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Thermodynamic Dissection of the Intrinsically Disordered N-terminal Domain of Human Glucocorticoid Receptor*

  • Jing Li
    Affiliations
    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

    T. C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218
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  • Hesam N. Motlagh
    Affiliations
    T. C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218
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  • Carolyn Chakuroff
    Affiliations
    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218
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  • E. Brad Thompson
    Affiliations
    Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5056

    Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1068
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  • Vincent J. Hilser
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

    T. C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218
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  • Author Footnotes
    * This work was supported by National Science Foundation Grants MCB-0446050 (to V. J. H.) and DBI-1005027 (to C. C.).
    This article contains supplemental Figs. 1 and 2, Table S1, and Appendix.
Open AccessPublished:June 04, 2012DOI:https://doi.org/10.1074/jbc.M112.355651
      Intrinsically disordered (ID) sequence segments are abundant in cell signaling proteins and transcription factors. Because ID regions commonly fold as part of their intracellular function, it is crucial to understand the folded states as well as the transitions between the unfolded and folded states. Specifically, it is important to determine 1) whether large ID segments contain different thermodynamically and/or functionally distinct regions, 2) whether any ID regions fold upon activation, 3) the degree of coupling between the different ID regions, and 4) whether the stability of ID domains is a determinant of function. In this study, we thermodynamically characterized the full-length ID N-terminal domain (NTD) of human glucocorticoid receptor (GR) and two of its naturally occurring translational isoforms. The protective osmolyte trimethylamine N-oxide (TMAO) was used to induce folding transitions. Each of the three NTD isoforms was found to undergo a cooperative folding transition that is thermodynamically indistinguishable (based on m-values) from that of a globular protein of similar size. The extrapolated stabilities for the NTD isoforms showed clear correlation with the known activities of their corresponding GR translational isoforms. The data reveal that the full-length NTD can be viewed as having at least two thermodynamically coupled regions, a functional region, which is indispensable for GR transcriptional activity, and a regulatory region, the length of which serves to regulate the stability of NTD and thus the activity of GR. These results suggest a new functional paradigm whereby steroid hormone receptors in particular and ID proteins in general can have multiple functionally distinct ID regions that interact and modulate the stability of important functional sites.

