Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how ...active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.
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•RIM and RIM-BP mixture forms liquid-liquid phase-separation-mediated condensates•Specific multivalent interaction between RIM and RIM-BP is essential for the LLPS•RIM and RIM-BP condensates cluster Ca2+ channels in solution and on membrane surface•RIM and RIM-BP are plausible organizers of presynaptic active zones
Clustering of Ca2+ channels at presynaptic active zones is critical for precise control of neurotransmitter release. Wu et al. show that the presynaptic active zone scaffold proteins RIM and RIM-BP form self-assembled condensates via liquid-liquid phase separations capable of clustering voltage-gated Ca2+ channels on lipid membrane bilayers.
Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated ...functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid-liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca
is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca
-induced liquid-liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity.
Necroptosis is a type of programmed cell death with great significance in many pathological processes. Tumour necrosis factor-α(TNF), a proinflammatory cytokine, is a prototypic trigger of ...necroptosis. It is known that mitochondrial reactive oxygen species (ROS) promote necroptosis, and that kinase activity of receptor interacting protein 1 (RIP1) is required for TNF-induced necroptosis. However, how ROS function and what RIP1 phosphorylates to promote necroptosis are largely unknown. Here we show that three crucial cysteines in RIP1 are required for sensing ROS, and ROS subsequently activates RIP1 autophosphorylation on serine residue 161 (S161). The major function of RIP1 kinase activity in TNF-induced necroptosis is to autophosphorylate S161. This specific phosphorylation then enables RIP1 to recruit RIP3 and form a functional necrosome, a central controller of necroptosis. Since ROS induction is known to require necrosomal RIP3, ROS therefore function in a positive feedback circuit that ensures effective induction of necroptosis.
A large number of inhibitory receptors recruit SHP1 and/or SHP2, tandem-SH2-containing phosphatases through phosphotyrosine-based motifs immunoreceptor tyrosine-based inhibitory motif (ITIM) and ...immunoreceptor tyrosine-based switch motif (ITSM). Despite the similarity, these receptors exhibit differential effector binding specificities, as exemplified by the immune checkpoint receptors PD-1 and BTLA, which preferentially recruit SHP2 and SHP1, respectively. The molecular basis by which structurally similar receptors discriminate SHP1 and SHP2 is unclear. Here, we provide evidence that human PD-1 and BTLA optimally bind to SHP1 and SHP2 via a bivalent, parallel mode that involves both SH2 domains of SHP1 or SHP2. PD-1 mainly uses its ITSM to prefer SHP2 over SHP1 via their C-terminal SH2 domains (cSH2): swapping SHP1-cSH2 with SHP2-cSH2 enabled PD-1:SHP1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to prefer SHP1 over SHP2 via their N-terminal SH2 domains (nSH2). The ITIM of PD-1, however, appeared to be de-emphasized due to a glycine at pY+1 position. Substitution of this glycine with alanine, a residue conserved in BTLA and several SHP1-recruiting receptors, was sufficient to induce PD-1:SHP1 interaction in T cells. Finally, structural simulation and mutagenesis screening showed that SHP1 recruitment activity exhibits a bell-shaped dependence on the molecular volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the SHP1/SHP2-binding specificities of PD-1 and BTLA, with implications for the mechanisms of a large family of therapeutically relevant receptors.
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurological disorders that share neurodegenerative pathways and features. The most prevalent genetic causes of ...ALS/FTD is the GGGGCC hexanucleotide repeat expansions in the first intron region of the chromosome 9 open reading frame 72 (C9orf72) gene. In this review, we comprehensively summarize the accumulating evidences elucidating the pathogenic mechanism associated with hexanucleotide repeat expansions in ALS/FTD. These mechanisms encompass the structural polymorphism of DNA and transcribed RNA, the formation of RNA foci via phase separation, and the cytoplasmic accumulation and toxicities of dipeptide-repeat proteins. Additionally, the formation of G-quadruplex structures significantly impairs the expression and normal function of the C9orf72 protein. We also discuss the sequestration of specific RNA binding proteins by GGGGCC RNA, which further contributes to the toxicity of C9orf72 hexanucleotide repeat expansions. The deeper understanding of the pathogenic mechanism of hexanucleotide repeat expansions in ALS/FTD provides multiple potential drug targets for these devastating diseases.
Ca
/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca
oscillations. Despite decades of extensive research, new mechanisms underlying ...CaMKIIα's function in synapses are still being discovered. Here, we discover that Shank3 is a specific binding partner for autoinhibited CaMKIIα. We demonstrate that Shank3 and GluN2B, via combined actions of Ca
and phosphatases, reciprocally bind to CaMKIIα. Under basal condition, CaMKIIα is recruited to the Shank3 subcompartment of postsynaptic density (PSD) via phase separation. Rise of Ca
concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca
removal. Protein phosphatases control the Ca
-dependent shuttling of CaMKIIα between the two PSD subcompartments and PSD condensate formation. Activation of CaMKIIα further enlarges the PSD assembly and induces structural LTP. Thus, Ca
-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.
