Bifunctional Rel stringent factors, the most abundant class of RSHs (RelA/SpoT Homologues), are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 of GTP/GDP to synthesize the bacterial alarmone (p)ppGpp, and also catalyse the 3 pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide switch mechanism controls catalysis by T. thermophilus Rel (Rel Tt ). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel Tt (Rel Tt NTD ) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation. Fondation Van Buren to A.G.-P.; the Molecular Infection Medicine Sweden (MIMS), Swedish Research council (grant 2017-03783), and Ragnar Söderberg foundation fellowship to V.H.; J. Hendrix and J. Hofkens are grateful to the Research Foundation Flanders (FWO Vlaanderen, G0B4915N) and large infrastructure grant ZW15_09 GOH6316N) and the KU Leuven Research Fund (C14/16/053); J. Hofkens thanks financial support of the Flemish government through long term structural funding Methusalem (CASAS2, Meth/15/04).

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

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The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to Cystic Fibrosis (CF). Here we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and related transporters. Crystallography studies reveal that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. However, under destabilizing conditions, such as the prominent disease-causing mutation F508el, this conformational flexibility enables unfolding of the βsubdomain. Our data indicate that in wild-type CFTR this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

30 31Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are 32 ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 33 3′ of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3′ pyrophosphate 34 hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems 35to be a complex allosteric mechanism, and despite decades of research, it is unclear how 36 the two opposing activities of Rel are controlled at the molecular level. Here we show 37 that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. 38 thermophilus Rel (Rel Tf ). The binding of GDP/ATP stretches apart the NTD catalytic 39 domains of Rel Tf (Rel Tt NTD ) activating the synthetase domain and allosterically blocking 40 the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain 41 and triggers recoil of both NTDs, which partially buries the synthetase active site and 42 precludes the binding of synthesis precursors. This allosteric mechanism acts as an 43 activity switch preventing futile cycles of alarmone synthesis and degradation. 44 The cellular level of the bacterial alarmone (p)ppGpp 1,2 -a key regulator of virulence and 45 antibiotic tolerance -is controlled by the action of RelA/SpoT Homologue (RSH) enzymes 3-46 5 . The synthesis of (p)ppGpp involves transfer of the pyrophosphate group of ATP onto the 3′ 47 hypothesis it is essential to solve the structures of Rel enzymes with catalytically engaged 58 SYN or HD domains. To understand how nucleotide binding stimulates the enzymatic 59 capacity of RSH enzymes, we took advantage of T. thermophilus Rel NTD (Rel Tt NTD , amino 60 acid positions 1-355) as an experimental system. Rel Tt NTD hydrolysis activity is virtually 61 undetectable at 4°C (Supplementary Fig. 1a and Supplementary Table 1), which is not 62 surprising given that T. thermophilus has an optimal growth temperature of about 65°C. This 63 enabled co-crystallization in the presence of the native ppGpp substrate. To generate the 64 structure of Rel Tt NTD engaged in ppGpp synthesis, we used APPNP, a β -γ phosphate non-65

7TM receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM receptors. Such 15 TM concatameric homo- and heterodimers of the beta(2)-adrenergic and the NK(1) receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two receptors. It is concluded that covalently joined 7TM receptor dimers with surprisingly normal receptor properties can be constructed with use of an artificial transmembrane connector, which perhaps can be used to fuse other membrane proteins.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.