An Amyloid-Like Pathological Conformation of TDP-43 Is Stabilized by Hypercooperative Hydrogen Bonds

TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates in vitro and in cells strongly suggest that TDP-43 aggregates have a self-templating, amyloid-like structure. Based on the importance of the Gln/Asn-rich 341–367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341–367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy.This work was supported by Grants SAF2013-49179-C2-2-R (DVL) and EU JPND AC14/00037 (DVL) and EU JPND RiModFTD, Italy, Ministero della Sanita’ (EB), and the Thierry Latran Foundation REHNPALS (EB).Peer reviewedPeer Reviewe

Estudios estructurales y computacionales de amiloides y plegamientos nocivos en biomoléculas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 20-11-2015El amiloide es un tipo de estructura de proteínas consistente en láminas beta muy largas y altamente estables, responsables de más de veinte enfermedades mortales en humanos, incluyendo Alzheimer, Parkinson y Esclerosis Lateral Amiotrófica (ELA). Paradójicamente los amiloides no siempre son patológicos, sino que también son esenciales en procesos vitales tales como la regulación del ARN o la consolidación de la memoria. A pesar de los avances en el campo, todavía hay muchos detalles desconocidos acerca de los aspectos energéticos y la formación de estas estructuras. En esta tesis presento resultados sobre la formación y los factores que estabilizan el amiloide del heptapéptido GNNQQNY de Sup35, un prion de levaduras. Mis resultados computacionales y experimentales muestran que el intermedio clave en la amiloidogénesis es una lámina beta paralela curvada compuesta por tres hebras. Esta lámina es capaz de dimerizar expulsando fácilmente las moléculas de agua de solvatación y así producir la estructura llamada columna beta cruzada (cross-β spine). La presencia de enlaces de hidrógeno híper-estables es crucial para la estabilidad tanto de este intermedio, como de oligómeros más largos y fibras de Sup35. Con estos datos, se propone un modelo para la formación de este núcleo que plantea la cuestión de si los oligómeros tóxicos son una consecuencia de columnas beta cruzadas en un plegamiento anómalo. La TDP-43 (Proteína de unión a ADN de respuesta transactiva de 43 kDa) es particularmente fascinante, puesto que su región C-terminal forma agregados de tipo amiloide implicados en la ELA, además de un hipotético amiloide funcional esencial para el transporte y la regulación del ARN. Puesto que existe un debate acerca de la naturaleza amorfa o amiloide de los agregados de TDP-43, en esta tesis se presentan una serie de ensayos bioquímicos y espectroscópicos que revelan la naturaleza de tipo amiloide de estos agregados. Entre ellos se incluyen experimentos de unión a cromóforos y fluoróforos, reconocimiento por anticuerpos conformacionales, dicroísmo circular, difracción de rayos-X, resonancia magnética nuclear (RMN) en estado líquido y sólido, así como microscopía electrónica. Mediante métodos espectroscópicos y computacionales, hemos investigado conformaciones permitidas para el posible amiloide patológico, elaborando modelos atómicos para el monómero constituyente. La estructura consiste en horquillas beta alineadas en una topología de giro beta y empaquetadas en paralelo para formar una estructura cuasi-amiloide con un plegamiento novedoso. Por otra parte, el dominio N-terminal (DNT) de la TDP-43 juega papeles clave en la regulación de la agregación funcional y patológica de la región C-terminal. La estructura de alta resolución del DNT ha sido obtenida por RMN y consiste en una hélice alfa y seis hebras beta, con un núcleo hidrófobo bien empaquetado, dos grupos de residuos cargados negativamente y dos cisteínas expuestas y distantes entre sí. La estabilidad conformacional, determinada por intercambio de hidrógeno/deuterio, es de 4 kCal/mol a pH 4 y 25 °C. Las propiedades dinámicas han sido elucidadas usando métodos de RMN y muestran que la hélice alfa y cinco de las seis hebras beta son rígidas. Una de las hebras del borde y los giros son más flexibles. Estos descubrimientos avanzan nuestra comprensión del DNT y proveen un medio para estudiar su interacción con la región C-terminalAmyloid is a class of protein structures composed of very long and highly stable β-sheets. Amyloid-like oligomers cause over twenty mortal human diseases, including Alzheimer’s, Parkinson’s diseases and Amyotrophic Lateral Sclerosis (ALS). Paradoxically, other amyloids are key to vital physiological processes, including RNA regulation and memory consolidation. Despite advances, many details on the formation and energetics of amyloids are still unknown. In this thesis, I present results on the formation and factors that stabilize the amyloid formed by the yeast prion Sup35 heptapeptide GNNQQNY. My computational and experimental results revealed that the key intermediate in amyloidogenesis is a twisted, three-stranded, parallel β-sheet. This sheet dimerizes with facile release of water molecules to yield the cross-β spine structure. Hyper-stable H-bonds are crucial to the stability of this intermediate and to that of larger Sup35 oligomers and amyloids. A model for this nucleus’ formation is proposed, raising the question of whether noxious oligomers are a consequence of misfolded cross-β spines. TDP-43 (Transactive response DNA-binding protein 43 kDa) is particularly fascinating because its C-terminal region forms amyloid-like aggregates implicated in ALS yet also a putative functional amyloid vital to RNA regulation and transport. Since a debate existed as to whether TDP-43 aggregates are amyloid-like or amorphous, a series of biochemical and spectroscopic assays were performed, which revealed the amyloid-like nature of these aggregates. This includes fluorophore and cromophore binding, recognition by conformational antibodies, circular dichroism, X-ray diffraction, solution and solid-state NMR, and electron microscopy. Using spectroscopic and computational methods, the conformation of the putative pathological amyloid was investigated and structural models for the monomeric amyloidogenic intermediate and amyloid-like oligomers were determined. The structure consists of β-hairpins aligned in a “β-turn” topology and packed in parallel to form an amyloid-like structure whose topology is novel. The N-terminal domain (NTD) of TDP-43 plays key roles in regulating the functional and pathological aggregation of the C-terminal region. The structure of the NTD has been solved at high resolution by NMR methods, and it consists of an α-helix and 6 β-strands featuring a well-packed hydrophobic core, two exposed clusters of negatively charged residues and two separated, exposed cysteine residues. The conformational stability, determined by hydrogen/deuterium exchange, is 4 kcal/mol at pH 4 and 25 °C. The dynamic properties were elucidated using NMR methods, and show that the α-helix and 5 of 6 β-strands are rigid. One edge strand and the loops are more flexible. These findings advance our understanding of the NTD and provide the means to study its interaction with the C-terminal regio

