Influence of an Intrinsically Disordered Region on Protein Domains Revealed by NMR-Based Electrostatic Potential Measurements

Abstract

Many human proteins possess intrinsically disordered regions containing consecutive aspartate or glutamate residues (“D/E repeats”). Approximately half of them are DNA/RNA-binding proteins. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we investigated the electrostatic properties of D/E repeats and their influence on folded domains within the same protein. Local electrostatic potentials were directly measured for the HMGB1 protein, its isolated D/E repeats, and DNA-binding domains by NMR. The data provide quantitative information about the electrostatic interactions between distinct segments of HMGB1. Due to the interactions between the D/E repeats and the DNA-binding domains, local electrostatic potentials of the DNA-binding domains within the full-length HMGB1 protein were largely negative despite the presence of many positively charged residues. Our NMR data on counterions and electrostatic potentials show that the D/E repeats and DNA have similar electrostatic properties and compete for the DNA-binding domains. The competition promotes dissociation of the protein–DNA complex and influences the molecular behavior of the HMGB1 protein. These effects may be general among the DNA/RNA-binding proteins with D/E repeats.

Gadolinium-Based NMR Spin Relaxation Measurements of Near-Surface Electrostatic Potentials of Biomolecules

“…Examples include electrostatics of single-stranded DNA/RNA, IDR-containing proteins, dynamic multidomain proteins, IDPs, and their complexes with other molecules. For instance, as recently demonstrated, 76 ϕ ENS potential measurements can reveal how protein domains are influenced by IDRs through electrostatics. The ϕ ENS method is also useful to investigate the potentially critical role of electrostatics in phase separation of IDPs as well.…”

“…Examples include electrostatics of single-stranded DNA/RNA, IDR-containing proteins, dynamic multidomain proteins, IDPs, and their complexes with other molecules. For instance, as recently demonstrated, ϕ ENS potential measurements can reveal how protein domains are influenced by IDRs through electrostatics. The ϕ ENS method is also useful to investigate the potentially critical role of electrostatics in phase separation of IDPs as well. , It is likely that measurement of ϕ ENS values can shed light on how post-translational modifications such as phosphorylation and acetylation within IDRs (e.g., histone tails) influence their structural dynamics and hence function.…”

Phosphorylation by Protein Kinase C Weakens DNA-Binding Affinity and Folding Stability of the HMGB1 Protein

“…Judging from prior studies on the intramolecular interactions involving the C-terminal acidic tail, ,− the low efficiency in phosphorylation for residues outside A-box may be partly due to the tail. For example, the low phosphorylation efficiency of S181, which is predicted to be a PKC site, may be due to strong interactions between the surrounding basic residues (K180, K182, K183, K184, and K185) and the acidic tail.…”