Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

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.

Author Summary

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.