Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Complexation Preferences of Dynamic Constitutional Frameworks as Adaptive Gene Vectors.

The growing applications of therapeutic nucleic acids requires the concomitant development of vectors that are optimized to complex one type of nucleic acid, forming nanoparticles suitable for further trafficking and delivery. While fine-tuning a vector by molecular engineering to obtain a particular nanoscale organization at the nanoparticle level can be a challenging endeavor, we turned the situation around and instead screened the complexation preferences of dynamic constitutional frameworks toward different types of DNAs. Dynamic constitutional frameworks (DCF) are recently-identified vectors by our group that can be prepared in a versatile manner through dynamic covalent chemistry. Herein, we designed and synthesized 40 new DCFs that vary in hydrophilic/hydrophobic balance, number of cationic headgroups. The results of DNA complexation obtained through gel electrophoresis and fluorescent displacement assays reveal binding preferences of different DCFs toward different DNAs. The formation of compact spherical architectures with an optimal diameter of 100-200 nm suggests that condensation into nanoparticles is more effective for longer PEG chains and PEI groups that induce a better binding performance in the presence of DNA targets.

Identification of new lead molecules against anticancer drug target TFIIH subunit P8 using biophysical and molecular docking studies.

The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.

Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy.

The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.

Switching Multivalent DNA Complexation using Metal-Controlled Cationic Supramolecular Self-Assemblies.

We provide a proof-of-principle that coordination chemistry drives the in situ self-assembly of an inactive ligand into a multivalent cluster capable of effectively complexing DNA. We show that metal coordination and scavenging can be used to switch the multivalency of the system. Thus, controlled DNA complexation and decomplexation could be achieved.

Multivalent Metallosupramolecular Assemblies as Effective DNA Binding Agents.

We report the implementation of coordination chemistry onto the generation of new types of metallosupramolecular complexes with laterally appended cationic moieties for DNA binding in buffered aqueous media. Utilization of an N,N,O-type coordination pocket along with an octahedral zinc(II) metal ion allowed us to obtain mono- and tetranuclear complexes in both solution and solid state, as confirmed by NMR spectroscopy and single-crystal X-ray diffraction, respectively. By using isothermal titration calorimetry and gel electrophoresis, multiply charged cationic assemblies were observed to effectively bind to DNA through multivalent electrostatic interactions. Furthermore, we observed a correlation between the multivalency of the compounds employed and the effectiveness of DNA binding.

The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization.

Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features.

Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters.

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.

Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1.

The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease.

NMR studies of a new family of DNA binding proteins: the THAP proteins.

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

Structural determinants of specific DNA-recognition by the THAP zinc finger.

Human THAP1 is the prototype of a large family of cellular factors sharing an original THAP zinc-finger motif responsible for DNA binding. Human THAP1 regulates endothelial cell proliferation and G1/S cell-cycle progression, through modulation of pRb/E2F cell-cycle target genes including rrm1. Recently, mutations in THAP1 have been found to cause DYT6 primary torsion dystonia, a human neurological disease. We report here the first 3D structure of the complex formed by the DNA-binding domain of THAP1 and its specific DNA target (THABS) found within the rrm1 target gene. The THAP zinc finger uses its double-stranded beta-sheet to fill the DNA major groove and provides a unique combination of contacts from the beta-sheet, the N-terminal tail and surrounding loops toward the five invariant base pairs of the THABS sequence. Our studies reveal unprecedented insights into the specific DNA recognition mechanisms within this large family of proteins controlling cell proliferation, cell cycle and pluripotency.

Characterization of plant microRNA-encoded peptides (miPEPs) reveals molecular mechanisms from the translation to activity and specificity.

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

TFIIH contains a PH domain involved in DNA nucleotide excision repair.

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.

A Cationic Tetraphenylethene as a Light-Up Supramolecular Probe for DNA G-Quadruplexes.

