Structural and functional relationships of the steroid hormone receptors' N-terminal transactivation domain

Abstract

Steroid hormone receptors are members of a family of ligand inducible transcription factors, and regulate the transcriptional activation of target genes by recruiting coregulatory proteins to the pre-initiation machinery. The binding of these coregulatory proteins to the steroid hormone receptors is often mediated through their two activation functional domains, AF1, which resides in the N-terminal domain, and the ligand-dependent AF2, which is localized in the C-terminal ligand-binding domain. Compared to other important functional domains of the steroid hormone receptors, our understanding of the mechanisms of action of the AF1 are incomplete, in part, due to the fact that, in solution, AF1 is intrinsically disordered (ID). However, recent studies have shown that AF1 must adopt a functionally active and folded conformation for its optimal activity under physiological conditions. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF1-mediated gene activation, focusing on AF1 conformation and coactivator binding. We further propose models for the binding/folding of the AF1 domains of the steroid hormone receptors and their protein:protein interactions. The population of ID AF1 can be visualized as a collection of many different conformations, some of which may be assuming the proper functional folding for other critical target binding partners that result in the ultimate assembly of AF1:coactivator complexes and subsequent gene regulation. Knowledge of the mechanisms involved therein will significantly help in understanding how signals from a steroid to a specific target gene are conveyed.

Figure 1. A general schematic representation of a SR showing functional domain arrangements

A/B = N-terminal domain (NTD); C = DNA-binding domain (DBD); D = hinge region (in some cases also known as Tau2); E/F = ligand binding domain (LBD). Not all SRs contain F domain, and this is not required for hormone binding. Precise location and size of each domain may differ in each SR. These differences are most pronounced in the A/B region. A typical SR is composed of several functional domains. The variable N-terminal region (A/B) contains the ligand-independent AF1 transactivation domain, which can constitutively activate transcriptional activity of the SR lacking the LBD. The precise location and size of AF1 is different in each SR, and is shown in Table 1. The conserved DBD is responsible for the recognition and binding of site-specific DNA sequences. A variable linker region D connects the DBD to the conserved LBD as well as the dimerization surface. LBD is also the site for the interaction of various heat shock and other chaperone proteins for the un-liganded receptor. The ligand-independent transcriptional activation AF1 domain is contained within the A/B region, and the ligand-dependent AF2 core transactivation sub-domain is within the C-terminal portion of the receptor. Most of the known coregulatory proteins interact with these two AF domains. Conformational alterations in these regions play an important role in the recruitment of proper and efficient binding partners under physiological conditions. Except for the NTD, the three-dimensional structure of both DBD and LBD is known for several SRs.

Figure 2. A schematic diagram showing various conformational states of an ID protein

Shown is a hypothetical ID protein that can exist in a number of probable conformations. A few such possible conformations are illustrated in this figure (not real conformations). These conformations can be highly dynamic and can adopt various shapes in a very rapid and inter-convertible fashion. In other words, these conformations are reversible in nature. Under physiological conditions, depending upon the cellular environment, some of these conformers could well have limited residual structure. Depending on the relative probabilities of each state prior to stabilizing conditions (such as interaction of target molecules), the change in free energy for one or more of the stable conformation(s) may vary. In the case of ID AF1 domain of the SRs, disorder-to-order transition should provide access to the interaction energies between different cooperative elements to identify the most efficient and stable interactions in the final form of the complex. Further, the disorder-to-order transition that occurs upon complex formation (between AF1 and other binding partner proteins) can be localized to binding interfaces.

Figure 3. A model for the regulation of transcription by SR:coregulator assemblies

The SR bound to its hormone response element (HRE) recruits certain specific coregulatory proteins (shown by different colors and shapes) mainly through AF1 and/or AF2 regions. Some of these coregulators may have been bound to both AF1 and AF2 through different binding motifs that may lead to formation of a bridge between AF1 and AF2 through these and/or other cofactor(s). By doing so, the signal is passed between AF1 and AF2, facilitating receptor activity. Precise relationships and locations of these cofactor proteins interactions are not accurate and may differ in different SRs. In certain cases, AF1 and AF2 also can interact directly through intra-molecular cross talk. The precise assembly of these coregulatory proteins may be determined by their levels of expression and concentrations in specific cell types and by the conformational state of the SR, particularly its ID AF1 domain. The complex may alter local chromatin structure, and affect the stabilization of the basal transcription pre-initiation complex through TBP bound to its TATA box and/or with other sub-units of the complex. This may well vary within different members of the SR family. The SR:coregulatory protein complex somehow bound to DNA enhancer sites, recruits and regulates Pol II via accumulations of specific proteins, which act as a functional bridge between the receptor and Pol II.

Figure 4. A hypothetical model of folding of the SRs’ AF1 domain in the context of full length receptor under physiological conditions

The ID AF1 domain interacts with one or more proteins from the basal transcription initiation machinery complex, e.g. TBP and in doing so acquires a set of conformations that allows AF1 surfaces to create a platform for interaction with other coregulatory proteins to carry out its function. The resulting structurally modified forms of AF1 may activate it for interactions with various other critical coregulatory proteins essential for gene regulation by the receptor. These interactions give the final folded structure to AF1 and form the basis for the multi-protein assemblies involved in SR-mediated regulation of transcription. This induced fit model of AF1 folding infers that AF1 is not fully structured in vivo until it binds one or another key partner molecule (shown by different colors and shapes). This induced-conformation or limited set of conformations in AF1 is needed in order for it to carry out its transcription function. Formally, induced fit could occur by initial non-specific interactions between the unfolded AF1 domain and the BP. In this version of the model, when such interactions occur, the proximity of the two proteins leads to rapid acquisition of the proper structure in AF1. Alternatively, there initially could be more specific interactions of coregulatory proteins with a partially folded AF1, or even with a tiny pool of fully folded AF1 molecules, creating a kinetic “sink” into which the general population falls.