N-terminal Coiled-coil Domain Research Articles

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants invivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Structure of the N-terminal coiled-coil domains of the ciliary protein Rpgrip1l.

Rpgrip1l is one of the key ciliary proteins located at the transition zone of the primary cilium, an important organelle for cells to sense the outer environment. Mutations in the RPGRIP1L gene are associated with various ciliopathies. Here, we focused on the N-terminal coiled-coil of Rpgrip1l. By comprehensive biochemical and structural characterizations, we demonstrated that the two predicted coiled-coil regions (CC12) located at Rpgrip1l N-terminus each can form a stable parallel dimer. We further showed that overexpression of Rpgrip1l CC12 in NIH/3T3 cells significantly shortened the length of primary cilia, and this effect depended on the dimer formation. In addition, we found that CC12 of the homolog protein Rpgrip1 in mouse and human were significantly different from Rpgrip1l. Finally, we confirmed that some disease-related mutations can alter the dimeric states of CC12 of Rpgrip1l or Rpgrip1, which might explain the pathogenic mechanisms.

PROTEIN S‐ACYL TRANSFERASE 13/16 modulate disease resistance by S‐acylation of the nucleotide binding, leucine‐rich repeat protein R5L1 in Arabidopsis

Nucleotide binding, leucine-rich repeat (NB-LRR) proteins are critical for disease resistance in plants, while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Of the five mutants that were hyper-susceptible to the pathogen, three (R5L1, R5L2 and RPS5) proteins contain the conserved S-acylation site in the N-terminal coiled-coil (CC) domain. In wild-type (WT) Arabidopsis plants, R5L1 was transcriptionally activated upon pathogen infection, and R5L1 overexpression lines had enhanced resistance. Independent experiments indicated that R5L1 localized at the plasma membrane (PM) via S-acylation of its N-terminal CC domain, which was mediated by PROTEIN S-ACYL TRANSFERASE 13/16 (PAT13, PAT16). Modification of the S-acylation site reduced its affinity for binding the PM, with a consequent significant reduction in bacterial resistance. PM localization of R5L1 was significantly reduced in pat13 and pat16 mutants, similar to what was found for WT plants treated with 2-bromopalmitate, an S-acylation-blocking agent. Transgenic plants expressing R5L1 in the pat13 pat16 double mutant showed no enhanced disease resistance. Overexpression of R5L1 in WT Arabidopsis resulted in substantial accumulation of reactive oxygen species after inoculation with Pst DC3000; this effect was not observed with a mutant R5L1 carrying a mutated S-acylation site. Our data suggest that PAT13- and PAT16-mediated S-acylation of R5L1 is crucial for its membrane localization to activate the plant defense response.

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Multi-Omics Analyses Reveal the Regulatory Network and the Function of ZmUGTs in Maize Defense Response

Maize is one of the major crops in the world; however, diseases caused by various pathogens seriously affect its yield and quality. The maize Rp1-D21 mutant (mt) caused by the intragenic recombination between two nucleotide-binding, leucine-rich repeat (NLR) proteins, exhibits autoactive hypersensitive response (HR). In this study, we integrated transcriptomic and metabolomic analyses to identify differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in Rp1-D21 mt compared to the wild type (WT). Genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were enriched among the DEGs. The salicylic acid (SA) pathway and the phenylpropanoid biosynthesis pathway were induced at both the transcriptional and metabolic levels. The DAMs identified included lipids, flavones, and phenolic acids, including 2,5-DHBA O-hexoside, the production of which is catalyzed by uridinediphosphate (UDP)-dependent glycosyltransferase (UGT). Four maize UGTs (ZmUGTs) homologous genes were among the DEGs. Functional analysis by transient co-expression in Nicotiana benthamiana showed that ZmUGT9250 and ZmUGT5174, but not ZmUGT9256 and ZmUGT8707, partially suppressed the HR triggered by Rp1-D21 or its N-terminal coiled-coil signaling domain (CCD21). None of the four ZmUGTs interacted physically with CCD21 in yeast two-hybrid or co-immunoprecipitation assays. We discuss the possibility that ZmUGTs might be involved in defense response by regulating SA homeostasis.

Dynamic localization of a helper NLR at the plant–pathogen interface underpins pathogen recognition

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. Here, we show that NRC4, from the "NRC" solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extrahaustorial membrane (EHM), presumably to mediate response to perihaustorial effectors that are recognized by NRC4-dependent sensor NLRs. However, not all NLRs accumulate at the EHM, as the closely related helper NRC2 and the distantly related ZAR1 did not accumulate at the EHM. NRC4 required an intact N-terminal coiled-coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that postactivation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection, however, NRC4 forms puncta mainly at the EHM and, to a lesser extent, at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation, whereas some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.

