Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation

Abstract

Many cell signaling events are coordinated by intrinsically disordered protein regions (IDRs) that undergo multisite Serine/Threonine phosphorylation. The conformational properties of these IDRs prior to and following multi-site phosphorylation are directly relevant to understanding their functions. Here, we present results from biophysical studies and molecular simulations that quantify the conformational properties of an 81-residue IDR from the S. cerevisiae transcription factor Ash1. We show that the unphosphorylated Ash1 IDR adopts coil-like conformations that are expanded and well-solvated. This result contradicts inferences regarding global compaction that are derived from heuristics based on amino acid compositions for IDRs with low proline contents. Upon phosphorylation at ten distinct sites, the global conformational properties of pAsh1 are indistinguishable from those of unphosphorylated Ash1. This insensitivity derives from compensatory changes to the pattern of local and long-range intra-chain contacts. We show that the conformational properties of Ash1 and pAsh1 can be explained in terms of the linear sequence patterning of proline and charged residues vis-à-vis all other residues. The sequence features of the Ash1 IDR are shared by many other IDRs that undergo multisite phosphorylation. Accordingly, we propose that our findings might be generalizable to other IDRs involved in cell signaling.

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…We observe no significant changes in LC secondary structure (i.e., folding) upon multiple phosphorylation, as shown for other heavily phosphorylated disordered domains (Martin et al , 2016). However, increasing negative charge at S/T sites across FUS LC decreases the propensity of those regions to form transient collapsed states and intermolecular contacts (Fig 3).…”

“…However, it is clear that low‐complexity domains specify and mediate functional protein–protein interactions and pathological self‐aggregation. Low‐complexity domains are well‐established targets of post‐translational modifications (Martin et al , 2016), and their sequences specify homotypic and heterotypic interactions mediating liquid–liquid phase separation (LLPS; Lin et al , 2015; Pak et al , 2016). Hence, cells might employ post‐translational modification to “change” the amino acid sequence of prion‐like domains and thus disrupt self‐interaction.…”

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

“…We performed all-atom Metropolis Monte Carlo thermal replica exchange simulations for five of the IDPs, using the ABSINTH implicit solvation model and force-field paradigm (24). This combination has proved to be useful for the analysis of conformationally heterogeneous IDPs (42,44). Details of the simulations are described in SI Appendix, Note S6.…”

“…This variance will encode the extent of convergence or divergence between measures of chain dimensions averaged across the entire sequence (R G ) and measures that probe specific length scales, such as R E . The blob-based analysis explains why despite water being a poor solvent for polypeptide backbones (29,70), we now know that the apparent solvent quality for real IDPs deviates from that for backbones and is actually governed by charge and proline contents as well as the patterning of charged and proline residues (3,17,41,42,68,69,71).…”

Structural evaluations of tau protein conformation: methodologies and approaches

“…IDPs undergo order-to-disorder or disorder-to-order transitions and are commonly present in humans and bacteria. Their structure and function may be modulated by protein chaperones, post-translational modifications (PTMs), and degradation processes (Martin et al 2016; Uversky et al 2014). Misregulation of modulators leads to dysfunction of the protein, most commonly resulting in unfolding, misfolding, aggregation, or loss of function (Goldberg et al 2003).…”