Large Multiprotein Complexes Research Articles

Solid-state biophotovoltaic cells containing photosystem I.

The large multiprotein complex, photosystem I (PSI), which is at the heart of light-dependent reactions in photosynthesis, is integrated as the active component in a solid-state organic photovoltaic cell. These experiments demonstrate that photoactive megadalton protein complexes are compatible with solution processing of organic-semiconductor materials and operate in a dry non-natural environment that is very different from the biological membrane.

Elution Profile Analysis of SDS-induced Subcomplexes by Quantitative Mass Spectrometry

Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.

Regulation of focal adhesion formation by a vinculin-Arp2/3 hybrid complex.

Focal adhesions (FAs) are large multi-protein complexes that act as transmembrane links between the extracellular matrix and the actin cytoskeleton. Recently, FAs were extensively characterized, yet the molecular mechanisms underlying their mechanical and signalling functions remain unresolved. To address this question, we isolated protein complexes containing different FA components, from chicken smooth muscle, and characterized their properties. Here we identified 'hybrid complexes' consisting of the actin-nucleating subunits of Arp2/3 and either vinculin or vinculin and α-actinin. We further show that suppression of p41-ARC, a central component of native Arp2/3, which is absent from the hybrid complexes, increases the levels of the Arp2/3-nucleating core in FA sites and stimulates FA growth and dynamics. In contrast, overexpression of p41-ARC adversely affects FAs. These results support the view that Arp2/3 can form modular 'hybrid complexes' containing an actin-nucleating 'functional core', and 'anchoring domains' (vinculin/p41-ARC) that regulate its subcellular localization.

A catalytic-independent role for the LUBAC in NF-κB activation upon antigen receptor engagement and in lymphoma cells

AbstractAntigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell–like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.

Phylogenetic Distribution and Evolution of the Linked RNA-Binding and NOT1-Binding Domains in the Tristetraprolin Family of Tandem CCCH Zinc Finger Proteins

In humans, the tristetraprolin or TTP family of CCCH tandem zinc finger (TZF) proteins comprises 3 members, encoded by the genes ZFP36, ZFP36L1, and ZFP36L2. These proteins have direct orthologues in essentially all vertebrates studied, with the exception of birds, which appear to lack a version of ZFP36. Additional family members are found in rodents, amphibians, and fish. In general, the encoded proteins contain 2 critical macromolecular interaction domains: the CCCH TZF domain, which is necessary for high-affinity binding to AU-rich elements in mRNA; and an extreme C-terminal domain that, in the case of TTP, interacts with NOT1, the scaffold of a large multi-protein complex that contains deadenylases. TTP and its related proteins act by first binding to AU-rich elements in mRNA, and then recruiting deadenylases to the mRNA, where they can processively remove the adenosine residues from the poly(A) tail. Highly conserved TZF domains have been found in unicellular eukaryotes such as yeasts, and these domains can bind AU-rich elements that resemble those bound by the mammalian proteins. However, certain fungi appear to lack proteins with intact TZF domains, and the TTP family proteins that are expressed in other fungi often lack the characteristic C-terminal NOT1 binding domain found in the mammalian proteins. For these reasons, we investigated the phylogenetic distribution of the relevant sequences in available databases. Both domains are present in family member proteins from most lineages of eukaryotes, suggesting their mutual presence in a common ancestor. However, the vertebrate type of NOT1-binding domain is missing in most fungi, and the TZF domain itself has disappeared or degenerated in recently evolved fungi. Nonetheless, both domains are present together in the proteins from several unicellular eukaryotes, including at least 1 fungus, and they seem to have remained together during the evolution of metazoans.

