HDX-MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker.

Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. 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 populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. 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.

Electrochemical cofactor recycling of bacterial microcompartments.

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.