      Introduction

      The classic view of protein structure-function relationships has been challenged by the increasing number of proteins, particularly cell signaling proteins and transcription factors, found to contain intrinsically disordered (ID)
      The abbreviations used are: ID
      intrinsically disordered
      GR
      glucocorticoid receptor
      NTD
      N-terminal domain
      TMAO
      trimethylamine N-oxide
      AF1
      activation function-1 domain
      R
      regulatory
      F
      functional.
      regions (
      • Tantos A.
      • Han K.H.
      • Tompa P.
      Intrinsic disorder in cell signaling and gene transcription.
      ,
      • Liu J.
      • Perumal N.B.
      • Oldfield C.J.
      • Su E.W.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in transcription factors.
      ,
      • Tompa P.
      Intrinsically unstructured proteins.
      ,
      • Uversky V.N.
      Intrinsically disordered proteins from A to Z.
      ). Many of these ID regions have binding sites for protein partners, and folding coupled with binding often occurs when these regions encounter such partners (
      • Tantos A.
      • Han K.H.
      • Tompa P.
      Intrinsic disorder in cell signaling and gene transcription.
      ,
      • Uversky V.N.
      Intrinsically disordered proteins from A to Z.
      ,
      • Dyson H.J.
      • Wright P.E.
      Intrinsically unstructured proteins and their functions.
      ,
      • Fink A.L.
      Natively unfolded proteins.
      ,
      • Dyson H.J.
      • Wright P.E.
      Coupling of folding and binding for unstructured proteins.
      ). Thus, in some cases, the conditionally folded states (which may be transient) are presumed to be among the functional states of ID proteins. Mutation, truncation, and translocation of ID regions have been implicated in a variety of diseases (
      • Dyson H.J.
      • Wright P.E.
      Intrinsically unstructured proteins and their functions.
      ,
      • Babu M.M.
      • van der Lee R.
      • de Groot N.S.
      • Gsponer J.
      Intrinsically disordered proteins: regulation and disease.
      ,
      • Midic U.
      • Oldfield C.J.
      • Dunker A.K.
      • Obradovic Z.
      • Uversky V.N.
      Unfoldomics of human genetic diseases: illustrative examples of ordered and intrinsically disordered members of the human diseasome.
      ). However, the mechanisms by which these ID regions regulate protein functions are largely unknown. Osmolyte-induced folding of some ID proteins has been reported, and the induced folded states were determined to be functionally relevant (
      • Kumar R.
      • Lee J.C.
      • Bolen D.W.
      • Thompson E.B.
      The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins.
      ,
      • Baskakov I.
      • Bolen D.W.
      Forcing thermodynamically unfolded proteins to fold.
      ). These results pave the way for more quantitative studies on the role of folding and stability of these ID regions in mediating function. However, the folding cooperativity is not well characterized (
      • Auton M.
      • Bolen D.W.
      Predicting the energetics of osmolyte-induced protein folding/unfolding.
      ) nor is the degree to which folded states of ID proteins resemble their folded globular counterparts. Although it is well known that amino acid composition of ID proteins differs significantly from that of globular proteins (
      • Tompa P.
      Intrinsically unstructured proteins.
      ,
      • Uversky V.N.
      Intrinsically disordered proteins from A to Z.
      ), the level at which these differences are manifested is not known. Are the folded states of ID sequence qualitatively different from globular proteins, or can the folded, native states of ID proteins be understood in terms of the same thermodynamic principles that describe globular proteins?
      To address these questions, we carried out thermodynamic characterization of the ID N-terminal domains (NTDs) of three human glucocorticoid receptor (GR/Nr3c1) translational isoforms. GR is a hormone-dependent nuclear transcription factor in the steroid hormone receptor family that contains three modular domains: the ID NTD (GR 1–420), the DNA binding domain (GR 421–486), and the ligand binding domain (GR 528–777). The ID NTDs for steroid hormone receptors are extremely important for transcription regulation, serving as hubs to recruit co-regulators that form the final transcription complex (
      • Griekspoor A.
      • Zwart W.
      • Neefjes J.
      • Michalides R.
      Visualizing the action of steroid hormone receptors in living cells.
      ,
      • McEwan I.J.
      • Lavery D.
      • Fischer K.
      • Watt K.
      Natural disordered sequences in the amino terminal domain of nuclear receptors: lessons from the androgen and glucocorticoid receptors.
      ,
      • Lavery D.N.
      • McEwan I.J.
      Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.
      ). Within the human GR NTD, mutational mapping has identified a subregion, termed the activation function-1 (AF1) region, which comprises residues 77–262 and is essential for the full transcriptional activity of the receptor (
