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

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

The nanomechanics of neurotoxina proteins reveals common features at the start of the neurodegeneration cascade.

1 pags. -- 56th Annual Meeting of the Biophysical-Society, FEB 25-29, 2012, San Diego, CAAmyloidogenic neurodegenerative diseases are incurable conditions caused by specific largely disordered proteins. However, the underlying molecular mechanism remains elusive. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these ‘‘neurotoxic proteins’’ triggers the pathogenic cascade. Using force spectroscopy with unequivocal singlemolecule identification we demonstrate a rich conformational polymorphism at their monomer level. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the monomeric b-conformational change and neurodegeneration. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt or reverse multiple neurodegenerative disease

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 °C in 0.1 M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge–dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds. A primary objective in this study was to investigate the influence of charge–charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the α-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge–dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge–charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pKSa−pK5K) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson–Boltzmann equation, which considers desolvation and charge–dipole interactions in addition to charge–charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge–charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.This work was supported by grants GM-37039 and GM-52483 from the National Institutes of Health (USA), grants BE-1060 and BE-1281 from the Robert A. Welch Foundation, and a grant PB-93-06777 to M.R. from the Dirección General de Investigación Cientı́fica y Técnica (Spain

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

AlphaFold 2 and NMR Spectroscopy: Partners to Understand Protein Structure, Dynamics and Function

12 pags., 5 figs.The artificial intelligence program AlphaFold 2 is revolutionizing the field of protein structure determination as it accurately predicts the 3D structure of two thirds of the human proteome. Its predictions can be used directly as structural models or indirectly as aids for experimental structure determination using X-ray crystallography, CryoEM or NMR spectroscopy. Nevertheless, AlphaFold 2 can neither afford insight into how proteins fold, nor can it determine protein stability or dynamics. Rare folds or minor alternative conformations are also not predicted by AlphaFold 2 and the program does not forecast the impact of post translational modifications, mutations or ligand binding. The remaining third of human proteome which is poorly predicted largely corresponds to intrinsically disordered regions of proteins. Key to regulation and signaling networks, these disordered regions often form biomolecular condensates or amyloids. Fortunately, the limitations of AlphaFold 2 are largely complemented by NMR spectroscopy. This experimental approach provides information on protein folding and dynamics as well as biomolecular condensates and amyloids and their modulation by experimental conditions, small molecules, post translational modifications, mutations, flanking sequence, interactions with other proteins, RNA and virus. Together, NMR spectroscopy and AlphaFold 2 can collaborate to advance our comprehension of proteins.This study was supported by project PID 2019-109306RB-I00/AEI/10.13039/501100011033 from the Spanish Ministry of Science and Innovation. The author declares no competing interests.Peer reviewe

A mechanism for protein recycling

VIII Congreso Nacional BIFI 2017. Instituto Universitario de Investigación en Biocomputación y Física de S. Complejos. Zaragoza, Campus Río Ebro (Actur), 31 de enero al 2 de febrero, 2017. .-http://www.unizar.es/actualidad/vernoticia_ng.php?id=3373

Structures and Applications of Antifreeze Polypeptides

Supported by SAF2016-76678-C2-2-R (DVL) from the Spanish Ministery of Economy and Competitivity. 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 (ICTS R-LRB)Peer reviewe