Interaction between isolated transcriptional activation domains of Sp1 revealed by heteronuclear magnetic resonance

Abstract

The promoter-specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N-terminal half of the protein are characterized by glutamine-rich (Q-rich) sequences. These Q-rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA-binding protein associated factors. Furthermore, the self-association of Sp1 via Q-rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a "distal" GC-box synergistically interacts with another Sp1 molecule at a "proximal" binding site. Although the formation of multimers via Q-rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q-rich domains. We analyzed the structural details of isolated glutamine-rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C-terminal half was responsible for the self-association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self-association between QB domains would be responsible, at least in part, for the formation of multimers by full-length Sp1 molecules that has been proposed to occur during transcriptional activation.

Figure 1

(A) Schematic representation of the transcription factor Sp1. Two glutamine-rich domains, QA and QB, and three zinc finger domains are indicated. (B) Amino acid sequence of the QB domain of Sp1 used in this study. Two fragment proteins, Bn (¹T–S⁸³, in blue) and Bc (⁸⁰Q–T¹⁴⁷, in red), were also used to facilitate the assignment. The proteins were expressed as a fusion protein with glutathione S-transferase (GST) at the N-terminus, which was cleaved during the purification. The residual linker amino acids at the N-terminus are indicated by small capitals in green. The proteins also contain anti-FLAG and hexahistidine tags at the C-terminal.

Figure 2

Far UV-CD spectra of QB domains measured at 4°C. Three traces recorded at different protein concentrations, 50 (red), 100 (blue), and 300 (black) μM, are overlaid.

Figure 3

¹H-¹⁵N HSQC spectra of QB domains measured 4°C. The assignments of signals are indicated by a single-letter code and residue number in the same colors as in Figure 1(B). A total of 129 residues among 138 nonproline residues (the N- and C-terminal extensions were not included) were unambiguously assigned.

Figure 4

(A) Overlay of the ¹H-¹⁵N HSQC spectra of ¹⁵N-QB domains in the absence (red) and presence (blue) of unlabeled QB domains at 4°C. Concentrations of ¹⁵N-QB were 50 μM in both spectra, and an excess amount (500 μM) of unlabeled QB was added. Expansion of the spectra in a representative region is shown in the right panel. The signal for Q113 showed a dramatic decrease in intensity and a slight upfield shift upon an increase in the total protein concentration. (B) The relative peak intensity of ¹H-¹⁵N HSQC spectra plotted against the residue number of the QB domain. The intensity in the presence of a 10-fold amount of ¹⁴N-protein relative to that in its absence is shown. The positions of glutamine residues as well as several characteristic amino acid residues, positively/negatively charged or hydrophobic, are indicated on the top of the graph. (C) Relative peak intensity of several representative residues plotted against total protein concentration. Samples containing a constant concentration (50 μM) of the ¹⁵N-QB domain and various concentrations (50–500 μM) of ¹⁴N-QB were prepared, and peak intensity relative to that in the absence of ¹⁴N-QB was plotted against the total concentration of protein (¹⁵N-QB + ¹⁴N-QB). A broken line indicates the relative fraction of monomer at a given total protein concentration on the assumption of a monomer–dimer equilibrium [Eq. (2)] at K_a = 4.5 × 10³ M^–1.

Figure 5

Sedimentation equilibrium analysis of the QB domain at protein concentrations of 50 (open circle), 100 (cross), and 150 μM (closed circle) and at 4°C. (A) ln(c) versus r² plot. The straight lines in the plot give a weight-averaged molecular weight of 21.3–24.4 kDa for each concentration (Supporting Information Table S1), which is apparently larger than that expected from the amino acid sequence (18.4 kDa). In addition, the data significantly deviated from the lines, indicating that proteins are not in a monodispersed state. (B) Plot of concentration versus radius with a theoretical fit using Eq. (3). Three independent sets of experimental data were globally fit to provide a K_a value of 4.5 × 10³ M^–1. The molecular weight of the monomer was fixed at 18.4 kDa during the fit.