Jianhui Song

Polycation-π interactions are a driving force for molecular recognition by an intrinsically disordered oncoprotein family.

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 Ewings 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 Ewings 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.

An Adequate Account of Excluded Volume Is Necessary To Infer Compactness and Asphericity of Disordered Proteins by Förster Resonance Energy Transfer

Single-molecule Förster resonance energy transfer (smFRET) is an important tool for studying disordered proteins. It is commonly utilized to infer structural properties of conformational ensembles by matching experimental average energy transfer ⟨E⟩exp with simulated ⟨E⟩sim computed from the distribution of end-to-end distances in polymer models. Toward delineating the physical basis of such interpretative approaches, we conduct extensive sampling of coarse-grained protein chains with excluded volume to determine the distribution of end-to-end distances conditioned upon given values of radius of gyration Rg and asphericity A. Accordingly, we infer the most probable Rg and A of a protein disordered state by seeking the best fit between ⟨E⟩exp and ⟨E⟩sim among various (Rg,A) subensembles. Application of our method to residues 1-90 of the intrinsically disordered cyclin-dependent kinase (Cdk) inhibitor Sic1 results in inferred ensembles with more compact conformations than those inferred by conventional procedures that presume either a Gaussian chain model or the mean-field Sanchez polymer theory. The Sic1 compactness we infer is in good agreement with small-angle X-ray scattering data for Rg and NMR measurement of hydrodynamic radius Rh. In contrast, owing to neglect or underappreciation of excluded volume, conventional procedures can significantly overestimate the probabilities of short end-to-end distances, leading to unphysically large smFRET-inferred Rg at high [GdmCl]. It follows that smFRET Sic1 data are incompatible with the presumed homogeneously expanded or contracted conformational ensembles in conventional procedures but are consistent with heterogeneous ensembles allowed by our subensemble method of inference. General ramifications of these findings for smFRET data interpretation are discussed.

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCF Cdc4 ubiquitin ligase

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

Random-phase-approximation theory for sequence-dependent, biologically functional liquid-liquid phase separation of intrinsically disordered proteins

Abstract Intrinsically disordered proteins (IDPs) are typically low in nonpolar/hydrophobic but relatively high in polar, charged, and aromatic amino acid compositions. Some IDPs undergo liquid-liquid phase separation in the aqueous milieu of the living cell. The resulting phase with enhanced IDP concentration can function as a major component of membraneless organelles that, by creating their own IDP-rich microenvironments, stimulate critical biological functions. IDP phase behaviors are governed by their amino acid sequences. To make progress in understanding this sequence-phase relationship, we report further advances in a recently introduced application of random-phase-approximation (RPA) heteropolymer theory to account for sequence-specific electrostatics in IDP phase separation. Here we examine computed variations in phase behavior with respect to block length and charge density of model polyampholytes of alternating equal-length charge blocks to gain insight into trends observed in IDP phase separation. As a real-life example, the theory is applied to rationalize/predict binodal and spinodal phase behaviors of the 236-residue N-terminal disordered region of RNA helicase Ddx4 and its charge-scrambled mutant for which experimental data are available. Fundamental differences are noted between the phase diagrams predicted by RPA and those predicted by mean-field Flory-Huggins and Overbeek-Voorn/Debye-Huckel theories. In the RPA context, a physically plausible dependence of relative permittivity on protein concentration can produce a cooperative effect in favor of IDP-IDP attraction and thus a significantly increased tendency to phase separate. Ramifications of these findings for future development of IDP phase separation theory are discussed.

Conformations of a Metastable SH3 Domain Characterized by smFRET and an Excluded-Volume Polymer Model

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.