Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions
Raj Kumar and
E. Brad Thompson
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, Texas 77555
Address all correspondence and requests for reprints to: E. Brad Thompson, Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, 301 University Boulevard, 605 Basic Science Building, Galveston, Texas 77555-0645. E-mail: bthompso{at}utmb.edu.
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ABSTRACT
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The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.
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INTRODUCTION
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IT IS GENERALLY acknowledged that the functions carried out by proteins depend upon the structures of those proteins. Thus, finding the structure of a protein can give important information about its function. As libraries of structures accumulate, solving a structure can lead to accurate prediction of its function. Often, parts of proteins expressed as independent domains form well folded, functional structures. This has been especially useful in studying the DNA-binding (DBD) and ligand-binding (LBD) domains of several members of the nuclear hormone receptor (NHR) family. It is known that the primary amino acid sequence of a protein or even a domain carries all the information necessary for that sequence to reach a fully folded structure (1). In recent years, however, it has become clear that in many proteins, significant functional regions contain amino acid sequences that do not automatically fold into their fully condensed, functional structures (2, 3, 4, 5). Long stretches of amino acids are likely to be either unfolded in solution or to exist as large steady-state arrays of nonglobular structures of unknown conformation. Yet it is generally believed that such natively unfolded sequences must achieve structure to carry out their functions. Characterization of the conformational propensities and functions of these nonglobular protein sequences represents a major challenge.
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CONFORMATION OF THE N-TERMINAL TRANSACTIVATION DOMAIN OF NHRs
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Striking among proteins with unfolded regions are a number of transcription factors, and among these, the NHRs (6, 7, 8, 9, 10, 11, 12, 13). In many cases, the unfolded or partially folded regions of such proteins take shape when the proteins interact with their proper binding partner(s), the molecules to which they must bind to carry out their functions (4, 10, 14, 15, 16). The N-terminal domain (NTD) of many members of the NHR family contains important functions responsible for regulation of transcription. These NTDs subsume smaller activation function (AF) regions. To distinguish them from the well known AF2 domain at the C terminus of the LBD of the NHRs, the NTD AFs are termed AF1, AF3, etc. In at least one NHR, an inhibitory function has also been found in the NTD (17). The AF1 (tau 1/enh2) domains of the glucocorticoid and some other steroid hormone receptors were discovered well before AF2 and, for a time, were thought to be the only transactivation function in these receptors (18, 19, 20, 21). Because of the recent focus on AF2, which has been greatly enhanced by the ability to solve the three dimensional structure of several receptor-recombinant LBDs (22, 23, 24, 25, 26, 27, 28, 29, 30, 31), and because the NTD AFs appear to be in a natively unfolded state, understanding their function has languished. Despite that, the early work that used molecular mapping structure/function techniques clearly established that in many receptors the NTD AF domains were quite powerful and, indeed in some, appeared to contain a large portion of the transcriptional transactivation activity. For example, mutants of the glucocorticoid receptor (GR) and other steroid hormone receptors truncated so as to contain only the NTD and the DBD were constitutively quite active in stimulating transcription from simple promoters containing their cognate binding sites (32, 33, 34). Constitutive activity may be cell/coactivator dependent (34). Table 1
lists some NHRs in which NTD AFs have been identified by mutational studies. As can be seen from the table, the regions are found both in the members of the steroid hormone receptor family, in which they are well known, and in several other NHRs. We believe it likely that many members of this family that contain a significant amino acid sequence N-terminal from the DBD have therein some AF activity.
The original model of the NHRs was that, once bound to DNA, they somehow directly contacted the nonspecific transcription machinery composed of the body of proteins found at the TATA box and the RNA polymerase complex. It is now clear that this simple model is insufficient to explain all data. In particular, the NHRs appear to contact directly or indirectly, through molecular bridges, a variety of other proteins, including general and receptor-specific coactivator and corepressor proteins as well as other transcription factors (41, 42, 43, 44, 45, 46, 47, 48, 49). We currently presume that, ultimately, some of the proteins forming the molecular bridges physically reach and regulate the primary transcription machinery. In addition, the bridges contain proteins that alter chromosome structure so as to enhance or repress transcription by changing the accessibility of the specified DNA region to the general transcription machinery. What has made the NTD AF regions difficult to understand functionally is that, unlike the DBDs and LBDs of the nuclear receptor family, the AFs of the NTDs do not closely resemble one another in sequence, exact location, or size. An additional major obstacle has been that when the AFs of the NTD appear expressed as independent proteins, they have failed to show significant structure. Whether the NTD AFs in their natural locations in holoreceptors utterly lack consistent structure is unknown. However, where they have been studied as free peptides, the NTD AFs are in random coil configuration (6, 7, 10, 14). That is to say, they are a large collection of forms without any clear dominant secondary or tertiary features. In the few cases in which NTD AFs have been studied in a two-domain molecule involving the NTD connected to a DBD, slightly more structure was seen, but clearly the AFs were not fully structured. This was found to be so for such two-domain peptides derived from the progesterone recptors (PRs) and GRs (8, 35).
