Base extrusion is a major structural feature at the junction between B- and Z-DNA (the B-Z junction) where a base pair is broken, and the two bases are extruded from the double helix. Despite the demonstration of base extrusion at the B-Z junction, it is not clear whether a similar base extrusion occurs at other types of junctions involving the left-handed Z conformation. Here, we investigate structural changes of bases at three Z-form junctions: DNA B-Z and Z-Z and RNA A-Z junctions. By monitoring fluorescently labeled duplex nucleic acids using 2-aminopurines at various positions relative to the junction point, we show that base extrusion occurs not only at the DNA B-Z junction, but also at the RNA A-Z and DNA Z-Z junctions. Our data suggest that base extrusion is a general feature of Z-form nucleic-acid junctions.

Synaptic adhesion molecules regulate synapse development and plasticity through mechanisms that include trans-synaptic adhesion and recruitment of diverse synaptic proteins. We found that the immunoglobulin superfamily member 11 (IgSF11), a homophilic adhesion molecule that preferentially expressed in the brain, is a dual-binding partner of the postsynaptic scaffolding protein PSD-95 and AMPA glutamate receptors (AMPARs). IgSF11 required PSD-95 binding for its excitatory synaptic localization. In addition, IgSF11 stabilized synaptic AMPARs, as determined by IgSF11 knockdown-induced suppression of AMPAR-mediated synaptic transmission and increased surface mobility of AMPARs, measured by high-throughput, single-molecule tracking. IgSF11 deletion in mice led to the suppression of AMPAR-mediated synaptic transmission in the dentate gyrus and long-term potentiation in the CA1 region of the hippocampus. IgSF11 did not regulate the functional characteristics of AMPARs, including desensitization, deactivation or recovery. These results suggest that IgSF11 regulates excitatory synaptic transmission and plasticity through its tripartite interactions with PSD-95 and AMPARs.

The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesion molecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesion ligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTP was capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network.

The abscisic acid (ABA) signaling pathway is regulated by clade A type 2C protein phosphatases (PP2CAs) in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors (PYL/RCARs) for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxGΦL motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2C50 with several OsPYL/RCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML ("FM") mutant, lowered the affinity to OsPYL/RCAR3 by ∼15-fold in comparison with the wild-type. Comparison of the crystal structures of wild-type OsPP2C50:ABA:OsPYL/RCAR3 with those composed of FM mutant revealed local conformational changes near the VxGΦL motif, further supported by hydrogen-deuterium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxGΦL motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C50FM showed altered ABA sensitivity. Taken together, the VxGΦL motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.

Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.

Synaptic adhesion molecules regulate synapse development through trans-synaptic adhesion and assembly of diverse synaptic proteins. Many synaptic adhesion molecules positively regulate synapse development; some, however, exert negative regulation, although such cases are relatively rare. In addition, synaptic adhesion molecules regulate the amplitude of post-synaptic receptor responses, but whether adhesion molecules can regulate the kinetic properties of post-synaptic receptors remains unclear. Here we report that Clmp, a homophilic adhesion molecule of the Ig domain superfamily that is abundantly expressed in the brain, reaches peak expression at a neonatal stage (week 1) and associates with subunits of AMPA receptors (AMPARs) and kainate receptors (KARs). Clmp deletion in mice increased the frequency and amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) and the frequency, amplitude, and decay time constant of KAR-mediated mEPSCs in hippocampal CA3 neurons. Clmp deletion had minimal impacts on evoked excitatory synaptic currents at mossy fiber-CA3 synapses but increased extrasynaptic KAR, but not AMPAR, currents, suggesting that Clmp distinctly inhibits AMPAR and KAR responses. Behaviorally, Clmp deletion enhanced novel object recognition and susceptibility to kainate-induced seizures, without affecting contextual or auditory cued fear conditioning or pattern completion-based contextual fear conditioning. These results suggest that Clmp negatively regulates hippocampal excitatory synapse development and AMPAR and KAR responses in the neonatal hippocampal CA3 as well as object recognition and kainate seizure susceptibility in mice.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Synaptic adhesion molecules regulate various aspects of synapse development, function and plasticity. These functions mainly involve trans-synaptic interactions and positive regulations, whereas cis-interactions and negative regulation are less understood. Here we report that SALM4, a member of the SALM/Lrfn family of synaptic adhesion molecules, suppresses excitatory synapse development through cis inhibition of SALM3, another SALM family protein with synaptogenic activity. Salm4-mutant (Salm4(-/-)) mice show increased excitatory synapse numbers in the hippocampus. SALM4 cis-interacts with SALM3, inhibits trans-synaptic SALM3 interaction with presynaptic LAR family receptor tyrosine phosphatases and suppresses SALM3-dependent presynaptic differentiation. Importantly, deletion of Salm3 in Salm4(-/-) mice (Salm3(-/-); Salm4(-/-)) normalizes the increased excitatory synapse number. These results suggest that SALM4 negatively regulates excitatory synapses via cis inhibition of the trans-synaptic SALM3-LAR adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in vivo. These findings collectively provide structural insights into the mechanism by which MDGAs negatively modulate synapse development governed by NLs/neurexins.

