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Conformational Selection in the Molten Globule State of the Nuclear Coactivator Binding Domain of CBP

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Conformational Selection in the Molten Globule State of the Nuclear Coactivator Binding Domain of CBP Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP Magnus Kjaergaard, Kaare Teilum, and Flemming M. Poulsen1 Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Kobenhavn N, Denmark Edited by Robert Baldwin, Stanford University, Stanford, CA, and approved June 3, 2010 (received for review February 23, 2010) Native molten globules are the most folded kind of intrinsically receptor coactivators (10), p53 (11), p73 (12), the interferon disordered proteins. Little is known about the mechanism by which regulatory proteins (IRF) 3 and 7 (13, 14) and the viral protein native molten globules bind to their cognate ligands to form fully Tax (15). In the ligand-free state, the NCBD is compact and has a folded complexes. The nuclear coactivator binding domain (NCBD) high degree of helicity, but apparently it lacks sigmoidicity in the of CREB binding protein is particularly interesting in this respect as unfolding curve (16–18). These observations suggest that NCBD structural studies of its complexes have shown that NCBD folds is a molten globule. The NCBD:ACTR complex was the first into two remarkably different states depending on the ligand documented example of an IDP that folds upon binding to an- being ACTR or IRF-3. The ligand-free state of NCBD was character- other IDP in a process termed mutual synergistic folding (16). ized in order to understand the mechanism of folding upon ligand Structural studies of the complexes of NCBD and three of its binding. Biophysical studies show that despite the molten globule ligands have shown that the complexes have fully compact and nature of the domain, it contains a small cooperatively folded core. well defined globular structures, and that NCBD adopts at least By NMR spectroscopy, we have demonstrated that the folded core two different ligand specific structures (16, 19, 20). This structural of NCBD has a well ordered conformer with specific side chain pack- adaptability is a functional hallmark of the IDPs. ing. This conformer resembles the structure of the NCBD in complex The present study combines biophysical studies of stability and with the protein ligand, ACTR, suggesting that ACTR binds to folding with structural studies by NMR spectroscopy to deter- prefolded NCBD molecules from the ensemble of interconverting mine the conformational properties and the binding mechanisms structures. of the promiscuous molten globule of murine NCBD. In spite of BIOPHYSICS AND the dynamic nature of the domain, it is being shown here that the COMPUTATIONAL BIOLOGY CREB binding protein ∣ folding upon binding ∣ NMR spectroscopy molten globule ensemble of NCBD contains a significant fraction of a conformer similar to NCBD in the NCBD:ACTR complex, he protein structure-function paradigm has dominated struc- suggesting that the ligand is able to select a prefolded conformer Ttural and molecular biology over the past five decades. This from the molten globule ensemble. paradigm has been challenged by the discovery of functional but intrinsically disordered proteins (IDPs) (1). IDPs are abundant in Results higher organisms and are necessary for many crucial biological Cooperativity in the Folding of NCBD. The equilibrium unfolding of functions such as cell cycle regulation, signal transduction, and NCBD was characterized in a series of urea denaturation experi- regulation of transcription (2, 3). Structural studies of IDPs have ments in the presence of varying concentrations of the stabilizing shown that despite the high degree of disorder, these proteins are osmolyte trimethylamine oxide (TMAO). The unfolding process far from randomly structured and form transient, yet specific, was followed by CD spectroscopy as the change in molar ellipti- secondary and tertiary structural elements (4, 5). The success city at 222 nm (Fig. 1). In the absence of TMAO, the unfolding of the structure-function paradigm in explaining the function curve lacks a pretransition baseline and thus appears nonsigmoi- of folded proteins suggests that the functions of the IDPs may dal (17). When urea denaturation of NCBD is performed in the also be understood by studying the transient structure of the presence of TMAO, the transition is shifted towards higher urea disordered state. concentrations, revealing a pretransition baseline and a sigmoidal The molten globule was originally discovered as a partially denaturation curve characteristic of a cooperative folding process folded state under mildly denaturing conditions and was shown (Fig. 1A). TMAO generally stabilizes the most folded states of to accumulate as an intermediate during protein folding reactions proteins, and thus shifts the unfolding transition towards higher (6, 7). With the emergence of the IDPs, proteins were discovered concentrations of denaturant (21). This stabilization happens where the molten globule is the