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 increasing 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 temperature 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. Data at three different concentrations of TMAO are included. The error bars are the standard errors from the fits. lation with a radius of gyration distributed around 15.5 Å. The highly skewed size distribution found in buffer and 1 M TMAO tration. In the absence of TMAO and at 25 °C, the free energy of resembles the distribution of the input library (Fig. S2), probably Δ ¼ 6 1 reflecting a bias towards the input ensemble rather than the true unfolding for the molten globule state of NCBD is GNU . 0.4 kJ∕mol, increasing to 8.7 0.5 kJ∕mol at 0.5 M TMAO, and conformational preferences of the ensemble. The data recorded 11.0 0.7 kJ∕mol at 1.0 M TMAO. The low stability of the mol- without TMAO or urea contains a shoulder, which indicates the ten globule state of NCBD in the absence of TMAO corresponds presence of an extended subpopulation, which can be suppressed to 8% of the NCBD molecules being unfolded in the absence of denaturant at 25 °C. The common m-value from the global fit is 2.9 0.1 kJ∕mol∕M, which corresponds to the complete unfolding of approximately 40 residues and is equivalent to the exposure 2 of 2;900 Å of solvent accessible surface area (22). A Attempting to thermally unfold NCBD resulted in a gradual loss of secondary structure as gauged from the change in ellipticity at 222 nm, which suggests the absence of a well defined cooperative transition (Fig. 1B). To further investigate the temperature B dependence of the free energy for folding, urea-unfolding experiments were performed at five temperatures ranging from 5–45 °C and in the presence of 0, 0.5, and 1.0 M TMAO (Fig. 1C). The free energy for folding is fairly insensitive to temperature C changes (Fig. 1D), suggesting that the change in heat capacity, Δ Cp, of the folding reaction is low. Like the m-value, the size Δ of Cp depends on the accessible surface area exposed upon un- Δ D folding. Consequently, the low Cp is consistent with the low m-values from the urea denaturation studies and the relatively small D R buried surface area in the folded state of NCBD (22). To verify max g that the apparently gradual unfolding was consistent with the Fig. 2. Small angle X-ray scattering of NCBD. Small angle X-ray scattering cooperativity observed in the urea unfolding transitions, we simu- profiles were recorded in (A) stabilizing conditions: 1 M TMAO; (B) in the ab- lated the fraction of folded protein and the accompanying differ- sence of urea or TMAO; (C) near the denaturation midpoint at 2 M urea and ential scanning calorimetry curve based on a two-state equilibrium (D) under conditions where the domain is almost completely unfolded 4 M and parameters estimated from the urea denaturation curves urea. All curves were recorded with a protein concentration of 1, 2, and 4 ∕ (SI Text). The simulation demonstrated that the gradual unfolding 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 and the lack of an observable DSC heat peak were consistent with using the ensemble optimization method (25). Histograms of the maximal a two-state folding model with a very low ΔC (Fig. S1). p intramolecular distance, Dmax,(middle) and the radius of gyration (right) The pretransition baselines increase relatively more rapidly show the geometric properties of the ensembles fitted to the SAXS data with urea concentration than expected for a well-folded protein. using EOM.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001693107 Kjaergaard et al. Downloaded by guest on September 29, 2021 by TMAO. In the presence of 2 and 4 M urea, an extended un- Hence there is only one aromatic group in the hydrophobic core folded state with an average radius of gyration of approximately (F2101) to account for. The chemical shifts of the methyl groups 22 Å is partly populated. The folded state appears to be popu- were predicted based on the structures deposited in the PDB lated even under conditions where nearly all of the molecules using SHIFTS 4.1 (29). The structures determined for the un- are unfolded, confirming that the extended unfolded state and bound NCBD structure published previously (1JJS) (30) and the compact folded state coexist even under highly denaturing the IRF-3 bound complex (1ZOQ) predict the ring current to conditions, and directly confirming the cooperativity of the fold- have the largest effect on L2071/I2102 and L2071/L2097, respec- ing process of NCBD. From the ensemble of NMR structures cal- tively. The ACTR bound complex (1KBH) correctly predicts the culated for the compact conformation of NCBD (vide infra), an ring current effect to be largest on V2087 and L2091 similar to ¼ 15 5 0 7 Rg . . Å was obtained using HydroPro (26). Using the observations for unbound NCBD. Both methyl groups from relationship between the chain length of an unfolded protein and L2091 are shifted upfield relative to the random coil values, the radius of gyration a value of 22.7 Å would be expected for an whereas V2087 has one methyl group shifted upfield and one unfolded protein chain of 59 residues (27). These values corre- shifted downfield. To get ring current contributions with opposite spond well to the mode of the simulated SAXS ensemble under signs on two methyl groups from the same side chain, the cone “ ” native-like conditions (15.2 Å) and