      • Hollenberg S.M.
      • Evans R.M.
      Multiple and cooperative trans-activation domains of the human glucocorticoid receptor.
      ). AF1 regions are found in all steroid hormone receptors but differ widely in size and primary sequence (
      • Lavery D.N.
      • McEwan I.J.
      Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations.
      ). Although constructs that lack NTD residues outside of the AF1 region are able to stimulate transcription in the context of simplified promoter-reporter systems (
      • Dahlman-Wright K.
      • Almlöf T.
      • McEwan I.J.
      • Gustafsson J.A.
      • Wright A.P.
      Delineation of a small region within the major transactivation domain of the human glucocorticoid receptor that mediates transactivation of gene expression.
      ), several pathogenic mutations have been mapped to these non-AF1 regions (
      • Bray P.J.
      • Cotton R.G.
      Variations of the human glucocorticoid receptor gene (NR3C1): pathological and in vitro mutations and polymorphisms.
      ), indicating that sequences outside AF1 do indeed play a role in maintaining or tuning function. This has also been demonstrated recently with the identification of several human GR NTD translational isoforms differing only in the lengths of their ID NTDs (
      • Lu N.Z.
      • Cidlowski J.A.
      Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.
      ) (see Fig. 1). These isoforms have varying activities, different tissue distributions, unique gene regulation sets and have been shown to be derived from a single GR mRNA through well known translational regulatory mechanisms (
      • Lu N.Z.
      • Cidlowski J.A.
      Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.
      ). With the exception of the GR isoforms that truncate the entire AF1 region and thus presumably ablate co-activator binding, all other isoforms are active and possess different potencies in transcription regulation (
      • Lu N.Z.
      • Cidlowski J.A.
      Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.
      ). Especially intriguing is the observation (discussed below) that the deletion of as few as eight amino acids results in an almost 2-fold increase in activity (
      • Lu N.Z.
      • Cidlowski J.A.
      Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.
      ).
      Figure thumbnail gr1
      FIGURE 1Schematic representation of the GR NTD sequence showing the positions of different GR translational isoform start sites (
      • Lu N.Z.
      • Cidlowski J.A.
      Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes.
      ). The N-terminal domains of the A (starting at position 1), C2 (starting at position 90), and C3 (starting at position 98) isoforms correspond to the constructs GR A-NTD, GR C2-NTD and GR C3-NTD, respectively (shown within squares). The transactivation domain AF1 (GR 77–262) is shown in gray.
      Here we investigate the thermodynamic basis for these results by examining the ID NTDs of three representative human GR translational isoforms: GR A-NTD, GR C2-NTD, and GR C3-NTD, which correspond to GR 1–420, GR 90–420, and GR 98–420 in the full-length GR, respectively (Fig. 1). To determine the stability of the folded conformations of these ID proteins, the naturally occurring protective osmolyte trimethylamine N-oxide (TMAO) was used to induce their unfolded to folded transitions. Protective organic osmolytes are small molecules in cells that function to stabilize and protect intracellular proteins against commonly occurring denaturing environmental stresses (
      • Bolen D.W.
      • Baskakov I.V.
      The osmophobic effect: natural selection of a thermodynamic force in protein folding.
      ,
      • Bolen D.W.
      Protein stabilization by naturally occurring osmolytes.
      ). TMAO has been demonstrated to force thermodynamically unstable proteins to fold and regain high functional activities (
      • Kumar R.
      • Lee J.C.
      • Bolen D.W.
      • Thompson E.B.
      The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins.
      ,
      • Baskakov I.V.
      • Kumar R.
      • Srinivasan G.
      • Ji Y.S.
      • Bolen D.W.
      • Thompson E.B.
      Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor.
      ,
      • Kumar R.
      • Baskakov I.V.
      • Srinivasan G.
      • Bolen D.W.
      • Lee J.C.
      • Thompson E.B.
      Interdomain signaling in a two-domain fragment of the human glucocorticoid receptor.
      ) and thus is an ideal compound to study folding/unfolding reactions.
      In summary, for each isoform, we found a TMAO-induced cooperative folding transition to an apparent globular protein-like folded conformation. Our results demonstrate that the GR NTD contains at least two thermodynamically coupled but functionally distinct regions, one regulatory (R) and the other functional (F). The use of alternative translation start sites in cells to vary the length of the R region results in GR translational isoforms of varying stability and activity. We show that this activity is correlated to the stability of each isoform, i.e. to its inherent propensity to fold.

