TR) and retinoic acid receptor (RAR) undergo a ligand-dependent conformation change. Since most of these results were obtained by either gel-shift assay or circular dichroism spectroscopic studies, it was not clear which part of the receptor bore the major conformational change. Moreover, it is not clear whether the formation of heterodimer between TR or RAR and retinoic X receptor (RXR) has any effects on this structural change. Utilizing partial proteolytic analysis, we demonstrated that thyroid hormone and retinoic acid induce a specific protease-resistant conformation to their cognate receptors. Studies of various deletion mutants reveal that the entire ligand binding domain of these receptors is involved in this change, and suggest that ligand may induce a more compact structure in its binding domain. Evidence from native gel electrophoresis supports this notion. This conformational change occurs in the absence of DNA and occurs indenpendently of other domains in the receptor. Heterodimerization between TR or RAR and the RXR has little effect on receptor conformation in the absence of hormone but does enhance the ligand-dependent structural change. Interestingly, dual hormone treatment, i.e. thyroid hormone and 9-cis RA, intensifies this enhancement. We suggest that the observed protease-resistant conformation may introduce a different configuration to the receptor and therefore may effect the receptor in various ways, but most likely is involved in converting the receptor from a negative regulator to a positive activator.     

Retinol-binding protein in human serum: conformational changes induced by retinoic acid binding

Polyacrylamide gel electrophoresis and isoelectrofocusing followed by immunoblotting technique with an anti-human retinol-binding protein (RBP) serum were used to study holo-RBP and apo-RBP in human plasma. Three observations were made the technique allowed for the first time to directly and quantitatively analyse holo- and apo-RBP. Holo-RBP represented 97.86 +/- 0.78% and apo-RBP 1.94 +/- 0.73% of the total RBP. All-trans-retinoic acid (RA) was found to bind to apo-RBP and to significantly modify the tertiary structure of the protein; this raises the question of RBP involvement in the transport of RA. reconstitution of holo-RBP using apo-RBP from delipidized serum was achieved only after its incubation with natural all-trans-retinoids such as retinol, 3-dehydroretinol and retinoic acid but not with synthetic analogs of retinoic acid (13-cis-retinoic acid, TMMP, 13-cis-TMMP, TTNPB). It appears that RBP has a structure specificity for natural retinoids.     

Retinoic acid receptor: a simulation analysis of retinoic acid binding and the resulting conformational changes.

The binding/escape mechanism of all- trans retinoic acid with respect to the ligand-binding domain of the nuclear receptor RARgamma has been studied by molecular dynamic simulations. The entry/exit channel was shown to be on the side of the activation helix by the use of multiple copy dynamics. Three independent minimum energy paths from the liganded structure to a model for the unliganded structure were calculated with the conjugate peak refinement method. Ligand escape takes place in the early steps of the transition during rearrangement of the binding pocket; the latter involves inward motion of the beta-sheet and outward motions of the Omega-loop and helix H6. The correlated rearrangements involved in the escape phase are similar and occur in the same order for the different paths. After the escape phase, the conformational changes affect primarily the C-terminal helices H11-H12 and the Omega-loop. The three paths are significantly different for this reorganization phase and reveal a multiplicity of possibilities, in agreement with the idea that the apo state is structurally less constrained. The present calculations extend the crystallographic results, confirming the "mouse trap" mechanism and stressing the importance of the helix H3 conformation and of the contacts between the Omega-loop and helices H11 and H6. They are in good agreement with known mutants and point to other functionally important residues, especially in helices H3 and H11, suggesting mutations that may affect the ligand-binding function and the associated conformational changes.     

Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry

Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.     

