Search

International audience; The controlled formation of filamentous protein complexes plays a crucial role in many biological systems and represents an emerging paradigm in signal transduction. The mitochondrial antiviral signaling protein (MAVS) is a central signal transduction hub in innate immunity that is activated by a receptor-induced conversion into helical superstructures (filaments) assembled from its globular caspase activation and recruitment domain. Solid-state NMR (ssNMR) spectroscopy has become one of the most powerful techniques for atomic resolution structures of protein fibrils. However, for helical filaments, the determination of the correct symmetry parameters has remained a significant hurdle for any structural technique and could thus far not be precisely derived from ssNMR data. Here, we solved the atomic resolution structure of helical MAVS(CARD) filaments exclusively from ssNMR data. We present a generally applicable approach that systematically explores the helical symmetry space by efficient modeling of the helical structure restrained by interprotomer ssNMR distance restraints. Together with classical automated NMR structure calculation, this allowed us to faithfully determine the symmetry that defines the entire assembly. To validate our structure, we probed the protomer arrangement by solvent paramagnetic resonance enhancement, analysis of chemical shift differences relative to the solution NMR structure of the monomer, and mutagenesis. We provide detailed information on the atomic contacts that determine filament stability and describe mechanistic details on the formation of signaling-competent MAVS filaments from inactive monomers.

Journal of molecular biology 385, 854-866 (2009). doi:10.1016/j.jmb.2008.10.034, Published by Elsevier, Amsterdam [u.a.]

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.

International audience; Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast, Podospora anserina and mammals can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218-289. Amyloid fibrils of HET-s(218-289) are necessary and sufficient for the induction and propagation of prion infectivity. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218-289). This approach revealed four beta-strands constituted by two pseudo-repeat sequences, each forming a beta-strand-turn-beta-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.

The concept of chemical shift-coding monitors chemical shifts in multi-dimensional NMR experiments without additional polarization transfer elements and evolution periods. The chemical shifts are coded in the line-shape of the cross-peak through an apparent scalar coupling dependent upon the chemical shift. This concept is applied to the three-dimensional triple-resonance experiment HNCA adding the information of (13)C(beta) or (13)C' chemical shifts. On average, the proposed TROSY-HNCA(coded)CB experiment is a factor of 2 less sensitive than the HNCA experiment. However, it contains correlations via the chemical shifts of both (13)C(alpha) and (13)C(beta), and provides up to three times higher resolution along the (13)C(alpha) chemical shift axis. Thus, it dramatically reduces ambiguities in linking the spin systems of adjacent residues in the protein sequence during the sequential assignment. The TROSY-HNCA(coded)CO experiment which is conceptually similar contains correlations via the chemical shifts of (13)C(alpha) and (13)C' without major signal losses. The proposed triple resonance experiments are applied to a approximately 70% (2)H, approximately 85% (13)C,(15)N labeled protein with a molecular weight of 25 kDa.

The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposi's sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.

We present an efficient method for the reduction of spectral complexity in the solid-state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally (13)C, (15)N-labeled prion domain of HET-s exhibits significantly reduced spectral overlap while retaining the wild-type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two-dimensional (13)C, (13)C cross-correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.