the average Rg in 4 M urea extending at the magic angle from the plane of the ring must intersect the side chain. The observed ring current shifts suggests (22.3 Å), respectively. The average of Rg under native-like conditions (18.8 Å) is in good agreement with the 10%–25% expan- the side chains in the hydrophobic core to have well defined or- sion that was observed for molten globules relative to a fully ientations and interactions, and that these are similar to those in compact state (28). the ACTR bound complex. The methyl groups of V2087, I2090, and L2091 all have well separated NMR signals, which allow NMR Study of the NCBD Molten Globule State. NMR studies of mol- probing of the dynamics of the hydrophobic core by comparing ten globule states are often hampered by exchange between the NOE patterns between methyl groups from the same side different conformations, which leads to broadening of the NMR each of the chain. All three side chains show different NOE pat- lines and consequently loss of signals. At low temperatures, terns for their methyl groups, which demonstrate that the orien- almost all signals in the heteronuclear single quantum coherence tations of the side chains are well defined in the conformer (HSQC) spectrum of NCBD are missing, which suggests ex- responsible for the NOEs (Fig. 4). change between multiple conformations. When the temperature The high degree of structure predicted from the chemical shift is increased the signals gradually begin to appear, and at 31 °C encouraged a thorough examination of the tertiary structure BIOPHYSICS AND

only signals of the first four residues and R2105 are missing. using NOEs. Assignment of the NOEs was hampered by the lack COMPUTATIONAL BIOLOGY The chemical shift dispersion is lower than in the complex with of dispersion particularly in the side chains. To avoid ambiguous ACTR (16), but significantly better than in the urea unfolded state (Fig. S3), suggesting an intermediate degree of structure. Secondary chemical shifts are the changes in chemical shifts relative to those expected for an unfolded polypeptide, and they report primarily on the conformation of the peptide backbone. In NCBD, the secondary shifts show that the domain contains three helices from 2066–2078, 2081–2093, and 2094–2104 (18), and their magnitude suggests that the helices are completely formed in the ligand-free state (18). The methyl region of the 13C HSQC shows, despite the lack of chemical shift dispersion, that individual peaks can be distinguished and assigned. Most methyl peaks have very similar proton chemical shifts close to the random coil values suggesting that these groups experience conformational averaging (Fig. 3A). V2087 and L2091 have methyl chemical shifts that deviate significantly from random coil values. The larger chemical shift dispersion from these side chains suggests the effect of a nearby aromatic ring. NCBD contains two aromatic groups, one of which is located in the flexible C terminus, Y2109, and thus unlikely to cause a large ring shift. AB

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 Fig. 4. Excerpts of NOEs to aliphatic protons originating from selected well shift predictions with ACTR reveals that the ring current is as predicted from resolved methyl groups. These methyl groups show unambiguous NOEs the ACTR bound complex. between helix 1 and 2 (T2074 and V2087) and helix 2 and 3 (L2091).

Kjaergaard et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 29, 2021 assignments from the crowded spectral regions, only NOEs suggests NCBD to be an atypical molten globule with a relatively originating from amides and well separated methyl groups were high degree of buried hydrophobic side chains. Similarly, NMR included in the analysis. A total of 455 NOE-based distance re- relaxation studies show that the core of NCBD is as folded as the straints were included in the analysis, 46 of these were long-range most folded part of the apomyoglobin molten globule (18, 32). NOEs. Analyses of NOEs in dynamic systems must be performed The folded core, however, is small compared to other protein do- with considerable care, as the NOE set may contain contributions mains without disulfide bonds. Consequently, the molten globule from different conformers. Therefore the compatibility of the state of NCBD is marginally stable and around 8% of the protein already published structures with the NOE dataset determined is fully unfolded at 25 °C in the absence of urea. The exact per- from the free form (Table S1) was examined. The structure of centage is, however, uncertain as the unfolding of NCBD might NCBD in complex with IRF-3 (1ZOQ) and the structure pub- be more complex than two-state. lished previously of the free NCBD (1JJS) are inconsistent with Theory predicts that under certain conditions proteins can fold a large fraction of the long-range NOEs in the present dataset. in a second-order transition without passing a free energy barrier The structures of NCBD in complex with ACTR (1KBH), and to (33). The molten globule of apomyoglobin, despite the sigmoidi- a smaller extent the structure in complex with SRC-1 (2C52), city of its unfolding curves unfolds via a second-order transition satisfy essentially all long-range NOEs, suggesting that all the (34). Folding via a first or second-order transition has different NOEs arise from a conformer that is similar to that found in com- predictions for the ensemble of structures