SummaryLiquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [ electron transfer ͉ encounter state ͉ transient complex ͉ spin label ͉ paramagnetic relaxation enhancement T he process of protein complex formation can be described by a two-step model, in which a short-lived, dynamic encounter complex precedes a dominant, well defined state (Fig. 1). The former enables proteins to undergo reduced-dimensionality search of the optimal binding geometry, thereby accelerating molecular association as compared with 3D diffusion (1). Fast molecular association is essential for protein-protein complexes that require high turnover rates, like those involved in electron transfer (ET) in photosynthesis, respiration, and other metabolic processes (2). The physiological complex of yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP) is a paradigm for the intermolecular ET (3) and is one of the few transient ET complexes for which a crystal structure has been solved (4). A recent study has confirmed that the protein-protein orientation observed in the crystal is ET-active (5). However, it has remained a matter of debate whether this structure represents the only form in solution (6-8). According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9-15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8, 16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are .Characterization of the binding interface in the dynamic encounter state (Fig. 1B) has so far proven to be elusive. Investigation of protein complexes by using x-ray crystallography or conventional NMR spectroscopy addresses only the singleorientation species (Fig. 1C), and the only way to visualize the dynamic state is offered by theoretical modeling studies (11,18). In the recent, elegant work of Clore and coworkers (19,20), it was shown how paramagnetic relaxation can be applied to study the dynamic state of protein-DNA complexes. We report on the application of an analogous experimental approach that allows us to define both the dominant protein-protein orientation (Fig. 1C) and the conformational space sampled by the proteins in the dynamic encounter complex (Fig. 1B). Results and DiscussionSolution Structure of the Complex. The concept of the approach is that NMR resonance intensities of one of the proteins in the complex are affected by a paramagnetic spin label covalently attached to the other protein (Fig. 2). The paramagnetic effects are converted into distance restraints (21-23), which can be used to calculate protein-protein orientations within the complex (24). Five ...

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for the shed APP ectodomain (sAPP). Here, we showed that the sAPP extension domain directly bound the sushi 1 domain specific to the gamma-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in hippocampal synapses via inhibition of synaptic vesicle release. A 17 amino acid peptide corresponding to the GABABR1a binding region within APP suppressed spontaneous neuronal activity in vivo. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission.

Recent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We use paramagnetic relaxation enhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize the ensemble of encounter orientations in the short-lived electron transfer complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). The complete conformational space sampled by the protein molecules during the dynamic part of the interaction was mapped experimentally. The encounter complex was described by an electrostatic ensemble of orientations based on Monte Carlo calculations, considering the protein structures in atomic detail. We demonstrate that this visualization of the encounter complex, in combination with the specific complex, is in excellent agreement with the experimental data. Our results indicate that Cc samples only about 15% of the surface area of CcP, surrounding the specific binding interface. The encounter complex is populated for 30% of the time, representing a mere 0.5 kcal/mol difference in the free energies between the two states. This delicate balance is interpreted to be a consequence of the conflicting requirements of fast electron transfer and high turnover of the complex.