TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

Abstract

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.

Fig. 1.

Proteins studied in this work. (A) Topological diagram of the human GR showing its major functional domains. The highlighted region represents the N-terminal activation domain AF1. LBD, ligand binding domain. (B) Coomassie blue-stained SDS/PAGE of 5 μg each of purified TBP (lane 1) and GR AF1 (lane 2) polypeptides.

Fig. 2.

Second derivative FTIR spectra showing that binding of TBP to AF1 enhances helical content in AF1. (A)—,AF1;–––, TBP; – ·· –, AF1:TBP complex. (B)—,AF1;–––, AF1:TBP complex after subtracting the contribution of TBP. (C)—,TBP;–––, AF1:TBP complex after subtracting the contribution of AF1. The spectra shown are one of the two independent experiments each carried out on an independent preparation of TBP and AF1.

Fig. 3.

¹H-¹⁵N HSQC NMR spectra. (A) ¹⁵N-labeled GR AF1 with (red) and without (blue) TBP bound. The numbering of amino acid residues indicates the positions of amino acids in the AF1_c.(B) Expanded view of Gly residues (boxed in A) of ¹⁵N-labeled GR AF1 in the absence (black) or presence (red) of TBP.

Fig. 4.

Near-UV CD spectra showing perturbation of W213 in the AF1 domain after AF1:TBP interaction. —, AF1;–––,TBP_C;– ·· –, AF1:TBP_C.

Fig. 5.

Limited proteolysis of AF1, TBP, and the AF1:TBP complex. (A) Coomassie blue-stained gel showing product of proteolytic digestion after 15 min. Lanes: 1, molecular weight markers; 2–4, undigested AF1, TBP, and AF1:TBP mixture, respectively; 5–7, trypsin-digested AF1, TBP, and AF1:TBP mixture, respectively; 8–10, chymotrypsin-digested AF1, TBP, and AF1:TBP complex, respectively. (B) Immunoreactions with an antibody raised against amino acids 150–175 in the human GR AF1 showing products of trypsin digestion. Lanes: 1, undigested TBP; 2, undigested AF1; 3–10, digested with trypsin for 10 min; 11–18, digested for 20 min at 4°C; 3, TBP; 4, AF1; 5–9 and 13–17, AF1:TBP mixture at a ratio of 1:0.25, 1:0.5, 1:1, 1:1.5, and 1:2, respectively; 10 and 18, AF1:TBP:TATA (1:1:1) mixture. TATA represents a double-stranded oligonucleotide containing TATA box sequences.