Tau protein misfolding and aggregation are pathological hallmarks of Alzheimer's disease and over twenty neurodegenerative disorders. However, the molecular mechanisms of tau aggregation in vivo ...remain incompletely understood. There are two types of tau aggregates in the brain: soluble aggregates (oligomers and protofibrils) and insoluble filaments (fibrils). Compared to filamentous aggregates, soluble aggregates are more toxic and exhibit prion-like transmission, providing seeds for templated misfolding. Curiously, in its native state, tau is a highly soluble, heat-stable protein that does not form fibrils by itself, not even when hyperphosphorylated. In vitro studies have found that negatively charged molecules such as heparin, RNA, or arachidonic acid are generally required to induce tau aggregation. Two recent breakthroughs have provided new insights into tau aggregation mechanisms. First, as an intrinsically disordered protein, tau is found to undergo liquid-liquid phase separation (LLPS) both in vitro and inside cells. Second, cryo-electron microscopy has revealed diverse fibrillar tau conformations associated with different neurodegenerative disorders. Nonetheless, only the fibrillar core is structurally resolved, and the remainder of the protein appears as a "fuzzy coat". From this review, it appears that further studies are required (1) to clarify the role of LLPS in tau aggregation; (2) to unveil the structural features of soluble tau aggregates; (3) to understand the involvement of fuzzy coat regions in oligomer and fibril formation.
CASK forms an evolutionarily conserved tripartite complex with Mint1 and Veli critical for neuronal synaptic transmission and cell polarity. The CASK CaM kinase (CaMK) domain, in addition to ...interacting with Mint1, can also bind to many different target proteins, although the mechanism governing CASK-CaMK/target interaction selectivity is unclear. Here, we demonstrate that an extended sequence in the N-terminal unstructured region of Mint1 binds to CASK-CaMK with a dissociation constant of ∼7.5 nM. The high-resolution crystal structure of CASK-CaMK in complex with this Mint1 fragment reveals that the C-lobe of CASK-CaMK binds to a short sequence common to known CaMK targets and the N-lobe of CaMK engages an α helix that is unique to Mint1. Biochemical experiments together with structural analysis reveal that the CASK and Mint1 interaction is not regulated by Ca2+/CaM. The CASK/Mint1 complex structure provides mechanistic explanations for several CASK mutations identified in patients with brain disorders and cancers.
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•CASK CaM kinase domain binds to Mint1 with a nanomolar affinity•An elongated Mint1 fragment wraps around the back side of CaMK•Ca2+/CaM does not affect CASK-CaMK binding to Mint1•The CASK/Mint1 structure explains some CASK variants found in patients
Wu et al. discover that an elongated fragment of Mint1/X11α binds to both the N- and C-lobes of CASK-CaMK domain and reveal the mechanism underlying a new mode of highly specific and stable scaffolding role of CASK-CaMK.
Calcium calmodulin-dependent kinase II (CaMKII) is critical for synaptic transmission and plasticity. Two major isoforms of CaMKII, CaMKIIα and CaMKIIβ, play distinct roles in synaptic transmission ...and long-term potentiation (LTP) with unknown mechanisms. Here, we show that the length of the unstructured linker between the kinase domain and the oligomerizing hub determines the ability of CaMKII to rescue the basal synaptic transmission and LTP defects caused by removal of both CaMKIIα and CaMKIIβ (double knockout DKO). Remarkably, although CaMKIIβ binds to GluN2B with a comparable affinity as CaMKIIα does, only CaMKIIα with the short linker forms robust dense clusters with GluN2B via phase separation. Lengthening the linker of CaMKIIα with unstructured “Gly-Gly-Ser” repeats impairs its phase separation with GluN2B, and the mutant enzyme cannot rescue the basal synaptic transmission and LTP defects of DKO mice. Our results suggest that the phase separation capacity of CaMKII with GluN2B is critical for its cellular functions in the brain.
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•CaMKII with a short linker is critical to support basal synaptic transmission and LTP•CaMKII with a short linker promotes phase separation with GluN2B•The different roles of the CaMKII linker on phase separation are sequence independent
CaMKIIα and CaMKIIβ play distinct roles in supporting synaptic transmission and LTP with unknown underlying mechanisms. Cai et al. show that the linker length of CaMKII determines its phase separation with GluN2B, which regulates its differential role in synaptic plasticity.
Pyruvate kinase (PYK) is an essential glycolytic enzyme that controls glycolytic flux and is critical for ATP production in all organisms, with tight regulation by multiple metabolites. Yet the ...allosteric mechanisms governing PYK activity in bacterial pathogens are poorly understood. Here we report biochemical, structural and metabolomic evidence that Mycobacterium tuberculosis (Mtb) PYK uses AMP and glucose-6-phosphate (G6P) as synergistic allosteric activators that function as a molecular "OR logic gate" to tightly regulate energy and glucose metabolism. G6P was found to bind to a previously unknown site adjacent to the canonical site for AMP. Kinetic data and structural network analysis further show that AMP and G6P work synergistically as allosteric activators. Importantly, metabolome profiling in the Mtb surrogate, Mycobacterium bovis BCG, reveals significant changes in AMP and G6P levels during nutrient deprivation, which provides insights into how a PYK OR gate would function during the stress of Mtb infection.
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