An NMR study on the intrinsically disordered core transactivation domain of human glucocorticoid receptor

摘要

A large number of transcriptional activation domains (TADs) are intrinsically unstructured, meaning they are devoid of a three-dimensional structure. The fact that these TADs are transcriptionally active without forming a 3-D structure raises the question of what features in these domains enable them to function. One of two TADs in human glucocorticoid receptor (hGR) is located at its N-terminus and is responsible for ~70% of the transcriptional activity of hGR. This 58-residue intrinsically-disordered TAD, named tau1c in an earlier study, was shown to form three helices under trifluoroethanol, which might be important for its activity. We carried out heteronuclear multi-dimensional NMR experiments on hGR tau1c in a more physiological aqueous buffer solution and found that it forms three helices that are ~30% pre-populated. Since pre-populated helices in several TADs were shown to be key elements for transcriptional activity, the three pre-formed helices in hGR tau1c delineated in this study should be critical determinants of the transcriptional activity of hGR. The presence of pre-structured helices in hGR tau1c strongly suggests that the existence of pre-structured motifs in target-unbound TADs is a very broad phenomenon. Although many globular proteins are three-dimensionally structured, they contain short flexible/disordered loops composed of less than 20 amino acid residues (1). This phenomenon of so-called protein disorder has been known for decades, but since the late 1990s, we have begun encountering some peculiar proteins that contain long unfolded/disordered regions (more than 40 and up to hundreds of residues) that do not form 3-D structures (2, 3). These proteins are now named as intrinsically disordered proteins (IDPs) (4, 5) and represent a special case of protein disorder. IDPs are highly unorthodox because even without three-dimensional structures, they are capable of performing specific biological functions (including transcription, translation, chaperoning, and cell cycle regulation) or are responsible for many fatal diseases (such as cancers, prion diseases, neurodegenerative diseases including Alzheimer’s and Parkinson’s, and so on) (6–9). Many viral proteins in HIV-1, HBV, HCV, SARS virus, and AI virus are also IDPs or contain intrinsically disordered regions (IDRs) (4, 10–13). The unexpected correlation between the “unstructured” nature of IDPs/IDRs and their functionality ended up nullifying the decades-old structure-function paradigm, 3-D structure = function, in protein science and structural biology. Approximately 40% of the entire protein kingdom is predicted to consist of IDPs/IDRs (11), and the proportion of IDPs/IDRs is much higher (~60%) in transcription factors (14). In the case of globular proteins, the collective structural features (secondary, tertiary and quaternary) provide a reasonable explanation of function. However, the absence of tertiary structures in IDPs makes it quite challenging to come up with an explanation on why and how they should function at all. For example, we still do not have a clear understanding on how IDPs bind to their targets. Initially, IDPs/IDRs were erroneously thought to be completely unstructured (CU) without any trace of secondary structures (15, 16). In contrast to this early view, a more quantitative structural picture on IDPs/IDR has emerged from many high-resolution multi-dimensional NMR investigations conducted over the last two decades. These studies have revealed that at least ~70% of IDPs/IDRs are not fully “unstructured”, but contain transient local structural elements in their free state that mediate binding of IDPs to targets (4). The IDPs/IDRs containing transient local structural elements are therefore described to be in a mostly unstructured (MU) state (3) since they are not completely unstructured in terms of secondary structure. Although these transient secondary structures in IDPs, which were recently named in 2012 as pre-structured motifs (Pr