Sequence grammar underlying unfolding and phase separation of globular proteins

Abstract

SummaryProtein homeostasis involves regulation of the concentrations of unfolded states of globular proteins. Dysregulation can cause phase separation leading to protein-rich deposits. Here, we uncover the sequence-grammar that influences the triad of folding, binding, and phase equilibria of unfolded proteins in cells. We find that the interactions that drive deposit formation of ALS-associated superoxide dismutase 1 mutations are akin to those that drive phase separation and deposit formation in variants of a model protein, barnase. We examined a set of barnase variants to uncover the molecular interactions that drive phase separation of unfolded proteins and formation of unfolded protein deposits (UPODs). The formation of UPODs requires protein destabilization, to increase the concentration of unfolded states, and a requisite sequence grammar to enable cohesive interactions among unfolded proteins. We further find that molecular chaperones, Hsp40 and Hsp70, destabilize UPODs by binding preferentially to and processing unfolded proteins in the dilute phase.HighlightsUnfolded states of globular proteins undergo aggregation-mediated phase separation to form unfolded protein deposits (UPODs) in cells.The threshold concentration of unfolded proteins required to drive phase separation is governed by a combination of protein stability and the requisite grammar of cohesive motifs known as stickers that provide cohesive intermolecular interactions.The sequence grammar that contributes to phase separation of unfolded proteins shares features with those of proteins that drive the formation of functional biomolecular condensates.Molecular chaperones regulate the concentrations of free unfolded proteins in cells by binding preferentially to unfolded states in dilute phases thereby destabilizing UPOD formation mainly via binding-mediated polyphasic linkage.

Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability

“…Specifically, this effect likely relies on interactions of hydrophobic residues accessible in unfolded SOD1 bar . This interpretation would be in line with prior assignments of homotypic intermolecular hydrophobic contacts as determinants for LLPS of unfolded barnase 31 and elastin. 51 Finally, we investigated diffusion properties of SOD1 bar in SGs to probe for a reduced mobility of the protein in comparison to the cytoplasm, suggesting stronger interactions and association.…”

“…18 In fact, the small but significant changes in the in-cell PC values of the SOD1 bar/FL mutants in comparison to those of Wt observed here support the need to integrate the PQC mechanisms 21,24 when generally establishing the main driving forces behind SGs-mediated sequestration of destabilized proteins (including diseaserelated mutations). In addition to SGs, unfolded and misfolded states also bind to chaperones, 17,31,82,83 are targeted for degradation, or are accumulated in aggresomes and further cleared by autophagy, 16,18,84,85 thus distributing the overall concentration of free unfolded species among these PQC centers. 31 As such previous studies revealed, chaperones can act as suppressors of phase separation of unfolded states by binding to them preferentially in the diluted phase 31 or can be recruited inside SGs to prevent accumulation of misfolded proteins.…”

“…In addition to SGs, unfolded and misfolded states also bind to chaperones, 17,31,82,83 are targeted for degradation, or are accumulated in aggresomes and further cleared by autophagy, 16,18,84,85 thus distributing the overall concentration of free unfolded species among these PQC centers. 31 As such previous studies revealed, chaperones can act as suppressors of phase separation of unfolded states by binding to them preferentially in the diluted phase 31 or can be recruited inside SGs to prevent accumulation of misfolded proteins. 18 Recent studies showed that many proteins involved in functional LLPS are expressed close to their solubility limits; thus, they are more prone to become less soluble with aging, leading to aggregation.…”

“…While lower multivalence and structural rigidity of globular proteins are thought to inherently reduce the propensity for LLPS, 30 their unfolded forms were recently shown to drive aggregationmediated phase separation via homotypic interactions. 31 Nevertheless, it remains unknown if unfolded states constitute an omnipotent driving force when considering heterotypic interactions in functional condensates 32,33 such as SGs. This is important as homotypic-driven aberrant phase transitions are bypassed inside MLOs by "heterotypic buffering", which refers to the ability of heterotypic interactions to suppress the excess of homotypic interactions within the condensates.…”

Sequence- and chemical specificity define the functional landscape of intrinsically disordered regions

“…Our results thus far identified two determinants of function: (i) the presence of a motif, and (ii) the presence of a sequence context that we interpret to mediate distributed multivalent interactions (16,17,67) as hydrophobic residues were important (Fig. 3I,J).…”