Retinoic acid and retinoic acid receptors in craniofacial development

Interest in retinoids and craniofacial development originated independently from nutritional and teratological studies; however, the site of action of retinoids in normal development remains contentious. Recent transgenic strategies have shown that retinoic acid and nuclear retinoid receptors are required for the morphogenetic specification of cranial neural crest cells and their mesenchymal derivatives during craniofacial development. Interestingly, while some aspects of the RA teratogenicity have been shown to be receptor-mediated, there is as yet no clear evidence that this is the case for the embryonic head and face. Hox genes are one important set of targets for RA in the developing neural primordium and cranial neural crest, but it remains unclear as to how retinoid-mediated regulation of such targets is realized as the morphogenetic specification of cell fate.     

Functional and conformational changes in the aspartic protease cardosin A induced by TFE

Conformational and functional changes of cardosin A, an aspartic protease of vegetal origin, in the presence of 2,2,2-trifluoroethanol (TFE), were assessed. TFE induced alterations of cardosin activity and conformation that differed with the solvent concentration. MD simulations showed that there are significant local alterations in protein flexibility and TFE molecules were found to replace several hydration molecules in the active site of the enzyme. This may explain some of the activity loss observed in the presence of TFE, especially at low TFE concentrations, as well as the recovery of enzyme activity upon aqueous dilution, indicating the release of the TFE molecules from the active site.     

Fluorescent and photoaffinity labeling probes for retinoic acid receptors.

A fluorescent probe for retinoid receptors (RARs) was designed and prepared. The probe consists of a retinoid moiety and a dansyl moiety, i.e., 2-[3-(5-dimethylaminonaphthalene-1-sulfonyl)- aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid: DAM-3. DAM-3 specifically bound RARs. Additionally, a photoreactive RAR fluorescent probe was designed and prepared, i.e., 2-[3-(5-azidonaphthalene- 1-sulfonyl)aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (ADAM-3). ADAM-3 irreversibly and specifically bound RARs using ultraviolet irradiation.     

Cell entry-associated conformational changes in reovirus particles are controlled by host protease activity

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the μ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.     

Small-angle x-ray scattering study of metal ion-induced conformational changes in Serratia protease.

Metal ion-induced conformational changes in Serratia protease which contains one zinc ion per molecule were investigated by the small-angle x-ray scattering method. The molecule is an elongated ellipsoid of approximately 110 x 40 x 40 A with a large cleft in its central region. Comparisons of the native (zinc-enzyme) with the zinc-free (apoenzyme) enzyme and with the zinc-replated metalloenzyme show small but significant differences in their radii of gyration, maximum particle dimensions, and intraparticle pair-distance distributions. The radius of gyration and maximum particle dimension of the native enzyme are almost the same as those of the cobalt-enzyme but are shorter and longer, respectively, than those of the apo- and cadmium-enzymes. Simulation analysis based on the intraparticle pair-distribution function showed that these modified enzymes are comparable with the native enzyme in overall structure, and, except for the cobalt-enzyme, differ in cleft size. The residual enzymatic activity of the cobalt-enzyme is the same as that of the native enzyme, but the apo- and cadmium-enzymes have considerably less activity. The size of the cleft therefore is strictly controlled to ensure optimal enzyme activity, and the position and coordination behavior of the zinc ion in the cleft appears to be essential both for biological functioning and for the maintenance of the gross tertiary structure.     

Different sedimentation properties of agonist- and antagonist-labelled platelet alpha2 adrenergic receptors

Alpha₂ adrenergic receptors were solubilized from human platelet particulate preparations with digitonin. The solubilized alpha₂ receptors retained the essential binding specificity characteristics of the membrane-bound receptors. The alpha₂ receptors could be labelled in platelet membranes with either agonist ([³H]epinephrine) or antagonist ([³H]yohimbine) radioligands. When these membranes were solubilized with digitonin and centrifuged on sucrose density gradients, the sedimentation coefficient of the agonist-labelled receptor (14.6S) was greater than that of the antagonist-labelled receptor (12.9S). This observation may provide insight into the mechanism of adenylate cyclase inhibition by alpha₂ adrenergic receptors.