near the denaturation plex with ACTR. To assess how well the NOE dataset defines the midpoint. A cooperative, first-order transition predicts a bimodal tertiary structure, the data were subsequently submitted to ensemble, where the folded state is depopulated and the un- ARIA2.2 for structure calculation (See Table S2 for structural folded state is populated with increasingly denaturing conditions. statistics). The resulting ensemble of structures is represented In contrast, a second-order transition predicts a unimodal ensem- in Fig. 5. The tertiary structure of NCBD in solution resembles ble, where the midpoint of the ensemble gradually shifts from the that of NCBD in complex with ACTR (rmsd ¼ 1.4 Å), except folded to the unfolded state with increasingly denaturing condi- that the second half of the third helix is disordered. The ensemble tions. The ensemble distribution of NCBD during unfolding was has a different overall fold from NCBD in complex with IRF-3 obtained from SAXS profiles in urea and TMAO. Remarkably, (1ZOQ, rmsd ¼ 5.2 Å) and the NCBD structure published for even under highly denaturing conditions a compact state remains the free domain (1JJS, rmsd ¼ 5.8 Å). The chemical shifts of populated with dimensions similar to those of the core of the the ring current shifted methyl groups were predicted using both ACTR-complex-like structure. Hence, the folded state remains the present NOE derived NCBD structure and the structure of populated even at highly denaturing conditions, and the protein the NBCD/ACTR complex. The predictions showed that the unfolds in a barrier limited first-order process. observed chemical shifts were better predicted by the structure Having established that NCBD molten globule has a coopera- of the complex than for those of the NOE derived NMR struc- tively folded core, the molecular interactions underlying the ture. Apparently, the actual side chain packing in the free form cooperativity was probed. The intrinsic conformational averaging of NCBD is more similar to that in the complex structure. The of molten globules makes NMR studies difficult by broadening observation indirectly suggests that the NOE-based structure cal- peaks and reducing their chemical shift dispersion, particularly culation is indeed hampered by the dynamics of the molecule. for side chain peaks (8). The small size of NCBD, however, is an advantage for overcoming the obstacles that normally prevent Discussion high resolution analysis of the tertiary structure of molten glo- Molten globules unfold with very different degrees of cooperativ- bules. The presence of a highly specific ring current effect and ity, however, generally with lower cooperativity than fully folded the observation of different NOE patterns between geminal proteins (31). Our urea denaturation studies demonstrate that methyl groups demonstrate that the hydrophobic side chains have NCBD unfolds in a cooperative transition that fits well to a preferred rotamers and make specific contacts in the molten glo- two-state process. The apparent two-state behavior in urea- bule of NCBD. This result shows that the hydrophobic core has unfolding experiments does not exclude the existence of a hetero- specific packing in a large fraction of the NCBD molten globule geneous native state, because an ensemble of interconverting ensemble, and it is in contradiction to the common description of structures with similar helicities and stabilities is likely to be in- molten globules, where the hydrophobic core is liquid-like. distinguishable from a true two-state reaction. The size of the From the NMR spectra of the ligand-free state of NCBD 455 m-value suggests the unfolding of a dehydrated hydrophobic core NOEs have been assigned, 46 of these were long range. Finding corresponding to a 40 residue protein, which is comparable to so many NOEs is surprising since the absence of long-range the size of the helical region determined by NMR. Since NCBD NOEs previously has been suggested to be a characteristic feature has an m-value similar to a folded protein of the same size this of molten globules (8, 35). The presence of long-range NOEs confirm that the molten globule of NCBD contains well defined tertiary structure, and this is a remarkable difference from the archetypical molten globule α-lactalbumin. Violation analyses of existing NCBD structures suggested that the NOE set arise from one dominant conformer. All three helices of NCBD are pair wise connected by long-range NOEs suggesting that they define the fold well enough to allow a structure calculation. The structures calculated from the NOEs resemble that of NCBD in complex with ACTR, but they are less well defined. The intensity of most long-range NOEs are weaker than predicted from the distances found in the structure presumably a result of the dynamic properties of the NCBD molten globule, and the underlying motions may be both fast and slow. Motions faster than the rotational correlation time of the vector between an interacting Fig. 5. Structural comparison of NCBD in different states. The structural ensemble of 10 structures determined by NOEs recorded in the free state of set of protons will reduce the NOE transfer efficiency. Slower NCBD (gray), is aligned with the structure of NCBD (A) in complex with ACTR; motions will result in ensemble averaging of the observed NOE (B) in complex with IRF-3 (red) and the structure previously reported for the and hence to reduced intensity (36). Alternative conformations free state (blue). are very likely to exist in the molten globule ensemble, even

