SARS-CoV-2 Nsp8 N-terminal domain folds autonomously and binds dsRNA.

The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long α-helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the α-helix population progressively declines towards the C-termini but surprisingly binds dsRNA while preserving structural disorder.

NMR characterization of an assembling RHIM (RIP homotypic interaction motif) amyloid reveals a cryptic region for self-recognition.

The RIP homotypic interaction motif (RHIM) is an essential protein motif in inflammatory signaling and certain cell death pathways. RHIM signaling occurs following the assembly of functional amyloids, and while the structural biology of such higher-order RHIM complexes has started to emerge, the conformations and dynamics of nonassembled RHIMs remain unknown. Here, using solution NMR spectroscopy, we report the characterization of the monomeric form of the RHIM in receptor-interacting protein kinase 3 (RIPK3), a fundamental protein in human immunity. Our results establish that the RHIM of RIPK3 is an intrinsically disordered protein motif, contrary to prediction, and that exchange dynamics between free monomers and amyloid-bound RIPK3 monomers involve a 20-residue stretch outside the RHIM that is not incorporated within the structured cores of the RIPK3 assemblies determined by cryo-EM or solid-state NMR. Thus, our findings expand on the structural characterization of RHIM-containing proteins, specifically highlighting conformational dynamics involved in assembly processes.

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

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.

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.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state.

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/ß proteins both on fast ps/ns as well as slower µs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

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 13Cß 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.

Aromatic and aliphatic residues of the disordered region of TDP-43 are on a fast track for self-assembly.

The C-terminal, intrinsically disordered, prion-like domain (PrLD) of TDP-43 promotes liquid condensate and solid amyloid formation. These phase changes are crucial to the normal biological functions of the protein but also for its abnormal aggregation, which is implicated in amyotrophic lateral sclerosis (ALS) and certain dementias. We and other previously found that certain amyloid forms emerge from an intermediate condensed state that acts as a nucleus for fibrillization. To quantitatively ascertain the role of individual residues within TDP-43's PrLD in its early self-assembly we have followed the kinetics of NMR 1H-15N HSQC signal loss to obtain values for the lag time, elongation rate and extent of condensate formation at equilibrium. The results of this analysis represent a robust corroboration that aliphatic and aromatic residues are key drivers of condensate formation.

SUMOylation Regulates TDP-43 Splicing Activity and Nucleocytoplasmic Distribution.

The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif (SIM, 106-110 residues), respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.

Do polyproline II helix associations modulate biomolecular condensates?

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.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

NMR assignments for the C-terminal domain of human TDP-43.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a 414-residue protein whose aberrant aggregation is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Intriguingly, TDP-43 has also been shown to functionally oligomerize to carry out physiological functions. TDP-43 also exists in mixed condensates or granules with other proteins (e.g. neuronal or stress granules), and its large C-terminal domain (CTD, residues 267-414) seems responsible for TDP-43 both homo- and heterotypic interactions underlying such diverse functional and pathological aggregation events. A myriad of distinct triggers may drive TDP-43 oligomerization, including interaction partners or changes in pH or salinity. In this Assignment Note, we report the complete backbone and a wealth of side chain chemical shift assignments for the CTD of TDP-43 at pH 4. The assignments presented here provide a solid starting point to study the aggregation pathway of TDP-43 at pH values below those considered physiological but relevant in pathological settings, and to contrast the aggregation behaviour under distinct conditions and in the presence of interacting partners.

Molecular mechanism of the inhibition of TDP-43 amyloidogenesis by QBP1.

Transactive Response DNA-Binding Protein of 43 kDa (TDP-43) is an essential human protein implicated in Amyotrophic Lateral Sclerosis (ALS) and common dementias. Its C-terminal disordered region, composed of residues 264-414 includes a hydrophobic segment (residues 320-340), which drives physiological liquid/liquid phase separation and a Q/N-rich segment (residues 341-357), which is essential for pathological amyloid formation. Due to TDP-43's relevance for pathology, identifying inhibitors and characterizing their mechanism of action are important pharmacological goals. The Polyglutamine Binding Peptide 1 (QBP1), whose minimal active core is the octapeptide WGWWPGIF, strongly inhibits the aggregation of polyQ-containing amyloidogenic proteins such as Huntingtin. Rather promiscuous, this inhibitor also blocks the aggregation of other glutamine containing amyloidogenic proteins, but not Aß, and its mechanism of action remains unknown. Using a series of spectroscopic assays and biochemical tests, we establish that QBP1 binds and inhibits amyloid formation by TDP-43's Q/N-rich region. NMR spectroscopic data evince that the aromatic rings of QBP1 accept hydrogen bonds from the HN groups of the Asn and Gln to block amyloidogenesis. This mechanism of blockage may be general to polyphenol amyloid inhibitors.

