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Title: Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family}
Article:
Abstract:
Molecular recognition by intrinsically disordered proteins( IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined “fuzziness”, often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family( EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs.
Introduction:
Understanding the sequence-function relationship of a protein and how it might malfunction is central to biomedical research. While many proteins function in their folded states, recently it became clear that intrinsically disordered proteins( IDPs) also play key functional roles[1],[2] in transcription, translation and cell cycle regulation that, when altered, frequently lead to cancer[3]. Indeed, ∼70% of proteins implicated in cancer are predicted to have significant disordered regions[3],[4]. Molecular recognition by IDPs typically involves target-induced folding. Intriguingly, however, certain IDPs engage in protein-protein interaction without coupled folding and binding[5] such that the IDP remains disordered even when bound to a globular target. This phenomenon has been termed “fuzziness”[6] and is characterised by IDP polyvalency, sequence-insensitivity and lack of strict geometric complementarity for binding[6]. Important examples of fuzziness include transcription factors[7], linker histones[8], prion-like proteins[9] and Sic1-Cdc4 in yeast[10]. To gain insight into “fuzzy” interactions, we have studied the Ewing's Sarcoma( EWS)-activation domain( EAD) in the TET family of RNA-binding proteins[11] and Ewing's family of oncoproteins( EFPs). EAD is a ∼280 residue polyvalent IDP comprised mainly of a degenerate repeat motif SYGQQS. Studies of EAD have mostly focused on its role in naturally occurring EFPs in which it is fused to various transcription factor partners. EFPs are potent EAD-dependent transcriptional activators, resulting in distinct phenotypes of the associated Ewing's family of tumors[12],[13] which are largely dictated by the DNA-binding domain of the EWS fusion partner. Progress in understanding EAD has been hindered by its IDP properties[14] and a general lack of biophysical/biochemical insights[15]. Another barrier is the paucity of information regarding cognate EAD-interacting proteins. Because native EWS interacts with a highly complex array of proteins at a network hub[16],[17] or potentially as a scaffold protein[18], it is probable that EAD has numerous partners. Functional studies of EFPs have provided a foundation for understanding sequence-function relationship of EAD. Most notably, the transcriptional and oncogenic activity of EAD is conferred by multiple tyrosine( Y) residues due to their aromaticity but not hydrophobicity[14]. EAD function is also markedly sequence-insensitive[14], although a permissive overall composition is apparently required. This type of interaction shares features with other systems that exploit polyvalent IDP phosphorylation, as in autoinhibition of CFTR[19], auto-regulation of Ets-1 transcription factor[20],[21] and interaction of Cdk inhibitor Sic1 with its E3 ubiquitin ligase Cdc4[10]. Sic1 has nine low-affinity Cdc4-binding sites and a threshold number of phosphorylated sites induces highly cooperative “polyelectrostatic” binding of Sic1 to a single positively charged pocket in Cdc4[10],[22]–[24]. Similarly, EAD activity requires cooperative action of multiple aromatic moieties in a disordered structure[14],[25], though it does not require phosphorylation. Thus molecular recognition by EAD was coined “polyaromatic”[26]. However, the physical basis for polyaromatic EAD function has not been elucidated. In light of the versatile roles of cation-π interactions in protein folding and protein-protein interactions[27]–[37], we hypothesized that a major contribution to molecular recognition by EAD( within EFPs) is the attraction between numerous unconstrained aromatic residues( π's) on the EAD and basic residues( cations) on the target. We tested this idea experimentally and also theoretically in molecular simulations that are based on cation-π contacts between the EAD and a generic folded target. We found broad agreement between EAD functionality and simulated EAD binding. Thus our findings strongly support the polycation-π model and suggest that similar mechanisms might also be exploited by other IDPs.
