Macromolecular interactions in vitro, comparing classical and novel approaches.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE.

Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.

switchSENSE Technology for Analysis of DNA Polymerase Kinetics.

The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme.

Different views of the dynamic landscape covered by the 5'-hairpin of the 7SK small nuclear RNA.

The 7SK small nuclear RNA (7SKsnRNA) plays a key role in the regulation of RNA polymerase II by sequestrating and inhibiting the positive transcription elongation factor b (P-TEFb) in the 7SK ribonucleoprotein complex (7SKsnRNP), a process mediated by interaction with the protein HEXIM. P-TEFb is also an essential cellular factor recruited by the viral protein Tat to ensure the replication of the viral RNA in the infection cycle of the human immunodeficiency virus (HIV-1). Tat promotes the release of P-TEFb from the 7SKsnRNP and subsequent activation of transcription, by displacing HEXIM from the 5'-hairpin of the 7SKsnRNA. This hairpin (HP1), comprising the signature sequence of the 7SKsnRNA, has been the subject of three independent structural studies aimed at identifying the structural features that could drive the recognition by the two proteins, both depending on arginine-rich motifs (ARM). Interestingly, four distinct structures were determined. In an attempt to provide a comprehensive view of the structure-function relationship of this versatile RNA, we present here a structural analysis of the models, highlighting how HP1 is able to adopt distinct conformations with significant impact on the compactness of the molecule. Since these models are solved under different conditions by nuclear magnetic resonance (NMR) and crystallography, the impact of the buffer composition on the conformational variation was investigated by complementary biophysical approaches. Finally, using isothermal titration calorimetry, we determined the thermodynamic signatures of the Tat-ARM and HEXIM-ARM peptide interactions with the RNA, showing that they are associated with distinct binding mechanisms.

Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes.

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

Double methylation of tRNA-U54 to 2'-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7.

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.

Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations.

We studied by native ESI-MS the binding of various DNA-polymerase-derived peptides onto DNA-polymerase processivity rings from Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. These homodimeric rings present two equivalent specific binding sites, which leads to successive formation during a titration experiment of singly- and doubly occupied rings. By using the ESI-MS free-ring spectrum as a ruler, we derived by robust linear regression the fractions of the different ring species at each step of a titration experiment. These results led to accurate Kd values (from 0.03 to 0.5 µM) along with the probability of peptide loss due to gas phase dissociation (GPD). We show that this good quality is due to the increased information content of a titration experiment with a homodimer. Isothermal titration calorimetry (ITC) led with the same binding model to Kd(ITC) values systematically higher than their ESI-MS counterparts and, often, to poor fit of the ITC curves. A processing with two competing modes of binding on the same site requiring determination of two (Kd, ΔH) pairs greatly improved the fits and yielded a second Kd(ITC) close to Kd(ESI-MS). The striking features are: (1) ITC detected a minor binding mode (~20%) of 'low-affinity' that did not appear with ESI-MS; (2) the simplest processing of ITC data with only one (Kd, ΔH) pair led wrongly to the Kd of the low-affinity binding mode but to the ΔH of the high-affinity binding mode. Analogous misleading results might well exist in published data based on ITC experiments. Graphical Abstract ᅟ.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches.

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Extending ITC to Kinetics with kinITC.

Isothermal titration calorimetry (ITC) has long been used for kinetic studies in chemistry, but this remained confined to enzymatic studies in the biological field. In fact, the biological community has long had the tendency of ignoring the kinetic possibilities of ITC considering it solely as a thermodynamic technique, whereas surface plasmon resonance is seen as the kinetic technique par excellence. However, the primary signal recorded by ITC is a heat power which is directly related to the kinetics of the reaction. Here, it is shown how this kinetic signal can be recovered by using kinITC, the kinetic extension of ITC. The theoretical basis of kinITC is detailed for the most common situation of a second-order reaction A+B Ω C characterized by kinetic parameters kon, koff. A simplified kinITC-ETC method based upon the determination of an "Equilibration Time Curve" (ETC) is presented. The ETC is obtained by automatic determination of the "effective end" of each injection. The method is illustrated with experimental results with a comparison to Surface Plasmon Resonance (SPR) data. kon values were obtained in a wide range, from 10(3) to 0.5×10(6) M(-1) s(-1). All procedures were implemented in the program AFFINImeter (https://www.affinimeter.com/).

Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.

kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods allow researchers to obtain a complete kinetic description of systems more diverse than previously thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction) and with the more complex case of the folding of a riboswitch triggered by the binding of its ligand. The originality of the new kinITC method lies in its ability to dissect, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method that can yield, in a simple way, such deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.

8-Modified-2'-deoxyadenosine analogues induce delayed polymerization arrest during HIV-1 reverse transcription.

The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2'-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2'-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein.

Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.

Discovery of chiral cyclopropyl dihydro-alkylthio-benzyl-oxopyrimidine (S-DABO) derivatives as potent HIV-1 reverse transcriptase inhibitors with high activity against clinically relevant mutants.

The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.

Effects of tRNA 3 Lys aminoacylation on the initiation of HIV-1 reverse transcription.

HIV-1 utilizes cellular tRNA(3)(Lys) to prime the initiation of reverse transcription. The selective incorporation of cytoplasmic tRNA(3)(Lys) into HIV-1 particles was recently shown to involve the lysyl-tRNA synthetase, and hence, the encapsidated tRNA(3)(Lys) is likely to be aminoacylated. Here, we tested the effect of aminoacylation on the initiation of reverse transcription. We show that HIV-1 reverse transcriptase is unable to extend lysyl-tRNA(3)(Lys). In addition, the viral polymerase does not significantly enhance the rate of tRNA deacylation, in contrast with previous studies on avian retroviruses. Thus, aminoacylation of the primer tRNA might prevent the initiation of HIV-1 reverse transcription from taking place before viral budding and maturation.