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001693107 Kjaergaard et al. Downloaded by guest on September 29, 2021 though the absence of unique NOEs from these conformers NCBD adopts a conformation similar to that in the complex with suggest that they either have too short a life time or too low a ACTR. Although many conformations of NCBD exist in the mol- population to give NOEs. The structure calculation protocol used ten globule state, the conformation that we describe is the only in this study is designed to find one unique structure as expected one that is stable and long lived enough to allow structural char- for a well folded protein. It is thus important to emphasize that acterization from NOEs. The present analysis does not allow for the heterogeneity in the structural ensemble does not reflect the the determination of the stability and life time of this conforma- true heterogeneity of the molten globule ensemble. Therefore it tion, but it permits the important conclusion that the conformation may be, that the structural ensemble determined here only of NCBD in the complex with ACTR is present even in the absence reports on the most well defined conformer in an ensemble of of ACTR. The existence of structured NCBD molecules implies a rapidly interconverting structures. conformational selection mechanism, where ACTR selectively A solution structure has previously been published for a very binds to the fraction of the prefolded ACTR binding conformation similar construct of NCBD (1JJS) (30). A large number of long- of the NCBD ensemble. range NOEs from the present dataset are violated in this struc- A key advantage of intrinsic disorder in proteins is believed to ture (Table S1) and no long-range NOEs unique to this structure be the ability to fold into different structures to accommodate are observed. This observation implies that the structure of structurally diverse ligands (2, 4). The structure of NCBD in com- unbound NCBD published previously is not representative of a plex with IRF-3 has an entirely different topology from the main major conformation in the molten globule ensemble, and it illus- conformation of free NCBD reported here. No NOEs unique to trates that automated NOE assignment programs must be used the IRF-3 bound structure were observed. If an IRF-3 bound- with caution to analyze datasets with limited signal dispersion. state structure is present in the molten globule of NCBD ob- viously it does not share those properties of the ACTR binding Is NCBD a Molten Globule? Originally the molten globule was de- conformation that makes this observable by NMR spectroscopy. fined as a “compact globule with native-like secondary structure and Still the data suggests that NCBD may sample other conforma- with slowly fluctuating tertiary structure” (7), where the side chains tions than the ACTR binding structure. The ability to sample dif- were believed to be “liquid-like” (6, 37). The experimental data ferent conformations is facilitated by the low structural stability, supporting this claim was the absence of a near-UV CD signal which allows the domain to have sizeable populations of unfolded and sharp NMR signals from the aromatic groups, indicating that or partially folded states. Future studies may reveal to what extent the aromatic side chains undergo conformational averaging the IRF-3 complex-like structure and other yet unknown ligand

(6, 37, 38). The data presented here demonstrate that the hydro- binding conformations play a role in the molten globule ensemble. BIOPHYSICS AND

phobic core is structured in the molten globule NCBD. We have IDPs encompass a wide range of degrees of residual structure COMPUTATIONAL BIOLOGY shown that the side chains form well defined interactions resem- formation, from random coils to molten globules. Functionally, bling those found in the NCBD∶ACTR complex in a significant however, they are united by their structural adaptability and pro- part of the ensemble. The lack of dispersion of the chemical miscuous ligand binding. Upon binding to their cognate ligands, shifts, the low intensity of the long-range NOEs, the broadening random coil like IDPs fold into extended structures with very few of NMR lines observed at low temperatures, and NMR relaxation intramolecular, tertiary interactions as observed for ACTR (16), analysis (18) all suggest that the domain undergoes extensive the chaperone Chz1 (47), the hypoxia inducible factor, HIF-1α dynamics throughout the domain. The present observation of (48, 49), and the kinase inducible domain of CREB (50). NCBD, a discrete folded state in this ensemble suggests that the dynamics by contrast, forms a compact state with extensive tertiary inter- might be caused by interconversion between discrete folded actions both in the free state and in its ligand bound states. While conformations similar to the multiple folded side chain confor- an extended random coil structure is needed to wrap around the mations observed in the molten globule state of α-lactalbumin binding partner, a molten globule ensemble of interconverting (9). Only one set of peaks were observed for NCBD, and at folded structures provides a target for conformational selection 31 °C the exchange broadening seen at lower temperature is re- by diverse ligands, so there may