CaMKII autophosphorylation is the only enzymatic event required for synaptic memory.

Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, autophosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory.

Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

AIDA-1/ANKS1B Binds to the SynGAP Family RasGAPs with High Affinity and Specificity.

AIDA-1, encoded by ANKS1B, is an abundant postsynaptic scaffold protein essential for brain development. Mutations of ANKS1B are closely associated with various psychiatric disorders. However, very little is known regarding the molecular mechanisms underlying AIDA-1's involvements under physiological and pathophysiological conditions. Here, we discovered an interaction between AIDA-1 and the SynGAP family Ras-GTPase activating protein (GAP) via affinity purification using AIDA-1d as the bait. Biochemical studies showed that the PTB domain of AIDA-1 binds to an extended NPx[F/Y]-motif of the SynGAP family proteins with high affinities. The high-resolution crystal structure of AIDA-1 PTB domain in complex with the SynGAP NPxF-motif revealed the molecular mechanism governing the specific interaction between AIDA-1 and SynGAP. Our study not only explains why patients with ANKS1B or SYNGAP1 mutations share overlapping clinical phenotypes, but also allows identification of new AIDA-1 binding targets such as Ras and Rab interactors.

The Enigma of Tau Protein Aggregation: Mechanistic Insights and Future Challenges.

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.

Super-Resolution Imaging of Tau Proteins in Isolated and Immobilized Brain Synaptosomes.

Tau protein has important physiological functions at both presynaptic and postsynaptic terminals. Pathological tau species are also associated with synaptic dysfunctions in several neurodegenerative disorders, especially Alzheimer's disease. To understand tau distribution inside synaptic compartments, super-resolution imaging is required. Here, we describe a facile protocol to immobilize and image brain synaptosomes without aggregation artefacts, by substituting the standard fixative paraformaldehyde with ethylene glycol bis(succinimidyl succinate) (EGS). Super-resolution imaging of tau proteins is achieved through three-color direct stochastic optical reconstruction microscopy (dSTORM). Tau protein is found to colocalize with synaptic vesicles as well as postsynaptic densities.

Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Role of C9orf72 hexanucleotide repeat expansions in ALS/FTD pathogenesis.

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.

The N-terminal region of Cdc6 specifically recognizes human DNA G-quadruplex.

Guanine (G)-rich nucleic acid sequences can form diverse G-quadruplex structures located in functionally significant genome regions, exerting regulatory control over essential biological processes, including DNA replication in vivo. During the initiation of DNA replication, Cdc6 is recruited by the origin recognition complex (ORC) to target specific chromosomal DNA sequences. This study reveals that human Cdc6 interacts with G-quadruplex structure through a distinct region within the N-terminal intrinsically disordered region (IDR), encompassing residues 7-20. The binding region assumes a hook-type conformation, as elucidated by the NMR solution structure in complex with htel21T18. Significantly, mutagenesis and in vivo investigations confirm the highly specific nature of Cdc6's recognition of G-quadruplex. This research enhances our understanding of the fundamental mechanism governing the interaction between G-quadruplex and the N-terminal IDR region of Cdc6, shedding light on the intricate regulation of DNA replication processes.

CaMKII autophosphorylation but not downstream kinase activity is required for synaptic memory.

CaMKII plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of kinase activity. Thus, autophosphorylation, together with binding to the GluN2B subunit, are the only two requirements for CaMKII in synaptic memory.

Differential roles of CaMKII isoforms in phase separation with NMDA receptors and in synaptic plasticity.

Calcium calmodulin-dependent kinase II (CaMKII) is critical for synaptic transmission and plasticity. Two major isoforms of CaMKII, CaMKIIα and CaMKIIß, 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 CaMKIIß (double knockout [DKO]). Remarkably, although CaMKIIß 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.

Molecular features underlying differential SHP1/SHP2 binding of immune checkpoint receptors.

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.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation.

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 Ca2+ 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, Ca2+-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.

CaMKIIα-driven, phosphatase-checked postsynaptic plasticity via phase separation.

Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) is essential for synaptic plasticity and learning by decoding synaptic Ca2+ 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 Ca2+ 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 Ca2+ concentration induces GluN2B-mediated recruitment of active CaMKIIα and formation of the CaMKIIα/GluN2B/PSD-95 condensates, which are autonomously dispersed upon Ca2+ removal. Protein phosphatases control the Ca2+-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, Ca2+-induced and phosphatase-checked shuttling of CaMKIIα between distinct PSD nano-domains can regulate phase separation-mediated PSD assembly and synaptic plasticity.

