Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Molecular details of the CPSF73-CPSF100 C-terminal heterodimer and interaction with Symplekin.

Eukaryotic pre-mRNA is processed by a large multiprotein complex to accurately cleave the 3' end, and to catalyse the addition of the poly(A) tail. Within this cleavage and polyadenylation specificity factor (CPSF) machinery, the CPSF73/CPSF3 endonuclease subunit directly contacts both CPSF100/CPSF2 and the scaffold protein Symplekin to form a subcomplex known as the core cleavage complex or mammalian cleavage factor. Here we have taken advantage of a stable CPSF73-CPSF100 minimal heterodimer from Encephalitozoon cuniculi to determine the solution structure formed by the first and second C-terminal domain (CTD1 and CTD2) of both proteins. We find a large number of contacts between both proteins in the complex, and notably in the region between CTD1 and CTD2. A similarity is also observed between CTD2 and the TATA-box binding protein (TBP) domains. Separately, we have determined the structure of the terminal CTD3 domain of CPSF73, which also belongs to the TBP domain family and is connected by a flexible linker to the rest of CPSF73. Biochemical assays demonstrate a key role for the CTD3 of CPSF73 in binding Symplekin, and structural models of the trimeric complex from other species allow for comparative analysis and support an overall conserved architecture.

Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B.

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

1H, 15N and 13C resonance assignments of a minimal CPSF73-CPSF100 C-terminal heterodimer.

The initial pre-mRNA transcript in eukaryotes is processed by a large multi-protein complex in order to correctly cleave the 3' end, and to subsequently add the polyadenosine tail. This cleavage and polyadenylation specificity factor (CPSF) is composed of separate subunits, with structural information available for both isolated subunits and also larger assembled complexes. Nevertheless, certain key components of CPSF still lack high-resolution atomic data. One such region is the heterodimer formed between the first and second C-terminal domains of the endonuclease CPSF73, with those from the catalytically inactive CPSF100. Here we report the backbone and sidechain resonance assignments of a minimal C-terminal heterodimer of CPSF73-CPSF100 derived from the parasite Encephalitozoon cuniculi. The assignment process used several amino-acid specific labeling strategies, and the chemical shift values allow for secondary structure prediction.

RNA G-quadruplex forming regions from SARS-2, SARS-1 and MERS coronoviruses.

COVID-19 (Corona Virus Disease 2019), SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome) are infectious diseases each caused by coronavirus outbreaks. Small molecules and other therapeutics are rapidly being developed to treat these diseases, but the threat of new variants and outbreaks argue for the identification of additional viral targets. Here we identify regions in each of the three coronavirus genomes that are able to form G-quadruplex (G4) structures. G4s are structures formed by DNA or RNA with a core of two or more stacked planes of guanosine tetrads. In recent years, numerous DNA and RNA G4s have emerged as promising pharmacological targets for the treatment of cancer and viral infection. We use a combination of bioinformatics and biophysical approaches to identify conserved RNA G4 regions from the ORF1A and S sequences of SARS-CoV, SARS-CoV-2 and MERS-CoV. Although a general depletion of G4-forming regions is observed in coronaviridae, the preservation of these selected G4 sequences support a significance in viral replication. Targeting these RNA structures may represent a new antiviral strategy against these viruses distinct from current approaches that target viral proteins.

Adaptive Binding of Alkyl Glycosides by Nonpeptidic Helix Bundles in Water: Toward Artificial Glycolipid Binding Proteins.

