Crystal structure of the tegument protein UL82 (pp71) from human cytomegalovirus.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects a majority of the world population. It may cause severe disease in immunocompromised people and lead to pregnancy loss or grave disabilities of the fetus upon congenital infection. For effective replication and lifelong persistence in its host, HCMV relies on diverse functions of its tegument protein UL82, also known as pp71. Up to now, little is known about the molecular mechanisms underlying the multiple functions of this crucial viral protein. Here, we describe the X-ray structure of full-length UL82 to a resolution of 2.7 Å. A single polypeptide chain of 559 amino acids mainly folds into three ß-barrels. We show that UL82 forms a dimer in the crystal as well as in solution. We identify point mutations that disturb the dimerization interface and show that the mutant protein is monomeric in solution and upon expression in human cells. On the basis of the three-dimensional structure, we identify structural homologs of UL82 from other herpesviruses and analyze whether their functions are preserved in UL82. We demonstrate that UL82, despite its structural homology to viral deoxyuridinetriphosphatases (dUTPases), does not possess dUTPase activity. Prompted by the structural homology of UL82 to the ORF10 protein of murine herpesvirus 68 (MHV68), which is known to interact with the RNA export factor ribonucleic acid export 1 (Rae1), we performed coimmunoprecipitations and demonstrated that UL82 indeed interacts with Rae1. This suggests that HCMV UL82 may play a role in mRNA export from the nucleus similar to ORF10 encoded by the gammaherpesviruses MHV68.

C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1ß cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

Crystal structure of the N-terminal domain of the effector protein SidI of Legionella pneumophila reveals a glucosyl transferase domain.

The Gram-negative bacterium Legionella pneumophila is an accidental human pathogen that can cause a life-threatening respiratory infection called Legionellosis. In the course of infection, L. pneumophila injects more than 300 effector proteins into the host cell. The effector proteins modify the intracellular environment in order to create a stable compartment for proliferation within the host cell. The effector protein SidI has been shown to potently inhibit host translation upon translocation. SidI is able to interact with the translation elongation factor eEF1A, which has been hypothesized to be a target of SidI. A postulated glycosyltransferase domain in the C-terminal half may be responsible for the toxic effect of SidI. Here, we present the crystal structure of an N-terminal fragment of SidI containing residues 37-573. The structure is divided into three subdomains, two of which display a novel fold. The third subdomain shows close structural homology to GT-B fold glycosyltransferases. Based on structural analysis we predict that the two previously identified residues R453 and E482 assume roles in the catalytic activity of SidI. Furthermore, we show that the N-terminal fragment of SidI is able to directly interact with a postulated target, the translation elongation factor eEF1A.

The Inflammasome Activity of NLRP3 Is Independent of NEK7 in HEK293 Cells Co-Expressing ASC.

The cytosolic immune receptor NLRP3 (nucleotide-binding domain, leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3) oligomerizes into the core of a supramolecular complex termed inflammasome in response to microbes and danger signals. It is thought that NLRP3 has to bind NEK7 (NIMA (never in mitosis gene a)-related kinase 7) to form a functional inflammasome core that induces the polymerization of the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain)), which is a hallmark for NLRP3 activity. We reconstituted the NLRP3 inflammasome activity in modified HEK293 (human embryonic kidney 293) cells and showed that the ASC speck polymerization is independent of NEK7 in the context of this cell system. Probing the interfaces observed in the different, existing structural models of NLRP3 oligomers, we present evidence that the NEK7-independent, constitutively active NLRP3 inflammasome core in HEK293 cells may resemble a stacked-torus-like hexamer seen for NLRP3 lacking its PYD (pyrin domain).

Development of an in vitro assay for the detection of polymerization of the pyrin domain of ASC.

Multicomponent protein complexes called inflammasomes play a major role in the innate immune system by activating proinflammatory cytokines and promoting a highly inflammatory form of programmed cell death, called pyroptosis. A hallmark of the function of the nucleotide-binding domain, leucine-rich repeat and NLRP3-mediated inflammasome assembly is the polymerization of ASC into large filaments. The ASC filaments recruit and activate procaspase-1 by induced proximity. We developed an in vitro assay for monitoring the polymerization of the pyrin domain of ASC by microscale thermophoresis. We have validated the assay by analyzing the effects of buffer conditions, mutations of ASC and the use of seeds on the polymerization behavior of ASC.

Crystal structure of the metaeffector MesI (Lpg2505) from Legionella pneumophila.