Pyruvate kinase from Plasmodium falciparum: Structural and kinetic insights into the allosteric mechanism.

During its intra-erythrocytic growth phase, the malaria parasite Plasmodium falciparum relies heavily on glycolysis for its energy requirements. Pyruvate kinase (PYK) is essential for regulating glycolytic flux and for ATP production, yet the allosteric mechanism of P. falciparum PYK (PfPYK) remains poorly understood. Here we report the first crystal structure of PfPYK in complex with substrate analogues oxalate and the ATP product. Comparisons of PfPYK structures in the active R-state and inactive T-state reveal a 'rock-and-lock' allosteric mechanism regulated by rigid-body rotations of each subunit in the tetramer. Kinetic data and structural analysis indicate glucose 6-phosphate is an activator by increasing the apparent maximal velocity of the enzyme. Intriguingly, the trypanosome drug suramin inhibits PfPYK, which points to glycolysis as a set of potential therapeutic targets against malaria.

Liquid-Liquid Phase Separation in Neuronal Development and Synaptic Signaling.

Formation of biomolecular condensates that are not enclosed by membranes via liquid-liquid phase separation (LLPS) is a general strategy that cells adopt to organize membraneless subcellular compartments for diverse functions. Neurons are highly polarized with elaborate branching and functional compartmentalization of their neurites, thus, raising additional demand for the proper subcellular localization of both membraneless and membrane-based organelles. Recent studies have provided evidence that several protein assemblies involved in the establishment of neuronal stem cell (NSC) polarity and in the asymmetric division of NSCs form distinct molecular condensates via LLPS. In synapses of adult neurons, molecular apparatuses controlling presynaptic neurotransmitter release and postsynaptic signaling transmission are also likely formed via LLPS. These molecular condensates, though not enclosed by lipid bilayers, directly associate with plasma membranes or membrane-based organelles, indicating that direct communication between membraneless and membrane-based organelles is a common theme in neurons and other types of cells.

Structural Basis for the High-Affinity Interaction between CASK and Mint1.

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.

Shank3 Binds to and Stabilizes the Active Form of Rap1 and HRas GTPases via Its NTD-ANK Tandem with Distinct Mechanisms.

Shank1/2/3, major scaffold proteins in excitatory synapses, are frequently mutated in patients with psychiatric disorders. Although the Shank N-terminal domain and ankyrin repeats domain tandem (NTD-ANK) is known to bind to Ras and Rap1, the molecular mechanism underlying and functional significance of the bindings in synapses are unknown. Here, we demonstrate that Shank3 NTD-ANK specifically binds to the guanosine triphosphate (GTP)-bound form of HRas and Rap1. In addition to the canonical site mediated by the Ras-association domain and common to both GTPases, Shank3 contains an unconventional Rap1 binding site formed by NTD and ANK together. Binding of Shank3 to the GTP-loaded Rap1 slows down its GTP hydrolysis by SynGAP. We further show that the interactions between Shank3 and HRas/Rap1 at excitatory synapses are promoted by synaptic activation. Thus, Shank3 may be able to modulate signaling of the Ras family proteins via directly binding to and stabilizing the GTP-bound form of the enzymes.

Pyruvate Kinase Regulates the Pentose-Phosphate Pathway in Response to Hypoxia in Mycobacterium tuberculosis.

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate-pyruvate-oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb.

The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA.

Human telomeric guanine-rich DNA, which could adopt different G-quadruplex structures, plays important roles in protecting the cell from recombination and degradation. Although many of these structures were determined, the chair-type G-quadruplex structure remains elusive. Here, we present a crystal structure of the G-quadruplex composed of the human telomeric sequence d[GGGTTAGG8GTTAGGGTTAGG20G] with two dG to 8Br-dG substitutions at positions 8 and 20 with syn conformation in the K+ solution. It forms a novel three-layer chair-type G-quadruplex with two linking trinucleotide loops. Particularly, T5 and T17 are coplanar with two water molecules stacking on the G-tetrad layer in a sandwich-like mode through a coordinating K+ ion and an A6•A18 base pair. While a twisted Hoogsteen A12•T10 base pair caps on the top of G-tetrad core. The three linking TTA loops are edgewise and each DNA strand has two antiparallel adjacent strands. Our findings contribute to a deeper understanding and highlight the unique roles of loop and water molecule in the folding of the G-quadruplex.