Guanine-quadruplexes (G4s) are targets for anticancer therapeutics. In this context, human telomeric DNA (HT-DNA) that can fold into G4s sequences are of particular interest, and their stabilization with small molecules through a visualizable process has become a challenge. As a new type of ligand for HT-G4, we designed a tetraimidazolium tetraphenylethene (TPE-Im) as a water-soluble light-up G4 probe. We study its G4-binding properties with HT-DNA by UV-Visible absorption, circular dichroism and fluorescence spectroscopies, which provide insights into the interactions between TPE-Im and G4-DNA. Remarkably, TPE-Im shows a strong fluorescence enhancement and large shifts upon binding to G4, which is valuable for detecting G4s. The association constants for the TPE-Im/G4 complex were evaluated in different solution conditions via isothermal titration calorimetry (ITC), and its binding modes were explored by molecular modeling showing a groove-binding mechanism. The stabilization of G4 by TPE-Im has been assessed by Fluorescence Resonance Energy Transfer (FRET) melting assays, which show a strong stabilization (ΔT 1/2 around +20°C), together with a specificity toward G4 with respect to double-stranded DNA.

Publisher Correction: Structural insights into chaperone addiction of toxin-antitoxin systems.

The original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the Mtb-SecBTA sequence was displayed incorrectly. In the inset panel within Fig. 4c, the y-axis of the graph incorrectly read (Q.Rg)2 × I(Q)//(0), and should have read (Q.Rg)2 × I(Q)/I(0). These errors have been corrected in both the PDF and HTML versions of the Article.

Structural insights into chaperone addiction of toxin-antitoxin systems.

SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB-SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions.

Structural insights on the pamoic acid and the 8 kDa domain of DNA polymerase beta complex: towards the design of higher-affinity inhibitors.

BACKGROUND: DNA polymerase beta (pol beta), the error-prone DNA polymerase of single-stranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. Several inhibitors have been identified, but none of them presents a sufficient affinity and specificity to become a drug. The fragment-based inhibitor design allows an important improvement in affinity of small molecules. The initial and critical step for setting up the fragment-based strategy consists in the identification and structural characterization of the first fragment bound to the target. RESULTS: We have performed docking studies of pamoic acid, a 9 micromolar pol beta inhibitor, and found that it binds in a single pocket at the surface of the 8 kDa domain of pol beta. However, docking studies provided five possible conformations for pamoic acid in this site. NMR experiments were performed on the complex to select a single conformation among the five retained. Chemical Shift Mapping data confirmed pamoic acid binding site found by docking while NOESY and saturation transfer experiments provided distances between pairs of protons from the pamoic acid and those of the 8 kDa domain that allowed the identification of the correct conformation. CONCLUSION: Combining NMR experiments on the complex with docking results allowed us to build a three-dimensional structural model. This model serves as the starting point for further structural studies aimed at improving the affinity of pamoic acid for binding to DNA polymerase beta.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

Functional roles of H98 and W99 and ß2α2 loop dynamics in the α-l-arabinofuranosidase from Thermobacillus xylanilyticus.

This study is focused on the elucidation of the functional role of the mobile ß2α2 loop in the α-L-arabinofuranosidase from Thermobacillus xylanilyticus, and particularly on the roles of loop residues H98 and W99. Using site-directed mutagenesis, coupled to characterization methods including isothermal titration calorimetry (ITC) and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and molecular dynamics simulations, it has been possible to provide a molecular level view of interactions and the consequences of mutations. Binding of para-nitrophenyl α-L-arabinofuranoside (pNP-α-l-Araf) to the wild-type arabinofuranosidase was characterized by K(d) values (0.32 and 0.16 mm, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA(3)XX (0.21 mm), and determination of the thermodynamic parameters of enzyme : pNP-α-L-Araf binding revealed that this process is driven by favourable entropy, which is linked to the movement of the ß2α2 loop. Loop closure relocates the solvent-exposed W99 into a buried location, allowing its involvement in substrate binding and in the formation of a functional active site. Similarly, the data underline the role of H98 in the 'dynamic' formation and definition of a catalytically operational active site, which may be a specific feature of a subset of GH51 arabinofuranosidases. Substitution of H98 and W99 by alanine or phenylalanine revealed that mutations affected K(M) and/or k(cat). Molecular dynamics performed on W99A implied that this mutation causes the loss of a hydrogen bond and leads to an alternative binding mode that is detrimental for catalysis. STD-NMR experiments revealed altered binding of the aglycon motif in the active site, combined with reduced STD intensities of the α-L-arabinofuranosyl moiety for W99 substitutions.

Solution state NMR structure and dynamics of KpOmpA, a 210 residue transmembrane domain possessing a high potential for immunological applications.

The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR.This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins(210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly 15N,13C,2H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 A for the main chain atoms in the core eight-stranded beta-barrel. The protein dynamics was assessed, in a residue-specific manner, by 1H-15N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and 1H-2H chemical exchange (hour-weeks).