Persistence and replication of the gram-negative bacterium Legionella pneumophila in the human host cell depend on so-called effector proteins that target diverse cellular functions and modulate them in favor of the pathogen. We solved the crystal structure of the L. pneumophila effector protein MesI de novo to a resolution of 2.2 Å. The 34 kDa polypeptide chain folds into two distinct α-helical domains. The larger C-terminal domain shows similarity to tetratricopeptide repeat proteins. Using size-exclusion chromatography, we confirmed that MesI binds tightly to full-length SidI and that deletion of either the N- or the C-terminus weakens the interaction. Based on the three-dimensional structure of MesI we suggest a possible binding mode for SidI and identified two homologs of MesI within the proteome of L. pneumophila that do not bind to SidI, but may act as specific inhibitors of other yet to be identified effectors.

Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex.

BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.

Modulation of dynamin function by small molecules.

Dynamins are essential as membrane remodelers in various cellular processes, like receptor-mediated endocytosis, synaptic vesicle recycling and spermatogenesis. Moreover, dynamin is involved in the internalization of numerous viruses and in the motility of several cancer cell lines. As tools for dissecting the underlying mechanisms of these important biological processes and as potential future therapeutics, small molecules have been developed in the last two decades that modulate the functions of dynamin. In this review we give an overview of the compound classes that are currently in use and describe how they affect dynamin function.

Protein kinase C ϵ stabilizes ß-catenin and regulates its subcellular localization in podocytes.

Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of ß-catenin, which promotes its degradation. ß-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with ß-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower ß-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, ß-catenin showed a reversed subcellular localization pattern: Although ß-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates ß-catenin expression and stabilization in a glycogen synthase kinase 3ß-independent manner. Further, ß-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the ß-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in ß-catenin that are required for full ß-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and ß-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of ß-catenin levels and define PKCϵ as an important ß-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.

Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.

Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.

Crystal structure of the GTPase domain and the bundle signalling element of dynamin in the GDP state.

Dynamin is the prototype of a family of large multi-domain GTPases. The 100 kDa protein is a key player in clathrin-mediated endocytosis, where it cleaves off vesicles from membranes using the energy from GTP hydrolysis. We have solved the high resolution crystal structure of a fusion protein of the GTPase domain and the bundle signalling element (BSE) of dynamin 1 liganded with GDP. The structure provides a hitherto missing snapshot of the GDP state of the hydrolytic cycle of dynamin and reveals how the switch I region moves away from the active site after GTP hydrolysis and release of inorganic phosphate. Comparing our structure of the GDP state with the known structures of the GTP state, the transition state and the nucleotide-free state of dynamin 1 we describe the structural changes through the hydrolytic cycle.

Crystal structure of the dynamin tetramer.

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.

Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly.

Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome cin vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: implications for binding of muramyl dipeptide.

The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain.

Changes in Apaf-1 conformation that drive apoptosome assembly.

Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate ß-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory ß-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by ß-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.

A molecular view on signal transduction by the apoptosome.

Apoptosomes are signaling platforms that initiate the dismantling of a cell during apoptosis. In mammals, assembly of the apoptosome is the pivotal point in the mitochondrial pathway of apoptosis, and is prompted by binding of cytochrome c to the apoptotic protease-activating factor 1 (Apaf-1) in the presence of ATP. The resulting wheel-like heptamer of seven molecules Apaf-1 and seven molecules cytochrome c binds and activates the initiator caspase-9, which in turn ignites the downstream caspase cascade. In this review we discuss the molecular determinants for the formation of the mammalian apoptosome and caspase activation and describe the related signaling platforms in flies and nematodes.

Crystal structure of full-length Apaf-1: how the death signal is relayed in the mitochondrial pathway of apoptosis.

The apoptotic protease-activating factor 1 (Apaf-1) relays the death signal in the mitochondrial pathway of apoptosis. Apaf-1 oligomerizes on binding of mitochondrially released cytochrome c into the heptameric apoptosome complex to ignite the downstream cascade of caspases. Here, we present the 3.0 Å crystal structure of full-length murine Apaf-1 in the absence of cytochrome c. The structure shows how the mammalian death switch is kept in its "off" position. By comparing the off state with a recent cryo-electron microscopy derived model of Apaf-1 in its apoptosomal conformation, we depict the molecular events that transform Apaf-1 from autoinhibited monomer to a building block of the caspase-activating apoptosome. Moreover, we have solved the crystal structure of the R265S mutant of full-length murine Apaf-1 in the absence of cytochrome c to 3.55 Å resolution and we show that proper function of Apaf-1 relies on R265 in the vicinity of the bound nucleotide.

A new model for the transition of APAF-1 from inactive monomer to caspase-activating apoptosome.

The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly.

Crystal structure of the GTPase domain of rat dynamin 1.

Here, we present the 1.9-A crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding in an assembly-dependent manner and the GTPase effector domain functions as an assembly-dependent GTPase activating protein in the fashion of RGS-type GAPs.