Discussion:
The significance of protein disorder in the bound state or “fuzziness” has only recently emerged[6]. Theoretical modeling of IDPs[22],[49]–[67], especially for fuzzy complexes[22], is also in its infancy but provides powerful tools for understanding dynamic conformer ensembles. Our integrated functional and computational approach has culminated in a distinctive model for fuzzy interactions( Fig. 5) that may contain the core features of a more general mode of protein-protein interaction. The model involves a simple biophysical contact( cation-π), strong cooperativity stemming from both IDP and target polyvalency, and a highly flexible and dynamic IDP conformer population in the bound state. Stable binding requires a sufficient number of cation-π contacts but allows kinetic exchanges between myriad bound states. Notably the molecular recognition events studied here are particular to the diseased state of EFP-induced malignancies and are therefore of immediate biomedical interest. Our hypothesis is intuitive given that cation-π interactions have wide and versatile biological roles, the interaction is strong[38] and EAD is highly polyvalent. The native intact EAD is also virtually devoid of cationic residues and thus especially amenable to trans cation-π interactions with target proteins. Here, our interrogation of the polycation-π model covered a wide range of EAD sequence properties( variations of Y number, cation-π strength, charge, Y density, and Y distribution) as well as simulation parameters( physically relevant variations of the cation-π, hydrophobic, and electrostatic interaction strengths; see Text S1). In all these tests, the polycation-π hypothesis provides a consistent biophysical account of the experiments. Other types of interactions are much less likely to contribute dominantly to EAD-target binding and our experiments address some of these. Of particular interest is the stoichiometric intramolecular blocking of Ys by Rs within EAD( Fig. 3C). This observation argues against alternative EAD-target aromatic interactions such as π-π stacking which are, in any event, probably of insufficient strength[68] in the absence of proximate metal ions[69] to account for the slope of Y number dependence of EAD activity( Fig. 1). One may also imagine a scenario in which EAD compaction is induced by Y-dependent hydrophobic interactions such that EAD-target contacts may involve poorly defined non-aromatic entities. But this possibility is strongly contraindicated by the high degree of EAD disorder[14] and also by our finding that EAD compaction by introduction of R residues( Text S1) actually decreases activity. Potential hydrogen bonding effects are not addressed in our model due to insufficient experimental data. Intuitively, hydrogen bonding involving prevalent EAD residues( Gln, Ser, and Thr) may well contribute to molecular recognition by EAD, although previous data[14],[25] together with the current study indicate that cation-π interactions are the essential driving force. More refined studies will be required to uncover secondary and more subtle contributions to EAD-target binding, including potential couplings between hydrogen bonding and cation-π interactions[70]. We also stress that our results do not preclude additional effects due to EAD posttranslational modifications, including tyrosine phosphorylation and O-GlcNAcylation[71], that might sometimes be manifest for particular EFPs and/or in specific physiological circumstances. We have assumed a globular target because the biophysical aspects of the proposed model strongly predict that a large number of real globular proteins interact with EAD. Nonetheless, a disordered( IDP) target that enables favorable cation-π contacts with the EAD is also possible( Fig. S8) although so far the fuzzy complexes known to involve two IDPs are homodimers[72],[73]. We cannot infer how many cation-π contacts are required for EAD binding to real targets. It is also likely that particular interactions will deviate in some manner from our generic model. One can envision a variety of target determinants that might have an impact, including, for example, number and/or density of cations, acute geometric constraints imposed by residues flanking target cations, and the contribution of other aromatic side chain interactions such as hydrogen bonding. Polyelectrostatic( Sic1/Cdc4) and polycation-π interactions share some similarities. Each may well reflect a general mode of interaction for polyvalent IDPs. In contrast to Sic1-Cdc4, however, the properties of the EAD studied herein are related to the diseased state[14] and our study points to several significant biophysical differences between EAD and Sic1/Cdc4. First, Sic1/Cdc4 binding involves a single Cdc4 site while EAD binding in our model invokes multiple simultaneous