Sequential Activation and Local Unfolding Control Poly(A)-Binding Protein Condensation

Abstract

Eukaryotic cells form biomolecular condensates to sense and adapt to their environment1,2. Poly(A)-binding protein (Pab1), a canonical stress granule marker3,4, condenses upon heat shock or starvation, promoting adaptation5. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogen-deuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1's condensation. We find that Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensate's interaction network. Our data yield a clear molecular picture of Pab1's stress-triggered condensation, which we term sequential activation, wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity. Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

An adaptive biomolecular condensation response is conserved across environmentally divergent species

“…We find that the patterns of ∆%D due to condensation are conserved across the Pab1 orthologs (r > 0.72, Figure 5e). Overall, the four RRMs undergo increased exchange for each ortholog upon condensation, consistent with local unfolding in the condensed structure, as reported for S. cerevisiae Pab1 (50). The disordered P domain, in contrast, becomes more protected in each ortholog after condensation (Figure 5f), consistent with its reported role in S. cerevisiae, in providing additional interactions in the condensate context and with the importance of its composition in regulating the temperature of condensation.…”

“…To investigate the structural conservation of the monomers and condensates for the three purified Pab1 orthologs, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS), building on our previous work on S. cerevisiae's Pab1 (50). HDX-MS reports on a protein's hydrogen bond network involving amide protons across the protein by monitoring the rate of deuterium exchange, typically obtained at the peptide level using in-line proteolysis and mass spectrometry.…”

“…Peptides with increased exchange in the condensate have positive ∆%D values, consistent with weaker or a reduced number of hydrogen bonds or increased exposure to solvent in the condensed structure for residues associated with that peptide, and vice versa for peptides with decreased exchange. We assumed unimodal fitting of likely bimodal data to calculate ∆%D which will affect the reported value (50).…”

Small Molecule Properties Define Partitioning into Biomolecular Condensates

“…This property differentiates the physical environment within condensates from that of the surrounding solution, leading to enrichment or exclusion. The physical basis of the difference in environment remains unclear but may involve solvent structure within condensates or perhaps a greater abundance of transient, partially unfolded states of proteins that could create hydrophobic surfaces to recruit small molecules non-stereospecifically 29,30 .…”

Quantitative reconstitution of yeast RNA processing bodies

“…5 ). The RNA recognition motifs of Poly(A)-binding protein (Pab1) undergo partial unfolding in condensates ( 90 ). Similar partial unfolding might be occurring here for P-body proteins in the absence of RNA.…”