A central dogma of biochemistry is that proteins must have structure to carry out their proper functions. Application of predictive algorithms for secondary structure suggests that in several NHRs there may be some helical structure in the NTD AFs (6, 10). Consistent with such predictions, in the presence of 30% trifluoroethanol, some helices formed in the GR AF1 (6). Putative helices were also suggested as important for the NTD AF function of the estrogen receptor (ER) (50). Nuclear magnetic resonance studies with the shorter AF1 core (AFc) of the GR in 40% trifluoroethanol showed three helices, and in independent experiments, helix-breaking amino acid substitutions in the helical regions interfered with function (6). Thus, some functional secondary structure was suggested, but three-dimensional structure was lacking. Other data suggested that the GR AF1 could weakly bind in solution with the TATA box binding protein (TBP), Ada2 proteins, and the general coactivator CBP [cAMP response element binding protein (51, 52, 53)]. The latter result was extended by recent studies of the presumptive helix-containing region in the GR AF1, showing that it can interact with a fragment of CBP lacking its N-terminal region, which has been mapped as important for interaction with AF2 (53). Thus, AF1 and AF2 may both bind CBP but differently, and conceivably, simultaneously. Constructs containing sequences coding for even one of the three potential helices of the GR AF1 could bind the CBP fragment; combinations with the potential for expressing two or three helices could activate transcription from a promoter-reporter construct in a yeast system (54). The contributions of specific key amino acids of the GR AF1 to its full functional structure(s) remain unclear. The core region (AFc) was defined by mutations in AF1 followed by transfections of the mutant GR and measurements of their ability to stimulate transcription from cotransfected promoter-reporter constructs. Despite this empiric, functional definition of an AFc, it is clear that mutations involving amino acids outside the core area can cause significant loss of activity (55, 56, 57). It is worth noting that most, if not all, of the analyses of AF1 function have used promoter-reporter constructs. These certainly are valuable, and they show what can happen. Tests of actual genes in chromatin will be essential to prove what actually does occur.
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MODELS FOR AF1 FOLDING
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Protein-Protein Interactions
Thus, the NTD AFs pose a paradox that must be solved before we can fully understand the functions of NHRs. How can these partially or fully unfolded regions carry out their transcription transactivation functions? Hypothetically, this paradox may be resolved in several ways. The first hypothesis is that the AF1 never needs to be well structured, but simply to present a cluster of charges, which is sufficient to activate the transactivation function (58, 59, 60, 61, 62). Alternatively, a cluster of hydrophobic residues could nucleate interactions with other proteins. In the case of the GR AF1, this hypothesis seems to have been ruled out by experiments in which the charged amino acids were systematically mutated without affecting the AF1 transcription function (56). Mutations of hydrophobic amino acids of the GR AF1, on the other hand, strongly reduced the domains activity (63). A second hypothesis states that the act of binding one or more of its cognate partners causes the AF domain of the NTD to fold. This induced-fit model assumes that nonspecific initial binding due to random interactions between the binding partner (BP) and the AF domain produces a rapid shift in structure of the AF, leading to collapse of the molecule into the correct functional shape and enhanced, specific AF1-BP binding. An alternative way of looking at this same concept is the selected-subset model: among the large number of random forms in which the AF is found, some are folded into structures that contain the specific binding site for particular BPs. When sufficient quantities of BP are in proximity to the properly folded subpopulation of AF, they bind with high affinity to these and thus, by mass action, shift the remainder of the AF1 into the fully formed functional AF-BP structure. Thermodynamically the pure induced-fit and the selected-subset models are equivalent (Fig. 1
). This idea of an unfolded domain in a protein is one consistent with findings in other proteins, particularly transcription factors. Table 1
includes as examples of non-NHRs, two transcription factors in which naturally unfolded or natively unfolded activation domains have been described. It is presumed that the multiarray of forms or states in which such domains exist allows for important recognition events to take place. Since thermodynamic principles that govern protein folding show that it is possible for fully folded forms of the same protein with varying degrees of stability to exist (64, 65), one could propose an explanation for the multiple proteins with which such domains can interact by assuming that they occur due to the choice or direction of folding imposed by the initial contacts in the induced-fit model or the selection of appropriate forms by the folded-subset model. This concept predicts that differing final folded forms of AF will be found, depending on the BP involved. On the other hand, if a single predominant fold is found, various BPs will have to be accommodated by various specific interactions with the folded AF surfaces. The behavior of the NTD AF domains also emphasizes the concept of protein flexibility. Although we are accustomed to seeing static structures for proteins as they are shown as solved in their crystal forms or diagrammed on the pages of journals and books, in fact nuclear magnetic resonance data as well as calculations and other direct methods indicate that proteins are flexible molecules (66). In this sense the unfolded domain represents only one end of the spectrum of protein flexibility.

