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1. Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

   https://doi.org/10.1038/s41467-020-14943-4  

   McClelland, Levi J.; Zhang, Kaiming; Mou, Tung-Chung; Johnston, Jake; Yates-Hansen, Cindee; Li, Shanshan; Thomas, Celestine J.; Doukov, Tzanko I.; Triest, Sarah; Wohlkonig, Alexandre; et al (February 2020, Nature Communications)

   Abstract Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα. 

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2. Regulatory links between the circadian clock and stress-induced biomolecular condensates

   https://doi.org/10.1038/s44323-025-00036-2  

   Brown, Gabriela; Nagel, Dawn H (December 2025, npj Biological Timing and Sleep)

   The circadian clock is a conserved timekeeping mechanism that is essential for integrating different environmental cues such as light and temperature to coordinate biological processes with the time of day. While much is known about transcriptional regulation by the clock, the role of post-transcriptional regulation, particularly through sequestration into biomolecular condensate such as stress granules, remains less understood. Stress granules are dynamic RNA-protein assemblies that play a critical role in the cellular response to stress by sequestering mRNAs to regulate translation during stressful conditions. In animals and fungi, the circadian clock regulates stress granule formation and mRNA translation by controlling key factors such as eIF2α, which orchestrates the rhythmic sequestration and translation of specific mRNAs. In plants, it has been shown that some transcripts, despite coming from arrhythmic expression, are rhythmically translated. In addition, some clock-controlled genes (CCGs) are induced in response to heat stress only at the transcriptional level and not at the translational level. Together this highlights a layer of clock regulation beyond transcription. This review discusses the intersection between the circadian clock and heat stress-related biomolecular condensates across eukaryotes, with a particular focus on plants. We discuss how the clock may regulate stress granule dynamics and preferential translation of mRNAs at specific times of the day or during stress responses, thereby enhancing cellular function and energy efficiency. By integrating evidence from animals, fungi, and plants, we highlight emerging questions regarding the role of biomolecular condensates as post-transcriptional mechanisms in controlling circadian rhythms and stress tolerance in plants. 

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3. Dissecting the Interaction between Cryptochrome and Timeless Reveals Underpinnings of Light-Dependent Recognition

   https://doi.org/10.1021/acs.biochem.3c00630  

   Schneps, Connor M; Dunleavy, Robert; Crane, Brian R (January 2024, Biochemistry)

   Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of Drosophila melanogaster, the clock is set by the light-induced degradation of the core oscillator protein timeless (TIM) by the principal light-sensor cryptochrome (CRY). The cryo-EM structure of CRY bound to TIM revealed that within the extensive CRY:TIM interface, the TIM N-terminus binds into the CRY FAD pocket, in which FAD and the associated phosphate-binding loop (PBL) undergo substantial rearrangement. The TIM N-terminus involved in CRY binding varies in isoforms that facilitate the adaptation of flies to different light environments. Herein, we demonstrate, through peptide binding assays and pulsed-dipolar electron spin resonance (ESR) spectroscopy, that the TIM N-terminal peptide alone exhibits light-dependent binding to CRY and that the affinity of the interaction depends on the initiating methionine residue. Extensions to the TIM N-terminus that mimic less light-sensitive variants have substantially reduced interactions with CRY. Substitutions of CRY residues that couple to the flavin rearrangement in the CRY:TIM complex have dramatic effects on CRY light activation. CRY residues Arg237 on α8, Asn253, and Gln254 on the PBL are critical for the release of the CRY autoinhibitory C-terminal tail (CTT) and subsequent TIM binding. These key light-responsive elements of CRY are well conserved throughout Type I cryptochromes of invertebrates but not by cryptochromes of chordates and plants, which likely utilize a distinct light-activation mechanism. 

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4. The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom

   https://doi.org/10.3390/biology9070155  

   Tan, Maxine H.; Smith, Sarah R.; Hixson, Kim K.; Tan, Justin; McCarthy, James K.; Kustka, Adam B.; Allen, Andrew E. (July 2020, Biology)

   null (Ed.)

   Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms. 

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5. An amino‐terminal threonine/serine motif is necessary for activity of the Crp/Fnr homolog, MrpC and for Myxococcus xanthus developmental robustness

   https://doi.org/10.1111/mmi.14378  

   Feeley, Brooke_E; Bhardwaj, Vidhi; McLaughlin, Maeve; Diggs, Stephen; Blaha, Gregor_M; Higgs, Penelope_I (September 2019, Molecular Microbiology)

   Summary The Crp/Fnr family of transcriptional regulators play central roles in transcriptional control of diverse physiological responses, and are activated by a surprising diversity of mechanisms. MrpC is a Crp/Fnr homolog that controls theMyxococcus xanthusdevelopmental program. A long‐standing model proposed that MrpC activity is controlled by the Pkn8/Pkn14 serine/threonine kinase cascade, which phosphorylates MrpC on threonine residue(s) located in its extreme amino‐terminus. In this study, we demonstrate that a stretch of consecutive threonine and serine residues, T₂₁T₂₂S₂₃S_24,is necessary for MrpC activity by promoting efficient DNA binding. Mass spectrometry analysis indicated the TTSS motif is not directly phosphorylated by Pkn14in vitrobut is necessary for efficient Pkn14‐dependent phosphorylation on several residues in the remainder of the protein. In an important correction to a long‐standing model, we show Pkn8 and Pkn14 kinase activities do not play obvious roles in controlling MrpC activity in wild‐typeM. xanthusunder laboratory conditions. Instead, we propose Pkn14 modulates MrpC DNA binding in response to unknown environmental conditions. Interestingly, substitutions in the TTSS motif caused developmental defects that varied between biological replicates, revealing that MrpC plays a role in promoting a robust developmental phenotype. 

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