Dysregulation of TDP-43 intracellular localization and early onset ALS are associated with a TARDBP S375G variant.

We investigated the Central Nervous System (CNS) and skeletal muscle tissue from A woman was clinically diagnosed with amyotrophic lateral sclerosis (ALS) at the age of 22. Neuropathologic evaluation showed upper and lower motor neuron loss, corticospinal tract degeneration and skeletal muscle denervation. Analysis of the patient's Deoxyribonucleic acid (DNA) revealed a AGT>GGT change resulting in an S375G substitution in the C-terminal region of TDP-43. This variant was previously reported as being benign. Considering the early onset and severity of the disease in this patient, we tested the effects of this genetic variant on TDP-43 localization, pre-mRNA splicing activity and toxicity, in parallel with the effects on known neighboring disease-associated mutations. In cell lines, expressed in culture, S375G TDP-43 appeared to be more significantly localized in the nucleus and to exert higher toxicity than wild-type TDP-43. Strikingly, a phosphomimic mutant at the same residue (S375E) showed a strong tendency to accumulate in the cytoplasm, especially under stress conditions, and molecular dynamics simulations suggest that phosphorylation of this residue can disrupt TDP-43 intermolecular interactions. The results of the current study highlight the importance of phosphorylation and regulation of TDP-43 nuclear-cytoplasmic shuttling/redistribution, in relation to the pathogenetic mechanisms involved in different forms of ALS.

The Singular NMR Fingerprint of a Polyproline II Helical Bundle.

Polyproline II (PPII) helices play vital roles in biochemical recognition events and structures like collagen and form part of the conformational landscapes of intrinsically disordered proteins (IDPs). Nevertheless, this structure is generally hard to detect and quantify. Here, we report the first thorough NMR characterization of a PPII helical bundle protein, the Hypogastrura harveyi "snow flea" antifreeze protein (sfAFP). J-couplings and nuclear Overhauser enhancement spectroscopy confirm a natively folded structure consisting of six PPII helices. NMR spectral analyses reveal quite distinct Hα2 versus Hα3 chemical shifts for 28 Gly residues as well as 13Cα, 15N, and 1HN conformational chemical shifts (Δδ) unique to PPII helical bundles. The 15N Δδ and 1HN Δδ values and small negative 1HN temperature coefficients evince hydrogen-bond formation. 1H-15N relaxation measurements reveal that the backbone structure is generally highly rigid on ps-ns time scales. NMR relaxation parameters and biophysical characterization reveal that sfAFP is chiefly a dimer. For it, a structural model featuring the packing of long, flat hydrophobic faces at the dimer interface is advanced. The conformational stability, measured by amide H/D exchange to be 6.24 ± 0.2 kcal·mol-1, is elevated. These are extraordinary findings considering the great entropic cost of fixing Gly residues and, together with the remarkable upfield chemical shifts of 28 Gly 1Hα, evidence significant stabilizing contributions from CαHα ||| O═C hydrogen bonds. These stabilizing interactions are corroborated by density functional theory calculations and natural bonding orbital analysis. The singular conformational chemical shifts, J-couplings, high hNOE ratios, small negative temperature coefficients, and slowed H/D exchange constitute a unique set of fingerprints to identify PPII helical bundles, which may be formed by hundreds of Gly-rich motifs detected in sequence databases. These results should aid the quantification of PPII helices in IDPs, the development of improved antifreeze proteins, and the incorporation of PPII helices into novel designed proteins.

The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

Pushing nuclear magnetic resonance sensitivity limits with microfluidics and photo-chemically induced dynamic nuclear polarization.