      DISCUSSION

      The results presented here show that thermodynamic coupling does exist between different regions of the disordered NTD of GR, allowing truncation of the extreme N terminus to modulate the stability of the functionally important AF1 region when the NTD is studied in isolation. Of course, how this stability tuning is manifested at the level of the full-length GR (which also contains the DNA binding and ligand binding domains) remains an open question. Nonetheless, the results presented here provide significant insight into the organizing principles describing how changes in one domain can affect structure, stability, and activity in other domains, principles that in many ways are clearer when presented in the context of allosteric communication in ID proteins.
      For more than 40 years, allostery has been described in terms of two classic models: Monod, Wyman, and Changeaux (
      • Monod J.
      • Wyman J.
      • Changeux J.P.
      On the nature of allosteric transitions: a plausible model.
      ) and Koshland, Némethy, and Filmer (
      • Koshland Jr., D.E.
      • Némethy G.
      • Filmer D.
      Comparison of experimental binding data and theoretical models in proteins containing subunits.
      ). Both models describe the quantitative relationship between ligand binding at two different coupled sites. A fundamental limitation of both models, however, is that they provide little insight into the energetic determinants for “how” the coupling is facilitated. Are there ground rules that determine whether binding at one site can facilitate an affinity change at the second site? In other words, are there quantitative energetic relationships that must exist to propagate signal from one site to another?
      Recently, this question was addressed by recasting allostery in terms of the ensemble of the high and low affinity states for each domain (
      • Hilser V.J.
      • Thompson E.B.
      Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins.
      ,
      • Motlagh H.N.
      • Hilser V.J.
      Agonism/antagonism switching in allosteric ensembles.
      ). The results revealed that allosteric phenomena (at least those under thermodynamic control) are a manifestation of a set of energetic ground rules that govern whether conformational changes can be propagated to distal sites (
      • Hilser V.J.
      • Thompson E.B.
      Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins.
      ,
      • Motlagh H.N.
      • Hilser V.J.
      Agonism/antagonism switching in allosteric ensembles.
      ). In the context of these ground rules, allostery is determined by (i) the local conformational equilibrium in each region, (ii) the intrinsic ligand affinity at each site, (iii) the ligand concentrations, and (iv) the coupling energies between those local equilibria (
      • Hilser V.J.
      • Thompson E.B.
      Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins.
      ,
      • Motlagh H.N.
      • Hilser V.J.
      Agonism/antagonism switching in allosteric ensembles.
      ). In essence, allostery can be described in terms of the same types of energetic parameters determined in this study.
      An important implication of this realization is the notion that allostery can evolve in systems that lack unique structure just as easily as it can evolve in ordered, folded structures. This observation stands in stark contrast to the classic structural view of allostery that has emerged over the past 50 years of structural biology research (
      • Hilser V.J.
      Biochemistry. An ensemble view of allostery.
      ). According to the structural view, site-to-site coupling results from ligand-induced structural changes that propagate from one site to the other presumably as a series (or pathway) of structural distortions that can be more or less ascertained through an inspection of the high resolution structure. The theoretical as well as experimental realization that simply changing the breadth of conformational distributions without significant changes in average position of atoms can nonetheless affect allostery (
      • Cooper A.
      • Dryden D.T.
      Allostery without conformational change. A plausible model.
      ,
      • Bai F.
      • Branch R.W.
      • Nicolau Jr., D.V.
      • Pilizota T.
      • Steel B.C.
      • Maini P.K.
      • Berry R.M.
      Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch.
      ,
      • Pan H.
      • Lee J.C.
      • Hilser V.J.
      Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble.
      ,
      • Popovych N.
      • Sun S.
      • Ebright R.H.
      • Kalodimos C.G.
      Dynamically driven protein allostery.
      ,
      • Reichheld S.E.
      • Yu Z.
      • Davidson A.R.
      The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor.
      ) reveals an entirely new spectrum of possible regulatory strategies available to proteins. Indeed, the regulatory strategy apparently at play in the GR system reveals an efficient mechanism of “overdesign.” As the stability and activity results show, the full-length GR is designed to operate at less than half of the potential maximum activity (Fig. 5) with activity being increased by the removal of sequence segments.
