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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. Phase separation-mediated formation of biological condensates are a powerful means of concentrating and localizing various molecular assemblies to specific subcellular regions in cells. Wu et al. review recent progresses of phase separation in forming membraneless biological condensates that regulate neuronal stem cell development as well as synaptic signaling in developed neurons. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • 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. [ABSTRACT FROM AUTHOR]

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. • 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. [ABSTRACT FROM AUTHOR]

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. • Shank3 binds to Rap1 with an unexpected mode and a 1:2 stoichiometry • Shank3 binds to HRas via a canonical binding mode and a 1:1 stoichiometry • Binding of Shank3 prevents Rap1 GTP hydrolysis by SynGAP • Binding of Shank3 to HRas or Rap1 is promoted upon synapse activation Cai et al. report that Shank3 N-terminal NTD-ANK tandem forms an integral structural supramodule, binds to two copies of Rap1 with distinct modes, and prevents Rap1 GTP hydrolysis catalyzed by SynGAP. [ABSTRACT FROM AUTHOR]

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,β-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 . [ABSTRACT FROM AUTHOR]