Regulation of vesicle trafficking: GTPases and friends

With the 2013 Nobel Prize awarded to scientists who uncovered mechanisms of vesicular trafficking, the next big question in the field is how this process is regulated. Talks in the Minisymposium on “Regulation of Vesicle Trafficking” highlighted ways in which GTPases from the highly conserved Ypt/Rab and Arf families regulate and coordinate vesicle trafficking steps. The roles of luminal phosphorylation and calcium signaling in this process were also underlined (see Figure 1). FIGURE 1: Regulation of vesicle trafficking. Vesicles carrying luminal or membrane protein cargo (blue) are formed on one compartment and fuse with the next. Regulation processes discussed here are shown at the top (see the text for details). Vesicular trafficking ... How do downstream effectors of GTPases mediate transport steps? When stimulated, GTPases recruit their effectors to mediate the various steps of vesicular transport. Three talks described how these effectors function. Bruno Goud (Institut Curie) talked about two molecular motors, an actin-based myosin and a microtubule-based kinesin, that act as Rab6 effectors to drive membrane fission during vesicle formation. Michael Ailion (University of Washington) described the identification of two Rab2 effectors in a genetic screen for Caenorhabditis elegans mutants defective in the maturation of neuronal dense-core vesicles. These effectors are part of a complex that contains Rab2, at least three Rab2 effectors, and a Rab2 GTPase-activating protein, underscoring the importance of large multiprotein complexes in vesicular trafficking. Anne Spang (University of Basel) talked about another protein complex, Exomer, which acts as an Arf effector to traffic cargo from the trans-Golgi network (TGN) to the plasma membrane (PM). The interesting twist in this story is the suggestion that prion domain–dependent aggregation might serve as a reversible signal for stalling of cargo in a compartment. GTPases and coordination GTPases are in a perfect position not only to regulate individual transport steps but also to coordinate them. Two talks described how different GTPases that function sequentially in the same pathway coordinate successive vesicular transport steps. In such cascades, the effector of one GTPase acts as a guanine-nucleotide exchange factor (GEF) of the next GTPase. Wei Guo (University of Pennsylvania) described two mechanisms that regulate recycling endosome-to-PM transport involving the stimulation of the GEF activity of Rabin8 on Rab8 by Rab11 and ERK1/2 phosphorylation. Chris Fromme (Cornell University) talked about a Rab-Arf cascade in which two Rabs regulate the Arf-GEF Sec7 at the TGN. Whereas Ypt1 (Rab1) helps recruit Sec7 to the Golgi, Ypt31/32 (Rab11) stimulates its GEF activity on Arf to stimulate vesicle formation. In another talk, Nava Segev (University of Illinois at Chicago) described how one Rab GTPase functions in two different GEF-GTPase-effector modules to coordinate transport from one compartment to two different destinations. When stimulated by one GEF, Ypt1 (Rab1) recruits certain effectors to regulate endoplasmic reticulum (ER)-to-Golgi transport in the secretory pathway. When stimulated by another GEF, the same Ypt recruits an autophagy-specific effector to shuttle membranes loaded with excess or misfolded proteins from the ER for degradation in the lysosome. Calcium signaling Calcium is a known signal of regulated secretion. However, its role in constitutive vesicular trafficking has been elusive. Jared Helm (Hay Laboratory, University of Montana) suggested a mechanism for the dependence of ER-to-Golgi transport on luminal calcium. On localized release of calcium from the ER, the calcium-binding protein ALG-2 interacts with the COPII coat component Sec31. This interaction ensures assembly of a functional vesicle and/or proper timing of its uncoating.

Distinct regions of triadin are required for targeting and retention at the junctional domain of the sarcoplasmic reticulum

Ca2+ release, which is necessary for muscle contraction, occurs at the j-SR (junctional domain of the sarcoplasmic reticulum). It requires the assembly of a large multiprotein complex containing the RyR (ryanodine receptor) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multiprotein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1 (targeting region 1), TR2 and TR3, that contribute to the localization of triadin at the j-SR. FRAP experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Taken together, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multiprotein complex associated with RyR.

