Binding-folding induced regulation of AF1 transactivation domain of the glucocorticoid receptor by a cofactor that binds to its DNA binding domain

Abstract

Intrinsically disordered (ID) regions of proteins commonly exist within transcription factors, including the N-terminal domain (NTD) of steroid hormone receptors (SHRs) that possesses a powerful activation function, AF1 region. The mechanisms by which SHRs pass signals from a steroid hormone to control gene expression remain a central unresolved problem. The role of N-terminal activation function AF1, which exists in an intrinsically disordered (ID) conformation, in this process is of immense importance. It is hypothesized that under physiological conditions, ID AF1 undergoes disorder/order transition via inter- and intra-molecular communications, which allows AF1 surfaces to interact with specific co-regulatory proteins, critical for the final outcome of target gene expression regulated by SHRs. However, the means by which AF1 acquires functionally folded conformations is not well understood. In this study, we tested whether binding of jun dimerization protein 2 (JDP2) within the DNA binding domain (DBD) of the glucocorticoid receptor (GR) leads to acquisition of functionally active structure in its AF1/NTD. Our results show that signals mediated from GR DBD:JDP2 interactions in a two domain GR fragment, consisting of the entire NTD and little beyond DBD, significantly increased secondary/tertiary structure formation in the NTD/AF1. This increased structure formation facilitated AF1's interaction with specific co-regulatory proteins and subsequent glucocorticoid response element-mediated AF1 promoter:reporter activity. These results support the hypothesis that inter- and intra-molecular signals give a functionally active structure(s) to the GR AF1, which is important for its transcriptional activity.

Figure 1. JDP2 binds to the C-terminal part of the GR DBD.

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 in vitro interactions using purified recombinant protein in each case.

Figure 2. JDP2 binding to the GR DBD induces secondary structure in ID NTD/AF1 domain.

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.

Figure 3. JDP2 binding to the GR DBD fails to induce any significant structural changes in DBD.

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.

Figure 4. JDP2:DBD binding-induced conformational changes facilitate interactions of AF1 with specific coregulatory proteins in cell as assessed by FRET analyses.

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 Materials and Methods). 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.

Figure 5. JDP2:DBD interaction-dependent cofactor-binding increases AF1-mediated transcriptional activity of a promoter containing 3xGRE as assessed by SEAP-based promoter:reporter assay.

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).

Figure 6. Proposed mechanism of effects of JDP2 binding/folding in the stimulation of GR’s AF1 activity.

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.