Amphipathic water-soluble helices formed from synthetic peptides or foldamers are promising building blocks for the creation of self-assembled architectures with non-natural shapes and functions. While rationally designed artificial quaternary structures such as helix bundles have been shown to contain preformed cavities suitable for guest binding, there are no examples of adaptive binding of guest molecules by such assemblies in aqueous conditions. We have previously reported a foldamer 6-helix bundle that contains an internal nonpolar cavity able to bind primary alcohols as guest molecules. Here, we show that this 6-helix bundle can also interact with larger, more complex guests such as n-alkyl glycosides. X-ray diffraction analysis of co-crystals using a diverse set of guests together with solution and gas-phase studies reveals an adaptive binding mode whereby the apo form of the 6-helix bundle undergoes substantial conformational change to accommodate the hydrocarbon chain in a manner reminiscent of glycolipid transfer proteins in which the cavity forms upon lipid uptake. The dynamic nature of the self-assembling and molecular recognition processes reported here marks a step forward in the design of functional proteomimetic molecular assemblies.

Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex.

To eliminate specific or aberrant transcripts, eukaryotes use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC, and its human counterpart PAXT, are key players in this mechanism but inner workings of these complexes are not understood in sufficient detail. Here, we present an NMR structure of an MTREC scaffold protein Red1 helix-turn-helix domain bound to the Iss10 N-terminus and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex explaining mutually exclusive interactions of hARS2 with various ED/EGEI/L motif-possessing RNA regulators, including hZFC3H1 of PAXT, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.

Principles and correction of 5'-splice site selection.

In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5'-splice site (5'ss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5'ss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5'ss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5'ss selectivity with antisense oligonucleotides and small-molecule splicing switches.

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function.

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531-1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals.

Crystal structures capture multiple stoichiometric states of an aqueous self-assembling oligourea foldamer.

We report here an oligourea foldamer able to self-assemble in aqueous conditions into helix bundles of multiple stoichiometries. Importantly, we report crystal structures of several of these stoichiometries, providing a series of high-resolution snap-shots of the structural polymorphism of this foldamer and uncovering a novel self-assembly.

Deciphering the Enigma of the Histone H2A.Z-1/H2A.Z-2 Isoforms: Novel Insights and Remaining Questions.

The replication independent (RI) histone H2A.Z is one of the more extensively studied variant members of the core histone H2A family, which consists of many replication dependent (RD) members. The protein has been shown to be indispensable for survival, and involved in multiple roles from DNA damage to chromosome segregation, replication, and transcription. However, its functional involvement in gene expression is controversial. Moreover, the variant in several groups of metazoan organisms consists of two main isoforms (H2A.Z-1 and H2A.Z-2) that differ in a few (3-6) amino acids. They comprise the main topic of this review, starting from the events that led to their identification, what is currently known about them, followed by further experimental, structural, and functional insight into their roles. Despite their structural differences, a direct correlation to their functional variability remains enigmatic. As all of this is being elucidated, it appears that a strong functional involvement of isoform variability may be connected to development.

Structural basis of UCUU RNA motif recognition by splicing factor RBM20.

The vertebrate splicing factor RBM20 (RNA binding motif protein 20) regulates protein isoforms important for heart development and function, with mutations in the gene linked to cardiomyopathy. Previous studies have identified the four nucleotide RNA motif UCUU as a common element in pre-mRNA targeted by RBM20. Here, we have determined the structure of the RNA Recognition Motif (RRM) domain from mouse RBM20 bound to RNA containing a UCUU sequence. The atomic details show that the RRM domain spans a larger region than initially proposed in order to interact with the complete UCUU motif, with a well-folded C-terminal helix encoded by exon 8 critical for high affinity binding. This helix only forms upon binding RNA with the final uracil, and removing the helix reduces affinity as well as specificity. We therefore find that RBM20 uses a coupled folding-binding mechanism by the C-terminal helix to specifically recognize the UCUU RNA motif.

Allosteric Recognition of Homomeric and Heteromeric Pairs of Monosaccharides by a Foldamer Capsule.

