Trehalose-induced conformation facilitates GR's interaction with GRE and SRC-1.

<p>(A) Gel mobility shift assay showing binding of GR500 with GRE. Left Panel, a native gel showing bands of GR500:GRE complex; and Right panel, data from densitometric analysis of band of complex plotted against increasing concentrations of trehalose. (B) Immuno-reaction to anti-AF1 antibody after immunoprecipitation (IP) from HeLa nuclear extracts (NE) with SRC-1 antibody (Upper Panel). Lane 1, AF1 + beads (negative control). Lane 2, AF1+NE+beads in absence of trehalose. Lane 3, AF1+NE+beads in presence of trehalose. An average densitometric analyses of band intensity showing relative fold induction (Lower Panel).</p

Effect of enhanced interaction of SRC-1 on GR AF1 activity.

<p>(A) CV-1 cell viability with or without trehalose treatment (24 h). (B) Upper Panel: AF1-dependent GR-mediated transcription activation of a promoter containing 3×GRE. CV-1 cells constitutively expressing AF1 in a two domain GR fragment containing entire N-terminal and DNA-binding domains (GR500) were cotransfected with DNA of the pGRE_SEAP plasmid alone or plus DNA for SRC-1 in the absence and presence of 50 mM trehalose. Lower Panel: Western blot showing the level of expression of GR500 in each case. In lanes 3 and 4, cells were treated with trehalose 4 h prior to transfections whereas in lanes 5 and 6, 4 h after transfection. Experiments were performed in triplicate. (C) Model for osmolyte-induced folding of GR AF1 domain. AF1U represents the assembly of unfolded conformers of AF1. AF1 could exist only in its unfolded state(s) or in equilibrium with a properly folded state (AF1F). SRC-1 could induce folding directly to AF1U by binding, shifting AF1 directly to the hetero-dimer AF1F·SRC-1. Alternatively, AF1U could be in equilibrium with its native folded state AF1F. Without SRC-1, [AF1F] is relatively small compared to [AF1U]; however, osmolyte could shift equilibrium in favor of AF1F and eventually shift AF1 to the complex, by the law of mass action.</p

Trehalose-induced folding protects AF1 against partial proteolysis.

<p>Products of proteolytic digestion of AF1 resolved on an SDS–PAGE gel after treatment with trypsin (Left Panel), Endo Gluc-C (Middle Panel), and chymotrypsin (Right Panel) in absence (lane 2) or presence of increasing concentrations of trehalose (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 M, lanes 3–8, respectively). MW = Molecular Weight Markers; and lane 1 = undigested AF1.</p

Trehalose induces secondary structural elements in the ID AF1.

<p>(A) Diagram of human GR protein showing major functional domains. Far-UV CD spectra of GR AF1 protein in the absence and presence of increasing concentrations of trehalose (B) or TFE (C). (D) Trehalose-induced conformational transition of AF1. The nonlinear least squares best fit of experimental data to two-state model of protein folding/denaturation using linear extrapolation methods gives apparent thermodynamic parameters of trehalose-induced folding: ΔG and m (expressed as kcal/mol; shown in the box). (E) A linear plot showing TFE-induced helical structure in AF1.</p

JDP2 binds to the C-terminal part of the GR DBD.

<p>A) A topological diagram of the GR500 fragment showing NTD (a.a. 1–420), AF1 (77–262), and DBD (421–481). B) Coomassie-stained SDS-PAGE gel showing the patterns of interactions between JDP2 or TBP_C and various fragments of the GR (indicated on the top). GST-pull down assay was performed to determine these <i>in vitro</i> interactions using purified recombinant protein in each case.</p

JDP2:DBD interaction-dependent cofactor-binding increases AF1-mediated transcriptional activity of a promoter containing 3xGRE as assessed by SEAP-based promoter:reporter assay.

<p>CV-1 cells cotransfected with vectors containing genes for GR500 with or without other cofactors (as indicated). Results are expressed as means ±SE. Experiments were repeated at least three times. Levels of significance were evaluated by a two-tailed paired Student's t test and P<0.05 was considered significant. Graphs were normalized to transfection efficiency of each construct assayed by immunoblot with specific antibody to GR (lower panel).</p

JDP2:DBD binding-induced conformational changes facilitate interactions of AF1 with specific coregulatory proteins in cell as assessed by FRET analyses.

<p>A) Representative same-cell images in the donor (CFP-GR500) and YFP-TBP, YFP-CBP, or YFP-SRC-1 channel before and after PB. The areas within the white boxes were photo bleached. 1 and 4 =  CFP- Pre PB; 2 and 5 =  CFP- Post PB; 3 and 6 =  YFP- Post PB. Upper panel, CFP-GR500 and YFP-TBP (left hand); and CFP-GR500 and YFP-TBP in the presence of JDP2 (right hand). Middle panel, CFP-GR500 and YFP-CBP (left hand); and CFP-GR500 and YFP-CBP in the presence of JDP2 (right hand). Lower panel, CFP-GR500 and YFP-SRC-1 (left hand); and CFP-GR500 and YFP-SRC-1 in the presence of JDP2 (right hand). Cells were also cotransfected with a promoter-reporter construct, GRE-SEAP (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025875#s2" target="_blank">Materials and Methods</a>). B) Panel displays calculated average FRET efficiencies for each condition. Experiments were carried out three independent times and were analyzed and calculated average FRET efficiencies ± SD of 15 cells were graphed for each of the conditions.</p

JDP2 binding to the GR DBD induces secondary structure in ID NTD/AF1 domain.

<p>A) Far-UV CD spectra of recombinant GR500, JDP2, and GR500:JDP2 mixture. B) Far-UV CD spectra of GR500:JDP2 mixture (experimental), and additive of GR500+JDP2 (theoretical sum of GR500 plus JDP2). Each spectrum presented is the average of five spectra recorded, corrected for the contribution of the buffer, and smoothed.</p

Proposed mechanism of effects of JDP2 binding/folding in the stimulation of GR’s AF1 activity.

<p>Compared to the highly structured GR’s DBD and LBD, the NTD/AF1 is mostly unstructured in solution (a). JDP2 interaction with the DBD/CTE transmits inter-domain signals to the AF1/NTD, resulting into secondary/tertiary structure formation in it (b). This induced structure in the AF1/NTD creates interaction surfaces for other coactivators (e.g., TBP and CBP) that mediate transcriptional activity of the AF1 (c). Undergoing conformational changes are indicated by different shapes and colors.</p

JDP2 binding to the GR DBD fails to induce any significant structural changes in DBD.

<p>A) Far-UV CD spectra of recombinant DBD (a.a. 398–500), JDP2, and DBD:JDP2 mixture. B) Far-UV CD spectra of DBD:JDP2 mixture (experimental), and additive of DBD+JDP2 (theoretical sum of DBD plus JDP2). Each spectrum presented is the average of five spectra recorded, corrected for the contribution of the buffer, and smoothed. C) Far-UV CD spectra of recombinant AF1 (a.a. 77–262), JDP2, and AF1:JDP2 mixture. D) Far-UV CD spectra of AF1:JDP2 mixture (experimental), and additive of AF1+JDP2 (theoretical sum of AF1 plus JDP2). Each spectrum presented is the average of five spectra recorded, corrected for the contribution of the buffer, and smoothed.</p