More pieces to the puzzle: recent structural insights into class II transcription initiation

Class II transcription initiation is a highly regulated process and requires the assembly of a pre-initiation complex (PIC) containing DNA template, RNA polymerase II (RNAPII), general transcription factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and Mediator. RNAPII, TFIID, TFIIH and Mediator are large multiprotein complexes, each containing 10 and more subunits. Altogether, the PIC is made up of about 60 polypeptides with a combined molecular weight of close to 4MDa. Recent structural studies of key PIC components have significantly advanced our understanding of transcription initiation. TFIID was shown to bind promoter DNA in a reorganized state. The architecture of a core-TFIID complex was elucidated. Crystal structures of the TATA-binding protein (TBP) bound to TBP-associated factor 1 (TAF1), RNAPII-TFIIB complexes and the Mediator head module were solved. The overall architectures of large PIC assemblies from human and yeast have been determined by electron microscopy (EM). Here we review these latest structural insights into the architecture and assembly of the PIC, which reveal exciting new mechanistic details of transcription initiation.

Arsenic Trioxide and Other Arsenical Compounds Inhibit the NLRP1, NLRP3, and NAIP5/NLRC4 Inflammasomes

Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain-like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1β from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin-mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years.

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Investigating The Role Of MLL2 (Mll4) In B Cell Development

BackgroundThe myeloid/lymphoid or mixed-lineage leukaemia 2 (MLL2) histone methyltransferase (referred to as Mll4 in mice) forms part of a large multiprotein complex, which catalyses the methylation of lysine 4 on histone H3 (H3K4). High levels of this histone modification are detected at promoter regions of actively transcribed genes. Loss-of-function mutations in MLL2 have been identified in 80-90% of follicular lymphoma (FL) cases. These mutations are distributed throughout the coding region and lead to the loss of the C-terminal catalytic SET domain and reduction in H3K4 methylation. We generated a Mll4 knockout (Mll4-/-) mouse model to elucidate the effects of Mll4 loss on B cell development and understand how mutations in this gene contribute to FL pathogenesis. Results and DiscussionCre:ERT2-mediated recombination was used to induce the deletion of exons 2–4 of Mll4 in adult mice. CD19+ B lymphocytes were purified from the spleens of wild-type (Mll4WT) and Mll4-/- mice and confirmed the loss of Mll4 mRNA and protein by quantitative RT-PCR (qRT-PCR) and immunoblotting, respectively. As expected, B cells lacking Mll4 (Mll4ΔB/ΔB) exhibited a global reduction in H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3), compared with normal B cells. Gene expression profiling (GEP) using the GeneChip® Mouse Genome 430 2.0 Array (n=5 Mll4WT + 5 Mll4-/- mice) was carried out to determine the transcriptional changes upon loss of Mll4. We identified >200 genes differentially expressed (>2-fold) between Mll4WT and Mll4ΔB/ΔB CD19+ B cells. The top 40 candidate genes (p<0.05) were verified in an independent series of experiments using qRT-PCR. We noted a significant decrease in the expression of the transcription factor lymphoid enhancer-binding factor 1 (Lef1) and the histone acetyltransferase nuclear receptor coactivator 3 (Ncoa3). The downregulation of these genes is consistent with published data, as the promoters of both are marked by H3K4me3 in normal mouse CD19+ B cells. Lef1 is a key regulator of lymphoid differentiation, while Ncoa3 depletion has been shown to induce B-cell lymphoma in mice as a result of constitutive NF-κB activation. A significant number of genes (p<0.001) involved in cell cycle and immune response were upregulated in Mll4ΔB/ΔB CD19+ B cells. Furthermore, loss of Mll4 led to an elevated expression (2.4-fold) of activation-induced cytidine deaminase (Aicda), an enzyme required for germinal centre-derived lymphomagenesis. Immunophenotyping was used to examine the role of Mll4 at different stages of B cell development. We detected a significant reduction in the number of pre-B cells (B220+CD43–IgM–) in the bone marrow of Mll4-/- mice (n=12), compared with their littermate controls (n=10; p<0.01), although this did not affect the number of peripheral mature splenic B cells (B220+IgM+). Mll4 loss had an adverse effect on immune response, with CD19+ B cells failing to induce expression of the MHC-II, CD86, CD40 and CD69 activation markers upon in vitro stimulation with lipopolysaccharide and interleukin 4. ConclusionOur findings provide the first insight into the potential mechanistic link between MLL2 loss and the onset of FL using a mouse model. Mll4-/- mice do not develop any lymphoproliferative disorders, but show defects in B cell development and an impaired immune response. Furthermore, loss of Mll4 leads to the global depletion of H3K4 methylation in mouse B lymphocytes, thus affecting the expression of Lef1, Ncoa3 and Aicda. Although these MLL2/Mll4 target genes have defined roles in B cell biology, their contribution to the pathogenesis of FL will depend on when MLL2 mutations arise during FL development. Disclosures:No relevant conflicts of interest to declare.

