Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP

Abstract

Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.

Fig. 1.

Denaturation of NCBD by urea and temperature at pH 7.4. (A) Urea denaturation curves in the presence of 0, 0.5, and 1 M of the kosmotrope TMAO at 25 °C measured by CD at 222 nm. Solid lines represent a global fit of the data to a two-state folding model with the m-value and slopes of both the pre and posttransition base lines as global parameters. (B) Temperature denaturation profile of NCBD in absence of urea and TMAO measured by CD at 222 nm with a temperature gradient of 1 °C/ min. (C) Urea denaturation profiles in 1 M TMAO at 5, 15, 25, 35, and 45 °C measured by CD at 222 nm. Solid lines represent a global fit of the data to a two-state folding transition performed as described under A. (D) Temperature stability diagram of NCBD. The free energy for folding, ΔG, obtained from two-state fits of urea denaturation curves as in A and C are plotted as a function of temperature. Data at three different concentrations of TMAO are included. The error bars are the standard errors from the fits.

Fig. 2.

Small angle X-ray scattering of NCBD. Small angle X-ray scattering profiles were recorded in (A) stabilizing conditions: 1 M TMAO; (B) in the absence of urea or TMAO; (C) near the denaturation midpoint at 2 M urea and (D) under conditions where the domain is almost completely unfolded 4 M urea. All curves were recorded with a protein concentration of 1, 2, and 4 mg/mL at pH 7.4, 150 mM NaCl and 25 °C. The left represents data points and a predicted scattering curve from an ensemble of structures selected using the ensemble optimization method (25). Histograms of the maximal intramolecular distance, D_max, (middle) and the radius of gyration (right) show the geometric properties of the ensembles fitted to the SAXS data using EOM.

Fig. 3.

Specific ring current effects in the hydrophobic core of the NCBD. (A) The methyl region of the ¹³C HSQC. The insert is an expansion of the cluster of peaks around 0.8 ppm and 24 ppm in the ¹H and ¹³C dimensions, respectively. (B) An excerpt of the structure of NCBD in complex and chemical shift predictions with ACTR reveals that the ring current is as predicted from the ACTR bound complex.

Fig. 4.

Excerpts of NOEs to aliphatic protons originating from selected well resolved methyl groups. These methyl groups show unambiguous NOEs between helix 1 and 2 (T2074 and V2087) and helix 2 and 3 (L2091).

Fig. 5.

Structural comparison of NCBD in different states. The structural ensemble of 10 structures determined by NOEs recorded in the free state of NCBD (gray), is aligned with the structure of NCBD (A) in complex with ACTR; (B) in complex with IRF-3 (red) and the structure previously reported for the free state (blue).