The mitochondrial antiviral signalling protein (MAVS) is a central signal transduction hub in the innate immune response against viral infections. Viral RNA present in the cytoplasm is detected by retinoic acid inducible gene I like receptors, which then activate MAVS via heterotypic interactions between their respective caspase activation and recruitment domains (CARD). This leads to the formation of active, high molecular weight MAVS complexes formed by homotypic interactions between the single N-terminal CARDs of MAVS. Filaments formed by the N-terminal MAVS(CARD) alone are sufficient to induce the autocatalytic conversion from a monomeric to an aggregated state in a prion-like manner. Here, we present the nearly complete spectroscopic (13)C and (15)N resonance assignments of human MAVS(CARD) filaments obtained from a single sample by magic angle spinning solid-state NMR spectroscopy. The corresponding secondary chemical shifts suggest that the filamentous form of MAVS(CARD) retains an exclusively alpha-helical fold that is very similar to the X-ray structure determined previously from monomeric MAVS(CARD)-maltose binding protein fusion constructs.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence., Author Summary Kaposi sarcoma-associated herpesvirus (KSHV) causes Kaposi Sarcoma, Primary Effusion lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Its oncogenic effect is linked to its ability to persist in a latent form for the life time of infected individuals. During latency viral genomes are replicated and passed to daughter cells in synchrony with the infected cell without the formation of new virions. A key viral protein in this process is the latency-associated nuclear antigen, LANA. In latently infected cells, viral genomes and LANA form characteristic nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin components. We have solved the crystal structure of the c-terminal, DNA-binding, domain (CTD) of KSHV LANA (kLANA) and its homologue mLANA of a related murine γ2-herpesvirus, which is frequently used as a model to study latent persistence in vivo. We also identified the binding site for two chromatin proteins, BRD2/4, by NMR spectroscopy. We demonstrate the functional importance of these structural features, and their contribution to latent replication and ‘LANA speckle’ formation, in cell culture and in vivo experiments. Our results provide a structural basis for the assembly of LANA-containing nuclear structures that are required for latent viral replication and persistence.

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen. In order to develop an anti-prion vaccine, we designed a novel DNA fusion vaccine composed of mouse PrP and immune stimulatory helper T-cell epitopes of the tetanus toxin that have previously been reported to break tolerance to other self-antigens. This approach provoked a strong PrP(C)-specific humoral and cellular immune response in PrP null mice, but only low antibody titres were found in vaccinated wild-type mice. Furthermore, prime-boost immunisation with the DNA vaccine and recombinant PrP protein increased antibody titres in PrP null mice, but failed to protect wild-type mice from mouse scrapie.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.

Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-β (Aβ) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Aβ(1–42), which was obtained by using hydrogen-bonding constraints from quenched hydrogen/deuterium-exchange NMR, side-chain packing constraints from pairwise mutagenesis studies, and parallel, in-register β-sheet arrangement from previous solid-state NMR studies. Although residues 1–17 are disordered, residues 18–42 form a β-strand–turn–β-strand motif that contains two intermolecular, parallel, in-register β-sheets that are formed by residues 18–26 (β1) and 31–42 (β2). At least two molecules of Aβ(1–42) are required to achieve the repeating structure of a protofilament. Intermolecular side-chain contacts are formed between the odd-numbered residues of strand β1 of the n th molecule and the even-numbered residues of strand β2 of the ( n – 1)th molecule. This interaction pattern leads to partially unpaired β-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Aβ fibril growth. It also provides a structural basis for fibrillization inhibitors.

