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Intrinsically disordered regions (IDRs) are important components of protein functionality, with their charge distribution serving as a key factor in determining their roles. Notably, many proteins possess IDRs that are highly negatively charged, characterized by sequences rich in aspartate (D) or glutamate (E) residues. Bioinformatic analyses indicate that negatively charged low-complexity IDRs are significantly more common than their positively charged counterparts rich in arginine (R) or lysine (K). For instance, sequences of 10 or more consecutive negatively charged residues (D or E) are present in 268 human proteins. In contrast, corresponding sequences of 10 or more consecutive positively charged residues (K or R) are present in only 12 human proteins. Interestingly, about 50% of proteins containing D/E tracts function as DNA-binding or RNA-binding proteins. Negatively charged IDRs can electrostatically mimic nucleic acids and dynamically compete with them for the DNA-binding domains (DBDs) or RNA-binding domains (RBDs) that are positively charged. This leads to a phenomenon known as autoinhibition, in which the negatively charged IDRs inhibit binding to nucleic acids by occupying the binding interfaces within the proteins through intramolecular interactions. Rather than merely reducing binding activity, negatively charged IDRs offer significant advantages for the functions of DNA/RNA-binding proteins. The dynamic competition between negatively charged IDRs and nucleic acids can accelerate the target search processes for these proteins. When a protein encounters DNA or RNA, the electrostatic repulsion force between the nucleic acids and the negatively charged IDRs can trigger conformational changes that allow the nucleic acids to access DBDs or RBDs. Additionally, when proteins are trapped at high-affinity non-target sites on DNA or RNA ("decoys"), the electrostatic repulsion from the negatively charged IDRs can rescue the proteins from these traps. Negatively charged IDRs act as gatekeepers, rejecting nonspecific ligands while allowing the target to access the molecular interfaces of DBDs or RBDs, which increases binding specificity. These IDRs can also promote proper protein folding, facilitate chromatin remodeling by displacing other proteins bound to DNA, and influence phase separation, affecting local pH. The functions of negatively charged IDRs can be regulated through protein-protein interactions, post-translational modifications, and proteolytic processing. These characteristics can be harnessed as tools for protein engineering. Some frame-shift mutations that convert negatively charged IDRs into positively charged ones are linked to human diseases. Therefore, it is crucial to understand the physicochemical properties and functional roles of negatively charged IDRs that compete with nucleic acids. 

Abstract In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition through intramolecular electrostatic interaction with functional domains. In this work, we investigated the impacts of D/E repeats on the target DNA search kinetics for the high-mobility group box 1 (HMGB1) protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats of varied lengths. Our experimental data showed that D/E repeats of particular lengths can accelerate the target association in the overwhelming presence of non-functional high-affinity ligands (‘decoys’). Our coarse-grained molecular dynamics (CGMD) simulations showed that the autoinhibited proteins can bind to DNA and transition into the uninhibited complex with DNA through an electrostatically driven induced-fit process. In conjunction with the CGMD simulations, our kinetic model can explain how D/E repeats can accelerate the target association process in the presence of decoys. This study illuminates an unprecedented role of the negatively charged IDRs in the target search process.