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Figure 1. A Hypothetical Model of AF1 Domain Folding and Recruitment of Cofactors
A, AF1 exists in equilibrium as an unfolded (AF1U) and properly folded (AF1N) conformation. Only AF1N can make physiologically functional interaction with cofactor BP. In the absence of BP, the equilibrium favors AF1U. B, The BP, by binding the small pool of AF1N, shifts the equilibrium to AF1N: BP. C, Alternatively, AF1U could interact nonspecifically at first with BP. These initial interactions could facilitate the folding of AFU directly to the heterodimer AF1N: BP. Both pathways are thermodynamically feasible. (Based on Ref.36 .)
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It is important to discover the BPs, which make physical and functional interactions with NTD AFs, and are either required or at least modulate the effect of NHRs on target gene transcription (51, 52, 53, 67, 68, 69, 70, 71, 72). Much progress has been made on this issue. These AF-bound BPs appear to function by either remodeling chromatin structure and/or acting as adapter molecules between the NHR and components of the basal transcription apparatus. Studies from several laboratories have identified a series of NHR NTD interacting proteins (14, 44, 51, 52, 53). The list also includes certain RNAs (73). The unfolded transactivation domains of a number of transcription factors have been shown recently to undergo a transition to a folded state upon interaction with either components of the general transcription machinery or with coactivators (4, 10, 14, 15, 16). Such transitions include members of the NHR superfamily, i.e. the ER (14), androgen receptor (AR) (10), and GR (our unpublished data). The TBP is reported to be a BP for the GR AF1 domain (36, 52, 53), and our structural data (unpublished) show that interaction of TBP with the GR AF1 induces secondary structure in the AF1. In the case of the AR, the transcription factor TFIIF showed similar effects (10). In the case of the ER
also, TBP is found to be a target for its NTD, although the interaction is weak (Kd = 10-6 to 10-5 M). The results further show that TBP binding induces and/or stabilizes ordered structure in otherwise intrinsically unstructured ER NTDs (14). These results clearly indicate that NTD AF-BP interactions/binding induces (induced-fit model) or selects for (subset selection) a structured AF domain.
Ultimately, function requires structure, and it will be necessary to determine the folded structure or structures of these NTD AF domains to fully understand and eventually modulate and control their function. It remains to be seen whether one or a few particular "folds" will be found, despite the varied size and sequences of the NTD AFs. Also to be determined is whether specific BPs will induce or select particular AF foldsor whether various surfaces of a single fold are used to accommodate the binding of multiple BPs. These results will be important for the general understanding of how these molecules regulate transcription and for the development of BP-like ligands that may modulate the activity. In addition to the binding of coactivators and corepressors, posttranslational modifications of receptors may contribute to the structure of the NTD AFs. Phosphorylations are well known to affect activity of certain steroid hormone receptors, and many of the phosphorylated amino acids are in the NTDs (74). In addition, interactions between the LBDs and the NTDs of several receptors have been suggested by mutational studies (75, 76, 77, 78). Studies show that when expressed in continuity with the DBD as a two-domain fragment, the NTD of the PRA shows resistance to proteolytic digestion (8). Thus, the PRA NTD AF region is influenced by the surrounding peptide context, specifically the DBD. DNA binding, however, strongly stabilizes the AF region of both PRA and PRB (8, 9).
