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.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

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.

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.

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.

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.

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.

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.

Fragment-wise design of inhibitors to 3C proteinase from enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD), which can spread its infection to central nervous and other systems with severe consequence. A key factor in the replication of EV71 is its 3C proteinase (3C(pro)), a significant drug target. Peptidomimetics were employed as inhibitors of this enzyme for developing antivirals. However, the peptide bonds in these peptidomimetics are a source of low bioavailability due to their susceptibility to protease digestion. To produce non-peptidomimetic inhibitors by replacing these peptide bonds, it would be important to gain better understanding on the contribution of each component to the interaction and potency. METHODS: A series of compounds of different lengths targeting 3C(pro) and having an α,ß-unsaturated ester as the warhead were synthesized and their interactions with the enzyme were evaluated by complex structure analyses and potency assays for a better understanding on the relationship between potency and evolution of interaction. RESULTS: The P2 moiety of the compound would need to be oriented to interact in the S2 site in the substrate binding cleft and the P3-P4 moieties were required to generate sufficient potency. A hydrophobic terminal group will benefit the cellular uptake and improve the activity in vivo. CONCLUSIONS AND GENERAL SIGNIFICANCE: The data presented here provide a basis for designing a new generation of non-peptidomimetics to target EV71 3C(pro).

Structural basis for targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide.

Pyrazinamide (PZA) is a first-line drug for tuberculosis (TB) treatment and is responsible for shortening the duration of TB therapy. The mode of action of PZA remains elusive. RpsA, the ribosomal protein S1 of Mycobacterium tuberculosis (Mtb), was recently identified as a target of PZA based on its binding activity to pyrazinoic acid (POA), the active form of PZA. POA binding to RpsA led to the inhibition of trans-translation. However, the nature of the RpsA-POA interaction remains unknown. Key questions include why POA exhibits an exquisite specificity to RpsA of Mtb and how RpsA mutations confer PZA resistance. Here, we report the crystal structures of the C-terminal domain of RpsA of Mtb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance. Structural analysis reveals that POA binds to RpsA through hydrogen bonds and hydrophobic interactions, mediated mainly by residues (Lys303, Phe307, Phe310 and Arg357) that are essential for tmRNA binding. Conformational changes induced by mutation or sequence variation at the C-terminus of RpsA abolish the POA binding activity. Our findings provide insights into the mode of action of PZA and molecular basis of PZA resistance associated with RpsA mutations.

Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71.

Enterovirus 71 (EV71) is the causative agent of hand, foot and mouth disease and can spread its infections to the central nervous and other systems with severe consequences. The replication of EV71 depends on its 3C proteinase (3Cpro ), a significant drug target. By X-ray crystallography and functional assays, the interactions between inhibitors and EV71 3Cpro were evaluated. It was shown that improved interactions at S4 for the substrate binding could significantly enhance the potency. A new series of potent inhibitors with high ligand efficiency was generated for developing antivirals to treat and control the EV71-associated diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Design, synthesis, and biological evaluation of anti-EV71 agents.

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), which can spread its infections to the central nervous and other systems with severe consequences. In this article, design, chemical synthesis, and biological evaluation of various anti-EV71 agents which incorporate Michael acceptors are described. Further SAR study demonstrated that lactone type of Michael acceptor provided a new lead of anti-EV71 drug candidates with high anti-EV71 activity in cell-based assay and enhanced mouse plasma stability. One of the most potent compounds (2K, cell-based anti-EV71 EC50=0.028µM), showed acceptable stability profile towards mouse plasma, which resulted into promising pharmacokinetics in mouse via IP administration.

Conformational plasticity of the 2A proteinase from enterovirus 71.

The 2A proteinase (2A(pro)) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A(pro) from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A(pro) was comprised of an N-terminal domain of a four-stranded antiparallel ß sheet and a C-terminal domain of a six-stranded antiparallel ß barrel with a tightly bound zinc atom. Unlike in other 2A(pro) structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A(pro) of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII ß strands. This was supported by molecular dynamic simulation. The structural variation among different 2A(pro) structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.

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.

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.

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 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.

Structures of Enterovirus 71 3C proteinase (strain E2004104-TW-CDC) and its complex with rupintrivir.

The crystal structure of 3C proteinase (3C(pro)) from Enterovirus 71 (EV71) was determined in space group C2221 to 2.2 Šresolution. The fold was similar to that of 3C(pro) from other picornaviruses, but the difference in the ß-ribbon reported in a previous structure was not observed. This ß-ribbon was folded over the substrate-binding cleft and constituted part of the essential binding sites for interaction with the substrate. The structure of its complex with rupintrivir (AG7088), a peptidomimetic inhibitor, was also characterized in space group P212121 to 1.96 Šresolution. The inhibitor was accommodated without any spatial hindrance despite the more constricted binding site; this was confirmed by functional assays, in which the inhibitor showed comparable potency towards EV71 3C(pro) and human rhinovirus 3C(pro), which is the target that rupintrivir was designed against.

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.

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.