functionally active state. Native without changing the baselines of the unfolding curve or the molten globules are the most compact conformational state con- m-value for the process (22). TMAO can therefore be used in sidered as IDPs (4). Molten globules have native-like secondary stability measurements of proteins that unfold at low denaturant structure, but are believed to lack the well-defined tertiary inter- concentrations (23). The urea denaturation curves at 0, 0.5, and actions found in folded proteins. Most high resolution NMR stu- 1.0 M TMAO were therefore analyzed globally assuming that the dies of the molten globule state have focused on the secondary pre- and posttransition baselines and the m-values are the same at structure of the backbone as this is more readily accessible from all TMAO concentrations. The urea concentrations of the transi- secondary chemical shifts (8). The degree of side chain packing, tion midpoints were fitted individually for each TMAO concen- however, is less well understood. Molten globules are believed to have dynamic hydrophobic cores due to the lack of signal in the Author contributions: M.K., K.T., and F.M.P. designed research; M.K. performed research; near-UV CD spectrum and the broadening of NMR signals of M.K., K.T., and F.M.P. analyzed data; and M.K., K.T., and F.M.P. wrote the paper. aromatic groups (6, 7). Recent studies have shown that the side The authors declare no conflict of interest. α chains in the -lactalbumin molten globule exchange between This article is a PNAS Direct Submission. several well defined conformations, representing a dynamic Data deposition: The atomic coordinates have been deposited in the BioMagResBank, ensemble of states with specific tertiary interactions (9). www.bmrb.wisc.edu, and the Protein Data Bank, www.pdb.org (BMRB accession A particularly interesting example of a molten globule is the no. 16363 and PDB ID code 2KKJ). nuclear coactivator binding domain (NCBD) of CREB binding 1To whom correspondence should be addressed. E-mail: [email protected]. protein (CBP). NCBD is responsible for the interaction of CBP This article contains supporting information online at www.pnas.org/lookup/suppl/ with a number of other important proteins such as the steroid doi:10.1073/pnas.1001693107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1001693107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 29, 2021 ABThis observation indicates increased dynamics or noncooperative loss of helicity in the molten globule form, in addition to the cooperative unfolding transition. This noncooperative loss of structure is similar to the behavior with increasing temperature, where the pretransition baseline reveals a loss of helicity with in- creasing temperature. The posttransition baselines are less steep than the pretransition baselines, and show a significant decrease in ellipticity with increasing temperature. This behavior has been observed previously for unfolded polypeptides and suggests a redistribution in the unfolded state from polyproline-II structure CDto extended β-structure (24). In combination, the urea and tem- perature stability analyses suggest that NCBD does indeed form a structure with a cooperatively folded core. Following NCBD Unfolding Using Small Angle X-ray Scattering. Small angle X-ray scattering (SAXS) is a powerful technique for char- acterizing the ensemble distribution of unfolded proteins. The time resolution of X-ray scattering experiments is high enough to resolve populations of protein molecules in exchange, which are averaged and hence indistinguishable in NMR experiments. Fig. 1. Denaturation of NCBD by urea and temperature at pH 7.4. (A) Urea SAXS experiments were performed at the following conditions: denaturation curves in the presence of 0, 0.5, and 1 M of the kosmotrope in the presence of 1 M TMAO; in aqueous buffer without TMAO TMAO at 25 °C measured by CD at 222 nm. Solid lines represent a global and urea; near the unfolding midpoint in 2 M urea; and in 4 M 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)Tem- urea, where the domain is almost entirely unfolded (Fig. 2). To perature denaturation profile of NCBD in absence of urea and TMAO mea- analyze the underlying ensemble of structures, 500 ensembles of sured by CD at 222 nm with a temperature gradient of 1 °C∕ min. (C) Urea 20 structures were selected each reproducing the experimental denaturation profiles in 1 M TMAO at 5, 15, 25, 35, and 45 °C measured scattering curve (25). The ensembles were selected using a genet- by CD at 222 nm. Solid lines represent a global fit of the data to a two-state ic algorithm, which was allowed to run until χ2 converged (50 gen- folding transition performed as described under A.(D) Temperature stability erations), resulting in an ensemble with the minimum number of diagram of NCBD. The free energy for folding, ΔG, obtained from two-state features and still reproducing the experimental data. fits of urea denaturation curves as in A and C are plotted as a function of The data recorded in 1 M TMAO have a single distinct popu- temperature.
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