The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome

Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1–6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis–driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.

Maize metacaspases modulate the defense response mediated by the NLR protein Rp1-D21 likely by affecting its subcellular localization.

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CCD21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CCD21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CCD21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Enterococcus NlpC/p60 Peptidoglycan Hydrolase SagA Localizes to Sites of Cell Division and Requires Only a Catalytic Dyad for Protease Activity.

Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase-secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a triseptal structure in dividing cells of enterococci and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases.

GALNT2 regulates ANGPTL3 cleavage in cells and in vivo of mice

Angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of lipoprotein lipase and endothelial lipase that plays critical roles in lipoprotein metabolism. It specifically expresses in the liver and undergoes proprotein convertase-mediated cleavage during secretion, which generates an N-terminal coiled-coil domain and C-terminal fibrinogen-like domain that has been considered as the activation step for its function. Previous studies have reported that the polypeptide GalNAc-transferase GALNT2 mediates the O-glycosylation of the ANGPTL3 near the cleavage site, which inhibits the proprotein convertase (PC)-mediated cleavage in vitro and in cultured cells. However, loss-of-function mutation for GALNT2 has no effect on ANGPTL3 cleavage in human. Thus whether GALNT2 regulates the cleavage of ANGPTL3 in vivo is unclear. In present study, we systematically characterized the cleavage of Angptl3 in cultured cells and in vivo of mice. We found that endogenous Angptl3 is cleaved in primary hepatocytes and in vivo of mice, and this cleavage can be blocked by Galnt2 overexpression or PC inhibition. Moreover, suppressing galnt2 expression increases the cleavage of Angptl3 in mice dramatically. Thus, our results support the conclusion that Galnt2 is a key endogenous regulator for Angptl3 cleavage both in vitro and in vivo.

ROP INTERACTIVE PARTNER b Interacts with RACB and Supports Fungal Penetration into Barley Epidermal Cells.

Rho of Plants (ROP) G-proteins are key components of cell polarization processes in plant development. The barley (Hordeum vulgare) ROP protein RACB is a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh). RACB also drives polar cell development, and this function might be coopted during the formation of fungal haustoria in barley epidermal cells. To understand RACB signaling during the interaction of barley with Bgh, we searched for potential downstream interactors of RACB. Here, we show that ROP INTERACTIVE PARTNER b (RIPb; synonym: INTERACTOR OF CONSTITUTIVE ACTIVE ROP b) directly interacts with RACB in yeast and in planta. Overexpression of RIPb supports the susceptibility of barley to Bgh RIPb further interacts with itself at microtubules. However, the interaction with activated RACB largely takes place at the plasma membrane. Both RIPb and RACB are recruited to the site of fungal attack around the neck of developing haustoria, suggesting locally enhanced ROP activity. We further assigned different functions to different domains of the RIPb protein. The N-terminal coiled-coil CC1 domain is required for microtubule localization, while the C-terminal coiled-coil CC2 domain is sufficient to interact with RACB and to fulfill a function in susceptibility at the plasma membrane. Hence, RIPb appears to be localized at microtubules and is then recruited by activated RACB for a function at the plasma membrane during formation of the haustorial complex.

Putative Coiled-Coil Domain-Dependent Autoinhibition and Alternative Splicing Determine SHTN1’s Actin-Binding Activity

The actin cytoskeleton plays a pivotal role in cell development, morphogenesis, and other cellular functions. Precise control of actin dynamics requires actin-binding proteins. Here, we characterize multifarious regulation of SHTN1 (shootin1) and show that, unlike known actin-binding proteins, SHTN1’s actin binding activity is intrinsically inhibited by a putative coiled-coil domain (CCD) and the autoinhibition is overcome by alternative splicing regulation. We found SHTN1 contains a noncanonical WH2 domain and an upstream proline-rich region (PRR) that by themselves are sufficient for actin interaction. Alternative splicing of Shtn1 at the C terminus and downstream of the WH2-PRR domain produces a long (SHTN1L or shootin1b) and a short (SHTN1S or shootin1a) isoform, which both contain the described PRR and WH2 domains. However, SHTN1S does not interact with actin due to inhibition mediated by an N-terminal CCD. A SHTN1L-specific C-terminal motif counters the intramolecular inhibition and allows SHNT1L to bind actin. A nuclear localization signal is embedded between PRR and WH2 and is subject to similar autoinhibition. SHTN1 would be the first WH2-containing molecule that adopts CCD-dependent autoinhibition and alternative splicing-dependent actin interaction.

