Structure determination of helical filaments by solid-state NMR spectroscopy.

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.

The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA.

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 Šresolution. 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 Šresolution 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.

Unambiguous assignment of short- and long-range structural restraints by solid-state NMR spectroscopy with segmental isotope labeling.

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.

A structural basis for BRD2/4-mediated host chromatin interaction and oligomer assembly of Kaposi sarcoma-associated herpesvirus and murine gammaherpesvirus LANA proteins.

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.

Correlation of structural elements and infectivity of the HET-s prion.

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 fold of alpha-synuclein fibrils.

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.

Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions.

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.

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie.

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.

Human prion diseases: molecular and clinical aspects.

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.

NMR structure of the human doppel protein.

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.

Solid-state NMR resonance assignments of the filament-forming CARD domain of the innate immunity signaling protein MAVS.

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.

Three-dimensional structures of the prion protein and its doppel.

This article discussed the implications of the structures of PrP and Dpl--with their unusual folds containing N-terminal flexible tails and C-terminal globular domains--to the physiologic functions of PrPC and Dpl, and investigations of a possible structural basis of familial human TSEs. Further relations between TSEs and the PrP structure would include the species barrier of TSEs (which seems to be associated with species-specific structural characteristics of PrPC [25,39,67]), and the conformational transition from PrPC to PrPSc using, for example, molecular dynamic simulations [68,69]. Due to the lack of knowledge on physiologic functions of PrPC, however, and the remaining uncertainty about the exact role of the PrP in TSE pathology, it appears that most or all of the physiologically relevant structure-function correlations of PrPC have yet to be identified.

Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA.

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.

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica.

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU(C)) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU(C) at 2.05 A and 1.55 A resolution, respectively. The globular domain is found to consist of a central, mixed beta-sheet surrounded by alpha-helices. The NPTH motif forms a type II beta-turn connecting two beta-strands. NMR analysis of cleaved and uncleaved YscU(C) indicates that the global structure of the protein is retained in cleaved YscU(C). The structure of YscU(C) variant N263D reveals that wild type YscU(C) is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely alpha-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.

The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro.

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.

3D structure of Alzheimer's amyloid-beta(1-42) fibrils.

Alzheimer's disease is the most fatal neurodegenerative disorder wherein the process of amyloid-beta (Abeta) amyloidogenesis appears causative. Here, we present the 3D structure of the fibrils comprising Abeta(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 beta-sheet arrangement from previous solid-state NMR studies. Although residues 1-17 are disordered, residues 18-42 form a beta-strand-turn-beta-strand motif that contains two intermolecular, parallel, in-register beta-sheets that are formed by residues 18-26 (beta1) and 31-42 (beta2). At least two molecules of Abeta(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 beta1 of the nth molecule and the even-numbered residues of strand beta2 of the (n - 1)th molecule. This interaction pattern leads to partially unpaired beta-strands at the fibrillar ends, which explains the sequence selectivity, the cooperativity, and the apparent unidirectionality of Abeta fibril growth. It also provides a structural basis for fibrillization inhibitors.

3D TROSY-HNCA(coded)CB and TROSY-HNCA(coded)CO experiments: triple resonance NMR experiments with two sequential connectivity pathways and high sensitivity.

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.

Analysis of the prion protein in primates reveals a new polymorphism in codon 226 (Y226F).

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.