The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by O-GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure–function relationships in other biological condensates. Significance Many important biological processes occur within biomolecular condensates. Unlike many organelles, these condensates lack a surrounding membrane but are formed by phase separation due to interactions of their molecular components. Although the important role of intrinsically disordered protein regions (IDRs) in condensates is appreciated and contributing amino acids have been identified, experimentally derived site-specific information reporting on interactions between IDRs in condensed phases is lacking. Using a suite of NMR experiments with improved resolution and sensitivity for studies of condensed phases, we have obtained quantitative and site-specific information on interactions and hot spots that govern phase separation of the C-terminal IDR of CAPRIN1 and how phase separation can be modulated by posttranslational modifications, mutations, and interactions with ATP.

Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials ( ϕ _ENS ) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ϕ _ENS values have been mapped along the adenosine triphosphate (ATP)–induced phase-separation trajectory. In the absence of ATP, ϕ _ENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral ( ϕ _ENS ≈ 0 mV), with ∼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Significance: The HtrA2 protease plays a critical role in ensuring proper cellular function and in regulating a variety of different processes, including programmed cell death. Although the three-dimensional structure of a trimeric form of the enzyme has been determined, the mechanism by which this important molecule is regulated remains to be elucidated. Our data reveal that this is accomplished through a second conformation of the enzyme in which it forms a hexamer with much lower affinity for substrate and by the fact that the enzyme remains in a low-activity state until substrate binding leads to domain reorientations, exposing the catalytic center. Together, these two regulatory features help to minimize proteolysis of nonsubstrate proteins that are required for cellular health.