Among the methods to enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy, small-diameter NMR coils (microcoils) are promising tools to tackle the study of mass-limited samples. Alternatively, hyperpolarization schemes based on dynamic nuclear polarization techniques provide strong signal enhancements of the NMR target samples. Here we present a method to effortlessly perform photo-chemically induced dynamic nuclear polarization in microcoil setups to boost NMR signal detection down to sub-picomole detection limits in a 9.4T system (400 MHz 1H Larmor frequency). This setup is unaffected by current major drawbacks such as the use of high-power light sources to attempt uniform irradiation of the sample, and accumulation of degraded photosensitizer in the detection region. The latter is overcome with flow conditions, which in turn open avenues for complex applications requiring rapid and efficient mixing that are not easily achievable on an NMR tube without resorting to complex hardware.

Structural insight into the XTACC3/XMAP215 interaction from CD and NMR studies on model peptides.

TACC3 is a centrosomal adaptor protein that plays important roles during mitotic spindle assembly. It interacts with chTOG/XMAP215, which catalyzes the addition of tubulin dimers during microtubule growth. A 3D coiled-coil model for this interaction is available but the structural details are not well described. To characterize this interaction at atomic resolution, we have designed a simplified version of the system based on small peptides. Four different peptides have been studied by circular dichroism and nuclear magnetic resonance both singly and in all possible combinations; namely, five peptide pairs and two trios. In cosolvents, all single peptides tend to adopt helical conformations resembling those of the full-length protein. However, neither the single peptides nor pairs of peptides form coiled coils. We show that the simultaneous presence of all preformed helices is a prerequisite for binding. The simplest 3D model for the interaction, based on the NMR results, is proposed. Interestingly, the peptide's structure remains unaffected by mutations at essential positions for TACC3 activity. This suggests that the lack of interaction of this TACC3 mutant with XMAP does not correlate with changes in the protein structure and that specific interactions are likely responsible for the interaction and stability of the complex.

Point mutations in the N-terminal domain of transactive response DNA-binding protein 43 kDa (TDP-43) compromise its stability, dimerization, and functions.

Transactive response DNA-binding protein 43 (TDP-43) performs multiple tasks in mRNA processing, transport, and translational regulation, but it also forms aggregates implicated in amyotrophic lateral sclerosis. TDP-43's N-terminal domain (NTD) is important for these activities and dysfunctions; however, there is an open debate about whether or not it adopts a specifically folded, stable structure. Here, we studied NTD mutations designed to destabilize its structure utilizing NMR and fluorescence spectroscopies, analytical ultracentrifugation, splicing assays, and cell microscopy. The substitutions V31R and T32R abolished TDP-43 activity in splicing and aggregation processes, and even the rather mild L28A mutation severely destabilized the NTD, drastically reducing TDP-43's in vitro splicing activity and inducing aberrant localization and aggregation in cells. These findings strongly support the idea that a stably folded NTD is essential for correct TDP-43 function. The stably folded NTD also promotes dimerization, which is pertinent to the protein's activities and pathological aggregation, and we present an atomic-level structural model for the TDP-43 dimer based on NMR data. Leu-27 is evolutionarily well conserved even though it is exposed in the monomeric NTD. We found here that Leu-27 is buried in the dimer and that the L27A mutation promotes monomerization. In conclusion, our study sheds light on the structural and biological properties of the TDP-43 NTD, indicating that the NTD must be stably folded for TDP-43's physiological functions, and has implications for understanding the mechanisms promoting the pathological aggregation of this protein.

Insights into protein-carbohydrate recognition: A novel binding mechanism for CBM family 43.

Despite the growing number of carbohydrate-binding modules (CBMs) that are being uncovered, information on the structural determinants for the sugar-binding regions at atomic resolution is scarce. It is widely accepted that aromatic and H-bonding interactions govern these processes, and reported simulations and theoretical calculations are valuable tools to quantify and understand these interactions. We present here a computational model derived from experimental data that provide a unique atomistic picture of an uncharacterized binding mode of laminarin to the CBM family 43. The present study, which is among the first describing an isolated CBM with the bound carbohydrate, is complemented with quantum mechanical calculations. This allows us to attribute certain experimental observations (binding affinities) to key interactions (H-bonds and aromatic stacking), on the basis of NMR-driven docking structure.