be a connection between the reduced. This observation suggests that the slowest exchange rates sidual structure in the free state and the architecture of the bound have rate constants just above the intermediate exchange regime, 4 −1 state, where the level of residual structure found in the free state i.e., with rate constants on the order of 10 s . This rate is on the is determined by the architecture of the final complex. same time scale as the folding of ultrafast folding small domains (39, 40), suggesting that NCBD has time for a complete rearrange- Materials and Methods ment of its tertiary structure. This picture contradicts the classical CD Spectroscopy and Denaturation Studies. All CD spectra were recorded using view of molten globules as having a liquid-like tertiary structure, a Jasco J-810 spectrometer using a 1 mm path length cuvette and a band- which implies a continuous distribution of partially folded states. width of 1 nm. All samples contained 150 mM NaCl and 20 mM phosphate Despite populating compact folded conformations NCBD still pH 7.4 and 16 μM protein and spectra were recorded at 5, 15, 25, 35, and has a slowly fluctuating tertiary structure and thus adheres to 45 °C. The concentrations of urea stock solutions were determined using the definition of a molten globule. the refractive index as described previously (51). The urea denaturation curves were fitted to a two-state transition with sloping base lines using the equation from (52) using m-values as global parameters at each tempera- Implications for Binding Mechanisms of IDPs. Two competing models ture. A temperature scan of the CD signal at 222 nm was recorded from have been proposed for protein association reactions where the 5–95 °C using a scan speed of 1 °C∕ min. binding is accompanied by a large structural change: induced fit and conformational selection. In the induced fit mechanism, the Small Angle X-ray Scattering. Samples for SAXS contained 150 mM NaCl, protein associates with the ligand and the conformational change 20 mM phosphate buffer pH 7.4 and either 1 M TMAO, no additives, 2 or happens after the association (41). The conformational selection 4 M urea. For each condition, three concentrations of NCBD were measured mechanism predicts that the bound-state conformation exists ranging from 4 mg∕mL to 1 mg∕mL. SAXS data were recorded at the Eur- in the free ensemble, and the ligand selectively associates with opean Molecular Biology Laboratory X33 beamline Hamburg using an expo- the fraction of the ensemble that is prefolded into the bound con- sure time of 2 min. As the data did not show any concentration dependent effects, we used the highest concentration for further analysis. Each of the formation (42). While the induced fit has been the dominating curves was used as input for the ensemble optimization method (25). The paradigm for decades, recent NMR experiments have shown that algorithm was only allowed to run for 50 generations, which was the point conformational selection plays an important role in enzyme cat- where the χ2 did not improve any further. For each scattering curve 500 alysis (43–45) and protein association reactions (46). Our data ensembles of 20 structures were selected and the distribution of radii of clearly demonstrate that within the molten globule ensemble gyration and maximal distances were extracted (Fig. 2).

Kjaergaard et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 29, 2021 NMR Spectroscopy. Lyophilized NCBD was resuspended into 20 mM phos- The network anchoring algorithm of this program was allowed to alter phate in 10% D2O and adjusted to pH 6.5 to a final concentration of the initial assignments in eight iterative cycles of structure calculation and ε ¼ 1;490 2 mM estimated by the absorbance of a single tyrosine ( 280 nm ). NOE assignment. 39 sets of ψ and ϕ torsion angle restraints generated using All spectra were recorded at 31 °C on Varian Unity Inova 750 or 800 MHz TALOS were included in the calculation. NMR spectrometers equipped with a room temperature or cryo probe, respectively. NMR spectra were processed using NMRPIPE (53) and analyzed in CCPNMR Analysis (54). Initial backbone assignments were adapted from ACKNOWLEDGMENTS. We thank the staff at the X33 beam line at EMBL BMRB entry 15398 using a HNCACB spectrum. Side chain assignments were Hamburg for help with the acquisition of the SAXS data and Peter E. Wright derived from HSQC tocsy, HCCH-tocsy, HccoNH and CcoNH spectra. Chemical (The Scripps Research Institute) for generously sharing the coexpression shift assignments were deposited in the BMRB database (16363). 15N-NOESY plasmid and for his help and constructive criticism of this study. We thank HSQC, 13C-NOESY HSQC and 13C-HSQC-NOESY-15N-HSQC were recorded with Penny von Wettstein-Knowles for critically reviewing the manuscript of this a mixing time of 100 ms. NOEs originating from amides and well resolved paper. This work was supported by the EliteForsk programme (to M.K.), The methyl groups were assigned interactively. Where the assignments were am- John and Birthe Meyer Foundation, the Carlsberg Foundation Grant 2008-01- biguous, no assignments were made. Manually assigned NOESY peak lists and 0368 and The Danish Natural Research Council Grants 272-08-0500 (to F.M.P) chemical shift list were used as input for a structure calculation in ARIA2.2. and 272-06-0251 (to K.T.)

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