An evolving understanding of nuclear receptor coregulator proteins

Nuclear receptors are transcription factors that regulate gene expression through the ligand-controlled recruitment of a diverse group of proteins known as coregulators. Most nuclear receptor coregulators function in large multi-protein complexes that modify chromatin and thereby regulate the transcription of target genes. Structural and functional studies are beginning to reveal how these complexes are assembled bringing together multiple functionalities that mediate: recruitment to specific genomic loci through interaction with transcription factors; recruitment of enzymatic activities that either modify or remodel chromatin and targeting the complexes to their chromatin substrate. These activities are regulated by post-translational modifications, alternative splicing and small signalling molecules. This review focuses on our current understanding of coregulator complexes and aims to highlight the common principles that are beginning to emerge.

Role of multifunctional coactivator complex SAGA in regulation of eukaryotic gene expression

Eukaryotic gene expression is known as a multistep process of high complexity. Transcription is one of cardinal and tightly regulated phase during gene expression. To provide accurate and precise work of gene regulation apparatus including a plethora of modification of chromatin structure and nucleosome dynamic turnover must be occurred. All transcription steps are under control of large multiprotein coactivator complexes. In this review we discuss an evolutionary conservative SAGA complex, which acetylates and deubiquitinates histones during transcription activation and furthermore is involved in subsequent stages of mRNP biogenesis and export.

Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k(off) values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.

A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed

Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multi-protein complexes that support multiple enzymatic activities. In this review, we will discuss single-molecule studies of the replisome, the protein complex responsible for the coordinated replication of double-stranded DNA. In particular, we will focus on new insights obtained into the dynamic nature of the composition of the DNA-replication machinery and how the dynamic replacement of components plays a role in the regulation of the DNA-replication process.

ARMC4 Mutations Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.

Delineating the mixed lineage leukemia gene expression network in hematopoietic stem cells

Hematopoiesis is maintained and sustained throughout life by rare hematopoietic stem cells (HSCs). The hematopoietic system is organized in a hierarchy with the primitive HSCs at the top, generating mature progeny through a series of intermediate and progressively restricted progenitor cells. Normal blood cell development is a tightly controlled process, regulated by transcription factors and epigenetic regulators internally and by cytokines and cellular interactions in the bone marrow microenvironment. Perturbation of the critical molecular regulators is associated often with malignancy of the hematopoietic system, such as leukemia. A key transcriptional regulator in HSCs, the mixed lineage leukemia (MLL) gene, is commonly mutated in both myeloid and lymphoid leukemia (1). MLL is the mammalian ortholog of Drosophila melanogaster Trithorax and widely expressed in multiple organs and tissues including the hematopoietic system and neuronal and vascular systems (2⇓–4). It belongs to a family of histone methyltransferases, where the MLL protein specifically methylates histone H3 on lysine 4 (H3K4), a mark associated with active transcription. MLL is included in a larger multiprotein complex containing protein with chromatin modification and remodeling functions (5). The MLL gene is frequently rearranged through chromosomal translocations, most notably in >70% of cases of infant leukemia and in 10% of all acute myelogeneous leukemia (AML) and acute lymphoblastic leukemia (ALL) (5, 6). The presence of MLL rearrangement in leukemia is generally associated with a poor prognosis. The chromosomal translocations result in MLL fusion proteins that lead to aberrant target gene expression. …

Analysis of Sequence Variation Underlying Tissue-specific Transcription Factor Binding and Gene Expression

Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.

Crystal Structure of NLRC4 Reveals Its Autoinhibition Mechanism

Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.

Structure of a bacterial type IV secretion core complex at subnanometre resolution.

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.