Site-Specific DNA Binding
Experiments with a similar two-domain construct from the human GR have carried this result further by showing that significant tertiary structure is acquired in the NTD when the DBD is bound to a DNA sequence containing the cognate palindromic glucocorticoid response element (GRE). Furthermore, once this two-domain portion of the GR is bound to the GRE, it can now strongly bind the ubiquitous coactivator CBP, as well as TBP (our unpublished data). A recent study proposes that binding of the ER to its response element can regulate the structure and biological activity of the receptor and influences the recruitment of coactivators to the ER at the target gene promoters (79).
These results suggest that we should reevaluate the consequences of site-specific DNA binding of these receptors. The classic models state that the DNA binding of the NHRs is a simple tethering event the function of which is to place them at the correct site in the regulatory regions of genes so that they may carry out their transactivation function. However, since binding to a GRE causes the AF1 to take shape and capability of binding specific BPs, the DBD-RE binding represents an active event in which intramolecular forces cause folding and confer function. The AF1 of the GR is approximately 200 amino acids removed from the DBD. It is well known that mutations in proteins at positions remote from functional sites can alter the structure at those sites. Thus such long-range structural consequences are not unfamiliar. In this case, however, not a mutation but a binding event causes the change. One conjecture as to how this happens is that the AF1 domain loops back and makes physical contact with the DBD. Another would be that no such contact is required, i.e. that intramolecular forces do the job. The resolution of these models will require future experiments.
Osmolyte-Induced Folding
A second important way in which functionally relevant structure can be caused to occur or be stabilized in AF domains is by use of osmolytes. Osmolytes are small molecules of several classes that are widely used in nature to promote or maintain protein folding. They are seen in creatures that are exposed to desiccation, denaturing chemicals, or other extremes that could threaten critical proteins with loss of structure and function or even outright denaturation. Osmolytes induce folding not by affecting the amino acid side chains, but as a result of their solvophobic effects on the backbone (80, 81, 82). Because osmolytes act by affecting the peptide backbone, they can provide an additional force for protein folding, while allowing the natural hydrophobic forces that drive protein folding to occur unhindered. Because the protein backbone comprises the most numerous functional groups in proteins, osmolyte-induced conformations are driven by very strong forces, and evidence from the several systems studied thus far indicates native folded functional species result (7, 36, 83, 84, 85). Reviews of the nature of osmolytes and how they promote folding without interfering with the normal forces involved are available (82, 86, 87, 88). It was shown recently that use of the osmolyte trimethylamine-N-oxide can result in folding of the AF1 domain of the GR (36). This folding was shown to have the characteristics of natural protein folding, which is a cooperative transition from an unfolded to a folded state. In the folded state the AF1 peptide resisted proteolysis and showed greatly enhanced binding to CBP, TBP, and a member of the steroid receptor coactivator 1 (SRC1) family. Two of these proteins had been previously shown to bind weakly to unfolded AF1. The thirdSRC1had not been detected. In the presence of quantities of trimethylamine-N-oxide sufficient to cause folding, there was strong binding in all cases. The generality of these results has recently been confirmed in very similar studies carried out on the AF1 of the androgen receptor (10). It seems that there are now tools that allow investigation of AF1 structure and function. In addition, the results with the PR and GR two-domain proteins (NTD + DBD) suggest that the standard model of DNA binding and its effects on NHRs need to be rewritten. We propose that the NTD AF1 domains are placed in a "cocked" structural condition by the act of the DBD binding to its cognate site (Fig. 2
). A corollary of this hypothesis is that slight variations in response elements could affect the nature of the fold and therefore the function of the NTD AFs. Data have already begun to emerge that this is so (79, 89, 90). When the probable influences of the LBD and its AF2 on NTD AFs are added (75, 76, 91, 92), the combined influences of specific ligands and variability in response element sequences may affect the structure assumed by NTD AFs. This may influence the choices of cofactors that the NTD AFs bind and consequently affect their function. Obviously the cell type-specific expression of sets of cofactors and their intracellular concentrations and locations all would be important influences as well. Furthermore, since it is now known that the NHRs may be tethered to DNA sites by way of other transcription factors, it is possible that additional configurational changes in the NTD AFs could be occurring by way of those connections (Fig. 3
). These would not involve direct DNA binding of the NHR and consequently may imply differing structures for the NTD AFs.