Unraveling the toxic effects mediated by the neurodegenerative disease-associated S375G mutation of TDP-43 and its S375E phosphomimetic variant

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein found in the nucleus that accumulates in the cytoplasm under pathological conditions, leading to proteinopathies, such as frontotemporal dementia and ALS. An emerging area of TDP-43 research is represented by the study of its post-translational modifications, the way they are connected to disease-associated mutations, and what this means for pathological processes. Recently, we described a novel mutation in TDP-43 in an early onset ALS case that was affecting a potential phosphorylation site in position 375 (S375G). A preliminary characterization showed that both the S375G mutation and its phosphomimetic variant, S375E, displayed altered nuclear-cytoplasmic distribution and cellular toxicity. To better investigate these effects, here we established cell lines expressing inducible WT, S375G, and S375E TDP-43 variants. Interestingly, we found that these mutants do not seem to affect well-studied aspects of TDP-43, such as RNA splicing or autoregulation, or protein conformation, dynamics, or aggregation, although they do display dysmorphic nuclear shape and cell cycle alterations. In addition, RNA-Seq analysis of these cell lines showed that although the disease-associated S375G mutation and its phosphomimetic S375E variant regulate distinct sets of genes, they have a common target in mitochondrial apoptotic genes. Taken together, our data strongly support the growing evidence that alterations in TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis and provide a further link between TDP-43 pathology and mitochondrial health

Structural Evidence of Amyloid Fibril Formation in the Putative Aggregation Domain of TDP-43

TDP-43 can form pathological proteinaceous aggregates linked to ALS and FTLD. Within the putative aggregation domain, engineered repeats of residues 341-366 can recruit endogenous TDP-43 into aggregates inside cells; however, the nature of these aggregates is a debatable issue. Recently, we showed that a coil to β-hairpin transition in a short peptide corresponding to TDP-43 residues 341-357 enables oligomerization. Here we provide definitive structural evidence for amyloid formation upon extensive characterization of TDP-43(341-357) via chromophore and antibody binding, electron microscopy (EM), solid-state NMR, and X-ray diffraction. On the basis of these findings, structural models for TDP-43(341-357) oligomers were constructed, refined, verified, and analyzed using docking, molecular dynamics, and semiempirical quantum mechanics methods. Interestingly, TDP-43(341-357) β-hairpins assemble into a novel parallel β-turn configuration showing cross-β spine, cooperative H-bonding, and tight side-chain packing. These results expand the amyloid foldome and could guide the development of future therapeutics to prevent this structural conversion.Peer Reviewe