Rpa1 mediates an immune response to avrRpm1Psa and confers resistance against Pseudomonas syringae pv. actinidiae.

The type three effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola (Pma) triggers an RPM1-mediated immune response linked to phosphorylation of RIN4 (RPM1-interacting protein 4) in Arabidopsis. However, the effector-resistance (R) gene interaction is not well established with different AvrRpm1 effectors from other pathovars. We investigated the AvrRpm1-triggered immune responses in Nicotiana species and isolated Rpa1 (Resistance to Pseudomonas syringae pv. actinidiae 1) via a reverse genetic screen in Nicotiana tabacum. Transient expression and gene silencing were performed in combination with co-immunoprecipitation and growth assays to investigate the specificity of interactions that lead to inhibition of pathogen growth. Two closely related AvrRpm1 effectors derived from Pseudomonas syringae pv. actinidiae biovar 3 (AvrRpm1Psa ) and Pseudomonas syringae pv. syringae strain B728a (AvrRpm1Psy ) trigger immune responses mediated by RPA1, a nucleotide-binding leucine-rich repeat protein with an N-terminal coiled-coil domain. In a display of contrasting specificities, RPA1 does not respond to AvrRpm1Pma , and correspondingly AvrRpm1Psa and AvrRpm1Psy do not trigger the RPM1-mediated response, demonstrating that separate R genes mediate specific immune responses to different AvrRpm1 effectors. AvrRpm1Psa co-immunoprecipitates with RPA1, and both proteins co-immunoprecipitate with RIN4. In contrast with RPM1, however, RPA1 was not activated by the phosphomimic RIN4T166D and silencing of RIN4 did not affect the RPA1 activity. Delivery of AvrRpm1Psa by Pseudomonas syringae pv. tomato (Pto) in combination with transient expression of Rpa1 resulted in inhibition of the pathogen growth in N. benthamiana. Psa growth was also inhibited by RPA1 in N. tabacum.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

The polarity protein Dlg5 regulates collective cell migration during Drosophila oogenesis.

Collective migration plays critical roles in animal development, physiological events, and cancer metastasis. However, the molecular mechanisms of collective cell migration are not well understood. Drosophila border cells represent an excellent in vivo genetic model to study collective cell migration and identify novel regulatory genes for cell migration. Using the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, we screened 240 P-element insertion lines to identify essential genes for border cell migration. Two genes were uncovered, including dlg5 (discs large 5) and CG31689. Further analysis showed that Dlg5 regulates the apical-basal polarity and cluster integrity in border cell clusters. Dlg5 is enriched in lateral surfaces between border cells and central polar cells but also shows punctate localization between border cells. We found that the distribution of Dlg5 in border cell clusters is regulated by Armadillo. Structure-function analysis revealed that the N-terminal Coiled-coil domain and the C-terminal PDZ3-PDZ4-SH3-GUK domains but not the PDZ1-PDZ2 domains of Dlg5 are required for BC migration. The Coiled-coil domain and the PDZ4-SH3-GUK domains are critical for Dlg5’s cell surface localization in border cell clusters.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum.

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Calcium-induced dissociation of CIB1 from ASK1 regulates agonist-induced activation of the p38 MAPK pathway in platelets.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) β/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains.

Structural characterization of a putative diguanylate cyclase conserved in hyperthermophiles

C-di-GMP, bis-(3′-5′)-cyclic dimeric guanosine monophosphate, is a key signaling molecule that regulates many important physiological processes in bacteria. C-di-GMP is synthesized by diguanylate cyclase (DGC) containing the homodimeric GGDEF domain. There are many uncharacterized hypothetical proteins annotated as a putative DGC in bacteria including hyperthermophiles; however, their structures still remain unexplored. Here, we solved the crystal structure of the GGDEF-like domain of Tm0107 protein from Thermotoga maritima at a resolution of 2.1 Å, which shares sequence similarities with DGC proteins in other bacteria. Tm0107 consists of an N-terminal coiled-coil and C-terminal GGDEF-like domain. We showed that the GGDEF-like domain of Tm0107 exists as monomer in solution and is structurally similar to other GGDEF domains. Two zinc ions are coordinated at the interface between two Tm0107 monomers. Based on our measurements of the Stokes radii of Tm0107 by analytical gel filtration, we propose a dimer model of Tm0107 containing both the N-terminal coiled coil and C-terminal GGDEF-like domains. Based on the model, Tm0107 forms a homodimer in a manner different compared to other structurally characterized DGC proteins. These results provide useful structural information about putative DGC proteins containing protein sequences similar to that of Tm0107, which is widely conserved in hyperthermophiles.