BZ junctions, which connect B-DNA to Z-DNA, are necessary for local transformation of B-DNA to Z-DNA in the genome. However, the limited information on the junction-forming sequences and junction structures has led to a lack of understanding of the structural diversity and sequence preferences of BZ junctions. We determined three crystal structures of BZ junctions with diverse sequences followed by spectroscopic validation of DNA conformation. The structural features of the BZ junctions were well conserved regardless of sequences via the continuous base stacking through B-to-Z DNA with A-T base extrusion. However, the sequence-dependent structural heterogeneity of the junctions was also observed in base step parameters that are correlated with steric constraints imposed during Z-DNA formation. Further, circular dichroism and fluorescence-based analysis of BZ junctions revealed that a base extrusion was only found at the A-T base pair present next to a stable dinucleotide Z-DNA unit. Our findings suggest that Z-DNA formation in the genome is influenced by the sequence preference for BZ junctions.

The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease1-3. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown4-6. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages4,5. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca2+ concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.

KIF1A is a brain-specific anterograde motor protein that transports cargoes towards the plus-ends of microtubules. Many variants of the KIF1A gene have been associated with neurodegenerative diseases and developmental delay. Homozygous mutations of KIF1A have been identified in a recessive subtype of hereditary spastic paraplegia (HSP), SPG30. In addition, KIF1A mutations have been found in pure HSP with autosomal dominant inheritance. Here we report the first case of familial complicated HSP with a KIF1A mutation transmitted in autosomal dominant inheritance. A heterozygous p.T258M mutation in KIF1A was found in a Korean family through targeted exome sequencing. They displayed phenotypes of mild intellectual disability with language delay, epilepsy, optic nerve atrophy, thinning of corpus callosum, periventricular white matter lesion, and microcephaly. A structural modeling revealed that the p.T258M mutation disrupted the binding of KIF1A motor domain to microtubules and its movement along microtubules. Assays of peripheral accumulation and proximal distribution of KIF1A motor indicated that the KIF1A motor domain with p.T258M mutation has reduced motor activity and exerts a dominant negative effect on wild-type KIF1A. These results suggest that the p.T258M mutation suppresses KIF1A motor activity and induces complicated HSP accompanying intellectual disability transmitted in autosomal dominant inheritance.

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD+ binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.

Shank3, a postsynaptic scaffolding protein involved in regulating excitatory synapse assembly and function, has been implicated in several brain disorders, including autism spectrum disorders (ASD), Phelan-McDermid syndrome, schizophrenia, intellectual disability, and mania. Here we generated and characterized a Shank3 knock-in mouse line carrying the Q321R mutation (Shank3 Q321R mice) identified in a human individual with ASD that affects the ankyrin repeat region (ARR) domain of the Shank3 protein. Homozygous Shank3 Q321R/Q321R mice show a selective decrease in the level of Shank3a, an ARR-containing protein variant, but not other variants. CA1 pyramidal neurons in the Shank3 Q321R/Q321R hippocampus show decreased neuronal excitability but normal excitatory and inhibitory synaptic transmission. Behaviorally, Shank3 Q321R/Q321R mice show moderately enhanced self-grooming and anxiolytic-like behavior, but normal locomotion, social interaction, and object recognition and contextual fear memory. In addition, these mice show abnormal electroencephalogram (EEG) patterns and decreased susceptibility to induced seizures. These results indicate that the Q321R mutation alters Shank3 protein stability, neuronal excitability, repetitive and anxiety-like behavior, EEG patterns, and seizure susceptibility in mice.