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Intrinsically disordered domains, amyloids and protein liquid phases: Evolving concepts and open questions

Enzymes and structural proteins dominated thinking about protein structure and function for most of the twentieth century. In recent decades, however, we have begun to appreciate the significant physiological and pathological roles of nonglobular proteins. Amyloids first gained infamy from their implications in a score of human mortal diseases. However, they have recently been discovered to play vital physiological roles, such as memory consolidation in humans. This raises an important question: Can we inhibit pathological amyloids without affecting functional amyloids? Intrinsically disordered proteins (IDPs), many of which are prone to form amyloids, perform many essential functions, yet their importance has only been recognized in the last quarter century. A subclass of IDPs can form, under certain conditions, water immiscible liquid phases which serve to process, regulate, store or transport RNA. Perturbation of these remarkable liquid phases can lead to aggregates, such as those formed by the proteins TDP-43 and FUS, which are linked to ALS and other dementia. Here, we summarize our changing view of intrinsically disordered, liquid phase forming and amyloidogenic proteins and the uncertainties that will drive future research.Peer Reviewe

Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to “hack” these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13Cβ and backbone 13CO, 1HN, 13Cα, and 15N nuclei of Nsp2’s 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 from the Carlos III Institute of Health and the Spanish Ministry of Science and Innovation to MM and DVL.Peer reviewe

Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to hack these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13C,15N-labeled C-terminal region of Nsp2. The 13Cβ and backbone 13CO, 1HN, 13Cα and 15N nuclei were assigned by analysis of a series of 2D 1H-15N HSQC and 13C-15N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of β-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T1ρ measurements, the {1H}-15N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.M. Mompeán is a “Caixa” Foundation Junior Group Leader. Funded by project COV20/00764 from the Carlos III Institute of Health and the Spanish Ministry of Science and Innovation. NMR experiments were performed in the “Manuel Rico” NMR Laboratory (LMR) of the Spanish National Research Council (CSIC), a node of the Spanish Large-Scale National Facility for Biomolecular NMR (ICTS R-LRB). Inhibitor screening is being conducted within the COVID19-NMR consortium by Dr. S. Sreeramula, Prof. Dr. H. Schwalbe and co-workers.N

Do polyproline II helix associations modulate biomolecular condensates?

10 pags., 2 figs., 1 tab.Biomolecular condensates are microdroplets that form inside cells and serve to selectively concentrate proteins, RNAs and other molecules for a variety of physiological functions, but can contribute to cancer, neurodegenerative diseases and viral infections. The formation of these condensates is driven by weak, transient interactions between molecules. These weak associations can operate at the level of whole protein domains, elements of secondary structure or even moieties composed of just a few atoms. Different types of condensates do not generally combine to form larger microdroplets, suggesting that each uses a distinct class of attractive interactions. Here, we address whether polyproline II (PPII) helices mediate condensate formation. By combining with PPII-binding elements such as GYF, WW, profilin, SH3 or OCRE domains, PPII helices help form lipid rafts, nuclear speckles, P-body-like neuronal granules, enhancer complexes and other condensates. The number of PPII helical tracts or tandem PPII-binding domains can strongly influence condensate stability. Many PPII helices have a low content of proline residues, which hinders their identification. Recently, we characterized the NMR spectral properties of a Gly-rich, Pro-poor protein composed of six PPII helices. Based on those results, we predicted that many Gly-rich segments may form PPII helices and interact with PPII-binding domains. This prediction is being tested and could join the palette of verified interactions contributing to biomolecular condensate formation.JO is a Ramon y Cajal Fellow of the Spanish AEI- Ministry of Science and Innovation (RYC2018- 026042-I) and a Leonardo Fellow from the BBVA Foundation (Grant Number BBM-TRA-0203). MM is a Ramon y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I) and a ‘La Caixa’ Foundation Junior Leader Fellow (LCR/ BQ/PR19/11700003). This study was supported by Grant BTC-PID2019-109306RB-I00 (DVL) from the Spanish Ministry of Science and Innovation.Peer reviewe