The recognition of either homomeric or heteromeric pairs of pentoses in an aromatic oligoamide double helical foldamer capsule was evidenced by circular dichroism (CD), NMR spectroscopy, and X-ray crystallography. The cavity of the host was predicted to be large enough to accommodate simultaneously two xylose molecules and to form a 1:2 complex (one container, two saccharides). Solution and solid-state data revealed the selective recognition of the α-4 C1 -d-xylopyranose tautomer, which is bound at two identical sites in the foldamer cavity. A step further was achieved by sequestering a heteromeric pair of pentoses, that is, one molecule of α-4 C1 -d-xylopyranose and one molecule of ß-1 C4 -d-arabinopyranose despite the symmetrical nature of the host and despite the similarity of the guests. Subtle induced-fit and allosteric effects are responsible for the outstanding selectivities observed.

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.

Biophysical Insight on the Membrane Insertion of an Arginine-Rich Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this peptide toward zwitterionic lipids.

Structure Elucidation of Helical Aromatic Foldamer-Protein Complexes with Large Contact Surface Areas.

The development of large synthetic ligands could be useful to target the sizeable surface areas involved in protein-protein interactions. Herein, we present long helical aromatic oligoamide foldamers bearing proteinogenic side chains that cover up to 450 Å2 of the human carbonic anhydrase II (HCA) surface. The foldamers are composed of aminoquinolinecarboxylic acids bearing proteinogenic side chains and of more flexible aminomethyl-pyridinecarboxylic acids that enhance helix handedness dynamics. Crystal structures of HCA-foldamer complexes were obtained with a 9- and a 14-mer both showing extensive protein-foldamer hydrophobic contacts. In addition, foldamer-foldamer interactions seem to be prevalent in the crystal packing, leading to the peculiar formation of an HCA superhelix wound around a rod of stacked foldamers. Solution studies confirm the positioning of the foldamer at the protein surface as well as a dimerization of the complexes.

Assessing Interactions between Helical Aromatic Oligoamide Foldamers and Protein Surfaces: A Tethering Approach.

Helically folded aromatic foldamers may constitute suitable candidates for the ab initio design of ligands for protein surfaces. As preliminary steps toward the exploration of this hypothesis, a tethering approach was developed to detect interactions between a protein and a foldamer by confining the former at the surface of the latter. Cysteine mutants of two therapeutically relevant enzymes, CypA and IL4, were produced. Two series of ten foldamers were synthesized bearing different proteinogenic side chains and either a long or a short linker functionalized with an activated disulfide. Disulfide exchange between the mutated cysteines and the activated disulfides yielded 20 foldamer-IL4 and 20 foldamer-CypA adducts. Effectiveness of the reaction was demonstrated by LC-MS, by MS analysis after proteolytic digestion, and by 2D NMR. Circular dichroism then revealed diastereoselective interactions between the proteins and the foldamers confined at their surface which resulted in a preferred handedness of the foldamer helix. Helix sense bias occurred sometimes with both the short and the long linkers and sometimes with only one of them. In a few cases, helix handedness preference is found to be close to quantitative. These cases constitute valid candidates for structural elucidation of the interactions involved.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module.

Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.

Distinct roles of Pcf11 zinc-binding domains in pre-mRNA 3'-end processing.

New transcripts generated by RNA polymerase II (RNAPII) are generally processed in order to form mature mRNAs. Two key processing steps include a precise cleavage within the 3' end of the pre-mRNA, and the subsequent polymerization of adenosines to produce the poly(A) tail. In yeast, these two functions are performed by a large multi-subunit complex that includes the Cleavage Factor IA (CF IA). The four proteins Pcf11, Clp1, Rna14 and Rna15 constitute the yeast CF IA, and of these, Pcf11 is structurally the least characterized. Here, we provide evidence for the binding of two Zn2+ atoms to Pcf11, bound to separate zinc-binding domains located on each side of the Clp1 recognition region. Additional structural characterization of the second zinc-binding domain shows that it forms an unusual zinc finger fold. We further demonstrate that the two domains are not mandatory for CF IA assembly nor RNA polymerase II transcription termination, but are rather involved to different extents in the pre-mRNA 3'-end processing mechanism. Our data thus contribute to a more complete understanding of the architecture and function of Pcf11 and its role within the yeast CF IA complex.