Tumor Susceptibility Gene 101 Regulates the Glucocorticoid Receptor through Disorder-Mediated Allostery.

Tumor susceptibility gene 101 (TSG101) is involved in endosomal maturation and has been implicated in the transcriptional regulation of several steroid hormone receptors, although a detailed characterization of such regulation has yet to be conducted. Here we directly measure binding of TSG101 to one steroid hormone receptor, the glucocorticoid receptor (GR). Using biophysical and cellular assays, we show that the coiled-coil domain of TSG101 (1) binds and folds the disordered N-terminal domain of the GR, (2) upon binding improves the DNA binding of the GR in vitro, and (3) enhances the transcriptional activity of the GR in vivo. Our findings suggest that TSG101 is a bona fide transcriptional co-regulator of the GR and reveal how the underlying thermodynamics affect the function of the GR.

Ensemble allosteric model: energetic frustration within the intrinsically disordered glucocorticoid receptor.

Allostery is an important regulatory phenomenon enabling precise control of biological function. Initial understanding of allostery was gained from seminal work on conformational changes exhibited by structured proteins. Within the last decade, protein allostery has also been demonstrated to occur within intrinsically disordered proteins. This emerging concept of disorder-mediated allostery can be usefully understood in the context of a thermodynamic ensemble. The advantage of this ensemble allosteric model is that it unifies the explanations of allostery occurring within both structured and disordered proteins. One central finding from this model is that energetic coupling, the transmission of a signal between separate regions (or domains) of a protein, is maximized when one or more domains are disordered. This is due to a disorder-order transition that contributes additional coupling energy to the allosteric system through formation of a molecular interaction surface or interface. A second key finding is that multiple interfaces may constructively or destructively interfere with each other, resulting in a new form of allosteric regulation called 'energetic frustration'. Articulating protein allostery in terms of the thermodynamic ensemble permits formulation of experimentally testable hypotheses which can increase fundamental understanding and direct drug-design efforts. These ideas are illustrated here with the specific case of human glucocorticoid receptor, a medically important multi-domain allosteric protein that contains both structured and disordered regions and exemplifies 'energetic frustration'.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor.

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of 'energetic frustration'. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

Structural Stability of the Coiled-Coil Domain of Tumor Susceptibility Gene (TSG)-101.

The tumor susceptibility gene-101 coiled coil domain (TSG101cc) is an integral component of the endosomal maturation machinery and cytokinesis, and also interacts with several transcription factors. The TSG101cc has been crystallized as a homotetramer but is known to interact with two of its binding partners as a heterotrimer. To investigate this apparent discrepancy, we examined the solution thermodynamics of the TSG101cc. Here, we use circular dichroism, differential scanning calorimetry, analytical ultracentrifugation, fluorescence, and structural thermodynamic analysis to investigate the structural stability and the unfolding of the TSG101cc. We demonstrate that TSG101cc exists in solution primarily as a tetramer, which unfolds in a two-state manner. Surprisingly, no homodimeric or homotrimeric species were detected. Structural thermodynamic analysis of the homotetrameric structure and comparison with known oligomeric coiled-coils suggests that the TSG101cc homotetramer is comparatively unstable on a per residue basis. Furthermore, the homotrimeric coiled-coil is predicted to be much less stable than the functional heterotrimeric coiled-coil in the endosomal sorting complex required for transport 1 (ESCRT1). These results support a model whereby the tetramer-monomer equilibrium of TSG101 serves as the cellular reservoir of TSG101, which is effectively outcompeted when its binding partners are present and the heteroternary complex can form.

Insulins, leptin and feeding in a population of Peromyscus leucopus (white-footed mouse) with variable fertility.

This article is part of a Special Issue "Energy Balance". Natural populations display a variety of reproductive responses to environmental cues, but the underlying physiology that causes these responses is largely unknown. This study tested the hypothesis that heritable variation in reproductive traits can be described by heritable variation in concentrations of hormones critical to both energy balance and reproduction. To test this hypothesis, we used mouse lines derived from a wild population and selectively bred for response to short day photoperiod. Reproductive and metabolic traits of Peromyscus leucopus display heritable variation when held in short photoperiods typical of winter. Our two lines of mice have phenotypes spanning the full range of variation observed in nature in winter. We tested male and female mice for heritable variation in fasted serum concentrations of three hormones involved in energetic regulation: leptin, insulin-like growth factor 1 (IGF-1) and insulin, as well as the effects of exogenous leptin and a high energy diet on reproductive maturation. Exogenous leptin decreased food intake, but protected males from the reduction in testis mass caused by equivalent food restriction in pair-fed, saline-infused controls. A high energy diet resulted in calorie adjustment by the mice, and failed to alter reproductive phenotype. Concentrations of the three hormones did not differ significantly between selection lines but had correlations with measures of food intake, fertility, blood glucose, and/or body mass. There was evidence of interactions between reproductive traits and hormones related to energy balance and reproduction, but this study did not find evidence that variation in these hormones caused variation in reproductive phenotype.