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Figure 2. A Model for the Regulation of NHR Through the Recruitment of Cofactor Assemblies
The NTD AF1 recruits certain specific cofactors (BP1-n). AF2 in the LBD also interacts with the same or different specific cofactors (e.g. BP2). A bridge between AF1 and AF2 is formed by the assembly of additional cofactors (BPx). (AF1 and AF2 may also interact directly, indicated by the dashed line.) These interactions may be influenced by posttranscriptional modifications, e.g. phosphorylation or DNA binding of receptor and/or cofactor(s). Cell-specific expression of cofactors and specific NHR ligands influence the nature of the AF1 and AF2 interactions. In turn, this probably results in varying functional interactions with the basal transcription machinery to initiate transcription. RE, Response element; HRE, hormone response element.
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Figure 3. A Model for the Regulation of NHR Through Other Transcription Factors (TFs)
The specific TF bound to its site-specific DNA can physically interact with the NHR and can regulate transcription by the NHR.
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CROSS-TALK BETWEEN AF1 AND AF2
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Although in many cases either AF1 or AF2 is capable of regulating transcription alone, full transcriptional activation by NHRs requires functional synergy between the two transactivation function regions. One possibility is that this is achieved through a physical intramolecular association between the NTDs and LBDs. This has been supported by studies with PR-B, in which a direct interaction between the N-terminal and C-terminal domains has been shown (75). Interestingly, this interaction is ligand dependent. Such an interaction takes place only when an agonist is bound and antagonist binding prevents it, suggesting a conformational change taking place in the agonist-bound LBD, which is well suited for this interaction. Furthermore, coactivators such as SRC-1 and CBP/p300 are not required for this physical association, although they are capable of enhancing a functionally productive interaction. Direct interactions between the NTDs and C-terminal domains of the AR have also been reported. The FXXLF core sequence in the AR NTD binds the AF2 subdomain, whereas another motif WXXLF binds to the LBD outside AF2 (93). These interactions are ligand dependent. It is also reported that these WXXLF and FXXLF motifs inhibit recruitment of transcriptional intermediary factor 2 (TIF2) by the AR AF2 subdomain (94). It has been suggested that agonist-dependent interaction of the AR NTD and AF2 contributes to stabilization of helix 12 to slow down ligand dissociation (95). A second possibility is that AF1 and AF2 interact via mutual binding of other proteins, such as coactivators. The AR AFs interactions are also suggested to be a prerequisite for the efficient recruitment of coactivators, and for its transcriptional activity (96). Molecular genetic functional studies on peroxisome proliferator activator and ERs have also suggested avenues of intramolecular signaling between the LBD and the amino-terminal domains (77, 78). Mutations of certain amino acids in the LBD of the ER and deletion of amino acids from the N-terminal region resulted in altered transactivation activity, suggesting the possibility of intramolecular signaling between the two domains. The synergy between the ER AF1 and AF2 is reported to be due to cooperative recruitment of members of p160 coactivators (97). Similar studies with peroxisome proliferator receptor showed that mutations in the N-terminal region modulate ligand binding by altering the conformation of the unliganded receptor. These conformational changes have been correlated with the interaction of the receptor with cofactors such as silencing mediator of retinoid and thyroid hormone receptor, a further support of intramolecular signaling between the N-terminal and C-terminal part of the receptor.
It has been reported that SRC-1 is able to interact with both the AF1 and AF2 regions of several steroid receptors. Differing regions of SRC-1 seem to bind to the two AF sites. This type of bridge protein interaction has been observed between AF1 and AF2 regions and appears to be assisted by other cofactor proteins as well, such as TIF2 (42). Another coactivator complex, thyroid hormone receptor-associated protein/vitamin D receptor interacting protein, is reported to interact with both AF1 and AF2 regions in several of these receptors (69). Similar observations of synergism between AF1 and AF2 are also observed between CBP/p300 and ER (91). A recent study shows that TIF2 is able to bridge the two AFs of the retinoic acid receptor-
1, which results in synergistic transactivation of the receptor (98).
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FOOTNOTES
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This work was supported by grants from the NIH (NIDDK 1RO1-DK-58829; to E.B.T.) and the Muscular Dystrophy Association (to R.K.).
Abbreviations: AF, Activation function; AR, androgen receptor; BP, binding partner; CBP, cAMP response element binding protein; DBD, DNA binding domain; ER, estrogen receptor; GR, glucocorticoid receptor; GRE, glucocorticoid response element; LBD, ligand-binding domain; NHR, nuclear hormone receptor; NTD, N-terminal domain; PR, progesterone receptor; SRC, steroid receptor coactivator; TBP, TATA box binding protein; TIF, transcriptional intermediary factor.
Received for publication July 23, 2002.
Accepted for publication September 23, 2002.
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