      Much in the same way that the activity of a folded allosteric protein may be tuned by stabilization of its active conformation (through phosphorylation, binding of ligands, pH changes, etc.), disordered proteins can achieve the same ends by merely removing sequence segments. Interestingly, although seemingly much different from allosteric mechanisms utilized by folded proteins (i.e. a ligand-induced change in the structure of the active site), the ID domain-mediated strategy outlined here reveals the universality of the principles at play in all (thermodynamically controlled) allosteric systems. In the case of allostery within folded proteins, the allosteric ligand binds to the active state of the molecule, thus stabilizing it. For the mechanism described here, the active, folded state of the F region is affected not by mutational changes that directly stabilize the folded state but by removing residues that stabilize the unfolded state (i.e. that destabilize the folded state). In this respect, although the underlying principles governing signaling in folded and ID allosteric systems are the same (i.e. the relative stability of the active state must be increased), ID proteins appear to have capitalized on an alternative approach to achieve those ends. It should be noted, however, that traditional modes of inducing allosteric effects (i.e. binding ligands, post-translational modification, etc.) may also be at play in GR. In fact, recent work has shown that phosphorylation of the AF1 region of the NTD can also be used to tune the activity of GR (
      • Kobayashi Y.
      • Mercado N.
      • Barnes P.J.
      • Ito K.
      Defects of protein phosphatase 2A causes corticosteroid insensitivity in severe asthma.
      ), thus demonstrating that many of the same thermodynamic strategies utilized for signal propagation in folded proteins can also be applied to ID proteins. How classical and ID domain-mediated allosteric strategies combine to explain the complex regulation of GR controlled genes is an intriguing and as yet unanswered question.
      We also note once again that the analysis described here was performed on the isolated NTDs. The DNA binding and ligand binding domains may also be coupled to the R and F regions of the NTD as well as to each other. As described elsewhere, couplings between three or more domains can potentiate a wide range of regulatory strategies that can even involve a conversion from activation to repression and vice versa (
      • Motlagh H.N.
      • Hilser V.J.
      Agonism/antagonism switching in allosteric ensembles.
      ). Thus, an understanding of the energetics of the DNA binding and ligand binding domains and their coupling to the NTD is essential to an understanding of how the functional division within the NTD described here will be manifested in the overall functional response of the protein. These studies are currently underway.
      In conclusion, based on well established criteria for determining cooperativity of folding-unfolding transitions (i.e. insensitivity of the extrapolated thermodynamic parameters to the observables being monitored and the comparability of the m-value with that of a globular protein of similar size), the ID NTDs of three GR translational isoforms were found to cooperatively fold into stable folded structures when induced by TMAO. The folded conformations are thermodynamically similar to folded native states of globular proteins based on m-value comparison (although negative cooperativity within the NTD slightly affects this interpretation). The correlation between the activity of each isoform and the stability of their ID NTDs supports previous conclusions (
      • Kumar R.
      • Lee J.C.
      • Bolen D.W.
      • Thompson E.B.
      The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins.
      ) that the folded state is needed for transcriptional activation.
      Most importantly, our thermodynamic analysis reveals that within GR not all of the ID NTD serves the same functional role. There are at least two evolutionarily conserved, functionally distinct regions, which can be operationally defined as the F and R regions that may vary in their boundaries. For GR, the F and R regions are thermodynamically negatively coupled, a situation that provides a regulatory strategy possibly unique to ID proteins. Truncation of the R region results in a corresponding increase in the stability of the remaining ID NTD and an increase in its transcriptional activity. These results are suggestive of a general paradigm for allosteric control whereby the folding of disordered protein regions can control and be controlled by the folding of other ID regions (
      • Freiburger L.A.
      • Baettig O.M.
      • Sprules T.
      • Berghuis A.M.
      • Auclair K.
      • Mittermaier A.K.
      Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme.
      ,
      • Garcia-Pino A.
      • Balasubramanian S.
      • Wyns L.
      • Gazit E.
      • De Greve H.
      • Magnuson R.D.
      • Charlier D.
      • van Nuland N.A.
      • Loris R.
      Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity.
      ). This strategy provides proteins with a general mechanism that can be utilized within all disordered segments, perhaps answering why ID sequences are found in such abundance in transcription factors and other cell signaling proteins (
      • Liu J.
      • Perumal N.B.
      • Oldfield C.J.
      • Su E.W.
      • Uversky V.N.
      • Dunker A.K.
      Intrinsic disorder in transcription factors.
      ).

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