contacts. Second, Sic1/Cdc4 interaction is switch-like, reflecting the biological need for acute response to cell cycle kinase levels, whereas the EAD is constitutively polyvalent[14],[25]. Third, like most other polyvalent IDPs, Sic1 has short sequence-specific or linear motifs[74],[75], a single copy of which can mediate suboptimal or high-affinity Sic1/Cdc4 binding[10]. Such elements are almost certainly absent in EAD[14]. Fourth, the multiple cation-π contacts that underpin EAD binding in our model entail transient restrictions of EAD conformations( though they remain disordered), whereas a Sic1 bound to a single Cdc4 pocket at a given instant is not subject to such conformational restriction[22]–[24]. The molecular recognition events studied here are related to pathological EAD function and, accordingly, are not obviously shaped by evolution[14]. Some aspects of EAD malfunction are an indirect consequence of loss of the EWS RNA-binding domain( RBD) or gain of a foreign DNA-binding domain in EFPs. In relation to our study, it is intriguing that the EWS RBD contains highly disordered regions with reiterated RGG that autorepress EAD[76], quite possibly via intramolecular masking[40]. The polycation-π perspective may offer a rationalization for this behavior. The simulated binding between a disordered EWS peptide containing multiple RGG boxes and the 10Yn EAD indeed reveals a strong interaction( Fig. S8). Intramolecular cation-π interactions between EAD and RGG have high potential to impact native EWS function by competing out aberrant interactions between EAD and the putative globular proteins relevant to EAD malfunction in oncogenesis. In this regard, knowledge of EAD-target interface might provide therapeutic avenues[77] for Ewing's family tumors with poor prognosis. Several small molecule inhibitors of EWS/Fli1 have been identified. Interestingly, they all have aromatic character[78]–[80] or, in one case, a very basic short peptide sequence[81]. Whether any of them target the EAD portion of EWS/Fli is unknown. Due to their likely being effective cation-π competitors, it will be of great biomedical interest to explore this possibility. How may polyaromatic molecular recognition by EAD relate to normal EWS protein function? This is a challenging question given the strong evolutionary conservation of EWS[82] that includes several EAD properties: a positionally conserved Gln two residues C-terminal to Y, Y phosphorylation sites[83], and SH2/SH3 interaction sites. However, none of these features are required, at least in some cases, for oncogenic EAD function[14]. Perhaps the mode of EAD action in EFP oncoproteins reflects a primordial polyaromatic function that was subsequently tailored by evolution to fulfill normal cellular roles. For example, Y phosphorylation can dramatically increase the aromatic-cation interactions required for peptide inhibitors of Src[84], indicating that phosphorylation of only a limited number of Ys in EAD could have profound effects on EAD-target interactions that are important for normal EWS. To conclude, the proposed model for molecular recognition by EAD expands the seemingly endless modalities for IDP function and malfunction. The hitherto unrecognized polycation-π mode of IDP-target binding can be versatile. It offers a highly plausible biophysical basis for EAD and perhaps other scaffold/networking proteins to interact with many distinct target proteins[16]–[18]. The present methodology and results can also be extended to facilitate the exciting search for real EAD targets.
Understanding how proteins recognize each other is central to deciphering the inner workings of living things and for biomedical research . It has long been known that the sequence of a protein , which is a string of different amino acids , can dictate how a protein molecule folds into a well-defined shape required for biological tasks . Many folded proteins recognize and bind with each other by a tight geometric fit similar to that between a lock and its key . Recently , however , it has become clear that some proteins function as a flexible string , in constant motion , without forming a stable shape . Understanding how such “disordered” proteins work is challenging . To gain insight , we studied a disordered protein region that causes a large family of human cancers . Employing an innovative combination of experimental and theoretical techniques , we describe a new mode of protein interaction based on multiple simple contacts between one type of amino acid ( aromatic ) in the disordered protein and another type ( positively charged ) on the partner protein . Because this mechanism also underlies the ability of the disordered protein to cause cancer , further investigation of this unprecedented mode of protein-protein interaction may open up new avenues for cancer therapy .