Order within a Disordered Structure

Intrinsically disordered proteins (IDPs) have the plasticity to exhibit different structures. This flexibility permits multiple interaction motifs to form numerous complexes with proteins, DNA, or RNA, to regulate various cellular functions such as transcriptional activation, translation, and cell signaling pathways (Wright and Dyson, 2015). Such proteins can therefore act as hubs of interaction networks. A partially folded conformation is favored in many cellular processes for a variety of reasons. First, its flexibility allows different molecular interactions under different conditions. Second, it is able to weakly bind to targets with high specificity, resulting in transient complex formation and dynamic signaling regulation. Third, conformational change is induced upon binding to targets, leading to stabilization. Finally, it allows easy access for post-translational modification (Wright and Dyson, 2015).

A well-studied example of a protein with large regions of intrinsic disorder is the transcriptional co-activator CREB-binding protein (CBP). Its intrinsic flexibility and minimally structured regions enable interactions with disordered transactivation domains of many different transcription factors. The oncoprotein c-Myc is another example of an IDP that is over-expressed in many types of cancers and thereby of high interest as a therapeutic drug target. However, the disordered nature of the protein has made drug development challenging. To date, c-Myc has been coined “undruggable,” and no small molecule that is a direct inhibitor of c-Myc has entered clinical trials.

By contrast, an inhibitor specific to a disordered region within the androgen receptor (AR) is available. Ralaniten (aka EPI) is the first small molecule inhibitor that directly binds to an intrinsically disordered region of a protein to enter clinical trials. It binds directly to the intrinsically disordered transactivation unit 5 (Tau-5) within the activation function 1 (AF-1) region in the N-terminal domain (NTD) of AR (Figure 1) (De Mol et al., 2016; Myung et al., 2013). Transcriptionally active AR drives almost all prostate cancers including advanced metastatic castration-resistant prostate cancer (CRPC). Tau-5 is the hot-spot of AR transcriptional activity and is required for the activities of both full-length AR as well as truncated constitutively active AR splice variants that lack a ligand-binding domain (LBD), such as AR-V7. Antiandrogens bind to the LBD at the C-terminus of AR and do not inhibit AR-V7 or other truncated AR variants lacking LBD. As a result, expression of AR-V7 and other truncated AR variants lacking LBD is associated with clinical resistance to antiandrogens and to treatment by ablation of androgen production (Antonarakis et al., 2014; Scher et al., 2016). For this reason, there is much interest in developing molecular inhibitors such as ralaniten, as these molecules hold therapeutic promise for those patients with advanced prostate cancer whose disease is driven by AR and/or AR-V7 (Sadar, 2011; Myung et al., 2013; Antonarakis et al., 2014; Scher et al., 2016). Understanding the binding properties of Tau-5 will aid in the development of more potent inhibitors and specific therapeutic strategies.

As a step in this direction, De Mol et al. (2018), in the current issue of Structure, investigate the interaction between the disordered Tau-5 region of AR and the C-terminal domain (CTD) of RAP74, the subunit 1 of the general transcription regulator TFIIF. To do this, the authors first used solution nuclear magnetic resonance (NMR) to identify the motif responsible for the interaction with RAP74-CTD, leading to the identification of AR 423–446 (⁴²³GSPSAAASSSWHTLFTAEEGQLYG⁴⁴⁶) within Tau-5. This motif interacts with RAP74-CTD to initiate transcription. Interestingly, the AR423–446 motif contains hydrophobic (W433, L436, and F437) and acidic (E440 and E441) residues in a pattern similar to that in the motifs of FCP1, a phosphatase recruited by RAP74-CTD during termination of transcription (De Mol et al., 2018).

However, this interaction between the identified motif and RAP74 was weak, as the affinity was in the millimolar range. The authors then found that induction of local helical conformation within the motif is required for the recruitment of RAP74-CTD and for the enhancement of protein-protein interaction. Specifically, residues from S432 to T438 contribute to two turns of an α helix. Studies by Lavery and McEwan (2008) had shown that folding of helical conformation in the AF-1 region of AR (AR142–485) was induced upon binding to RAP74-CTD. An increase in α-helical structure contributed to the stabilization of the complex from an initial low-affinity protein-protein interaction to high-affinity protein-protein interaction. Through a combination of NMR and circular dichroism analyses, De Mol et al. (2018) further confirm that the increase in helical structure in the motif AR423–446 indeed contributes to the higher affinity for the stabilized binding to RAP74-CTD.

Since the motif AR423–446 is rich in serine residues, De Mol et al. (2018) hypothesized and then demonstrated that phosphorylation (specifically at S424) has an impact on the stabilization of binding to RAP74-CTD. These data support previous observations by others (Yang et al., 2005; Gioeli and Paschal, 2012) that the phosphorylation at this serine residue in the presence of androgen is important for AR transcriptional activity. More importantly, De Mol et al. (2018) showed that the deletion of motif AR 423–446 and mutation of serine to alanine at residue 424 reduced the amount of protein-protein interaction and the level of transcriptional activity in cells. Cell-based analysis provided strong evidence to support the therapeutic strategy for specifically targeting the direct interaction between the AR motif in the disordered NTD and RAP74-CTD.

In summary, this study demonstrates that an induced secondary structure in a disordered region, combined with post-translational modification, stabilizes a transcriptional complex in order to initiate or enhance transcriptional activity. From a therapeutic perspective, targeting the limited structure within the disordered region of Tau-5 may help avoid off-target effects. Furthermore, identification of the AR motif that interacts with RAP74-CTD provides guidance for finding novel inhibitors that could target the transient and weak interactions between AR and RAP74, perhaps via prevention of the stabilization of AR and its recruitment of the transcriptional machinery. Finally, development of inhibitors that specifically target the phosphorylation site on the AR motif may be an attractive approach to treat any disease driven by full-length AR or its truncated, constitutively active splice variants.