A soluble, oligomeric beta-sheet-rich conformational variant of recombinant full-length prion protein, PrP beta, was generated that aggregates into amyloid fibrils, PrP betaf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrP Sc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrP C to PrP beta occurs at pH 5.0 in bicellar solutions containing equimolar mixtures of dihexanoyl-phosphocholine and dimyristoyl-phospholipids, and a small percentage of negatively charged dimyristoyl-phosphoserine. The same protocol was applicable to human, cow, elk, pig, dog and mouse PrP. Comparison of full-length hPrP 23-230 with the N-terminally truncated human PrP fragments hPrP 90-230, hPrP 96-230, hPrP 105-230 and hPrP 121-230 showed that the flexible peptide segment 105-120 must be present for the generation of PrP beta. Dimerization of PrP C represents the rate-limiting step of the PrP C-to-PrP beta conformational transition, which is dependent on the amino acid sequence. The activation enthalpy of dimerization is about 130 kJ/mol for the recombinant full-length human and bovine prion proteins, and between 260 and 320 kJ/mol for the other species investigated. The in vitro conversion assay described here permits direct molecular characterization of processes that might be closely related to conformational transitions of the prion protein in transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE) are neurodegenerative fatal diseases in mammalian species that appear to occur as rare, ‘‘sporadic’’ disorders, but also have been traced to mutations and to infectious transmission, including iatrogenic transfer [1,2]. Scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and the human TSEs—kuru, Creutzfeldt-Jakob disease (CJD), and variants thereof—have attracted interest beyond the scientific community. The basic neuropathologic signs in TSEs are a progressive vacuolation of brain tissue, and, usually, extensive astroglial hyperthrophy and proliferation, which is sometimes described as ‘‘spongiform change’’ [3]. The ‘‘protein-only hypothesis’’ [4–6] suggests that TSEs are distinct from infectious diseases caused by bacteria, viruses, or viroids in that the origin of the disease is related to conformational alterations of an ubiquitous protein—the prion protein (PrP)—and that nucleic acids are not essential for the propagation of the infectious agent. Accordingly, the normal cellular form of PrP (PrP C ) is transformed into the diseaserelated, and possibly in itself infectious, scrapie form (PrP Sc ) [2,6].

The NMR structure of the recombinant human doppel protein, hDpl(24-152), contains a flexibly disordered "tail" comprising residues 24-51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four alpha-helices comprising residues 72-80 (alpha1), 101-115 (alpha2(a)), 117-121 (alpha2(b)), and 127-141 (alpha3), and a short two-stranded anti-parallel beta-sheet comprising residues 58-60 (beta1) and 88-90 (beta2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the beta-sheet is flipped by 180 degrees with respect to the molecular scaffold formed by the four helices, and the beta1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between beta2 and alpha3, which has no counterpart in hPrP. The helix alpha2 of hPrP is replaced by two shorter helices, alpha2(a) and alpha2(b). The helix alpha3 is shortened by more than two turns when compared with alpha3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144-149 folds back onto the loop connecting beta2 and alpha2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms.

Bovine spongiform encephalopathy has been epizootic in cows for the last two decades, and most probably causes variant Creutzfeldt-Jakob disease in humans. A thorough understanding of prion pathogenesis relies on suitable animal models. Modeling the transmission of BSE to primates is a crucial public health priority, necessary for determining the tissue distribution of the agent and for devising therapies. Susceptibility of humans to BSE is partly determined by polymorphism within the gene encoding the cellular prion protein, Prnp, a fact that must be taken into account in primate studies. However, no information is available on Prnp polymorphisms in primates. We have sequenced the Prnp open reading frames of 30 non-consanguineous Rhesus macaques. All macaques were homozygous for methionine at codon 129, which is polymorphic in humans and seems to modulate prion susceptibility. However, we identified a novel polymorphism in macaque Prnp, localized on codon 226 (Y226F). A modulatory effect of this polymorphism on the development of prion disease is possible because codon 226 is close to the suggested binding side of the factor X, which has been invoked as a determinant of the prion species barrier.

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90–230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered “tail.” The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–228 and a short anti-parallel β-sheet comprising the residues 128–131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219–228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219–226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.

Compared with that of other human pathogens, the proposed replicative cycle of prions is disarmingly simple. It encompasses misfolding of a single protein, the cellular prion protein (PrPC), into a disease-associated form called PrPSc. This is followed by PrPSc aggregation and possibly fragmentation of aggregates, which may augment the number of replicative units. Although there is no formal proof of the correctness of this model, a wealth of evidence indicates that pathogen-encoded informational nucleic acids are dispensable for prion replication. Despite the simplicity of the replicative process, the human phenotypic range of prion diseases is extremely variable and includes the sporadic, inherited, and acquired forms of Creutzfeldt-Jakob disease. In addition, prion diseases occur in a wide range of animals and can be propagated within and between animal species. The present review article discusses current concepts and controversies surrounding the basic biological features of prions.