Backbone 1H, 15N, and 13C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8 M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5 M urea and its fully unfolded state in 8 M urea.

Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2.

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid binding properties of AtGRP2-CSD. The 2D [1H,15N] HSQC spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of eleven residues at the C-terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a ß-barrel composed of five antiparallel ß-strands and a 310 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the ß3-ß4 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands ß2 and ß3, as well as the ß3-ß4 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the ß3-ß4 loop, evidenced by 15N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central ß-sheet, completely abolished DNA binding, highlighting the significance of π-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.

Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization.

The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membrane-less compartments through liquid-liquid phase separation, thereby facilitating the congregation of the replication and transcription complex. Considering the information available regarding the catalytic activities and binding signatures of the N protein-RNA interaction, this review focuses on the regulatory role of the SARS-CoV-2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities.

Late-stage Fluorination of Tyrosine Residues in Antiviral Protein Cyanovirin-N.

The applications of fluorinated molecules in chemical biology are rapidly expanding driven by the unique properties of C-F bonds, leading to increased interest in methodologies for controlled introduction of this atom. In this study, we present the first method for late-stage fluorination of tyrosine residues in proteins. Our results demonstrate that electrophilic fluorination using Selectfluor, a stable and non-toxic reagent, offers a straightforward and cost-effective method for labeling Cyanovirin-N (CVN), a 101-amino-acid lectin with effective antiviral activity. Uni- and bidimensional 1H, 13C and 19F NMR analyses, along with mass spectrometry, revealed chemoselective fluorination of the three tyrosine residues in CVN without affecting its overall structure or mannose-binding affinity. Additionally, we observed that other aromatic amino acids, such as tryptophan, phenylalanine, and histidine, are not fluorinated using this method. These findings advance our understanding of protein fluorination and its applications in studying structure, dynamics, and interactions, as well as other biological utilities.

1H, 15N, and 13C resonance assignments of the N-terminal domain and ser-arg-rich intrinsically disordered region of the nucleocapsid protein of the SARS-CoV-2.

The nucleocapsid (N) protein of SARS-CoV-2 is a multifunctional protein involved in nucleocapsid assembly and various regulatory functions. It is the most abundant protein during viral infection. Its functionality is closely related to its structure, which comprises two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by intrinsically disordered regions. The linker between the NTD and CTD includes a Serine-Arginine rich (SR) region, which is crucial for the regulation of the N protein's function. Here, we report the near-complete assignment of the construct containing the NTD followed by the SR region (NTD-SR). Additionally, we describe the dynamic nature of the SR region and compare it with all other available chemical shift assignments reported for the SR region.

Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.

Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.

Toward the mechanism of jarastatin (rJast) inhibition of the integrin αVß3.

Disintegrins are a family of cysteine-rich small proteins that were first identified in snake venom. The high divergence of disintegrins gave rise to a plethora of functions, all related to the interaction with integrins. Disintegrins evolved to interact selectively with different integrins, eliciting many physiological outcomes and being promising candidates for the therapy of many pathologies. We used NMR to determine the structure and dynamics of the recombinant disintegrin jarastatin (rJast) and its interaction with the cancer-related integrin αVß3. rJast displayed the canonical fold of a medium-sized disintegrin and showed complex dynamic in multiple timescales. We used NMR experiments to map the interaction of rJast with αVß3, and molecular docking followed by molecular dynamics (MD) simulation to describe the first structural model of a disintegrin/integrin complex. We showed that not only the RGD loop participates in the interaction, but also the N-terminal domain. rJast plasticity was essential for the interaction with αVß3 and correlated with the main modes of motion depicted in the MD trajectories. In summary, our study provides novel structural insights that enhance our comprehension of the mechanisms underlying disintegrin functionality.

NMR Relaxation Dispersion Experiments to Study Phosphopeptide Recognition by SH2 Domains: The Grb2-SH2-Phosphopeptide Encounter Complex.

Protein interactions are at the essence of life. Proteins evolved not to have stable structures, but rather to be specialized in participating in a network of interactions. Every interaction involving proteins comprises the formation of an encounter complex, which may have two outcomes: (i) the dissociation or (ii) the formation of the final specific complex. Here, we present a methodology to characterize the encounter complex of the Grb2-SH2 domain with a phosphopeptide. This method can be generalized to other protein partners. It consists of the measurement of 15N CPMG relaxation dispersion (RD) profiles of the protein in the free state, which describes the residues that are in conformational exchange. We then acquire the dispersion profiles of the protein at a semisaturated concentration of the ligand. At this condition, the chemical exchange between the free and bound state leads to the observation of dispersion profiles in residues that are not in conformational exchange in the free state. This is due to fuzzy interactions that are typical of the encounter complexes. The transient "touching" of the ligand in the protein partner generates these new relaxation dispersion profiles. For the Grb2-SH2 domain, we observed a wider surface at SH2 for the encounter complex than the phosphopeptide (pY) binding site, which might explain the molecular recognition of remote phosphotyrosine. The Grb2-SH2-pY encounter complex is dominated by electrostatic interactions, which contribute to the fuzziness of the complex, but also have contribution of hydrophobic interactions.

Backbone and sidechain NMR assignments of residues 1-81 from yeast Sis1 in complex with an Hsp70 C-terminal EEVD peptide.

Molecular chaperones aid proteins to fold and assemble without modifying their final structure, requiring, in several folding processes, the interplay between members of the Hsp70 and Hsp40 families. Here, we report the NMR chemical shift assignments for 1 H, 15 N, and 13 C nuclei of the backbone and side chains of the J-domain of the class B Hsp40 from Saccharomyces cerevisiae, Sis1, complexed with the C-terminal EEVD motif of Hsp70. The data revealed information on the structure and backbone dynamics that add significantly to the understanding of the J-domain-Hsp70-EEVD mechanism of interaction.

1 H, 15 N, and 13 C backbone and side chain resonance assignments of the cold shock domain of the Arabidopsis thaliana glycine-rich protein AtGRP2.

AtGRP2 (Arabidopsis thaliana glycine-rich protein 2) is a 19-kDa RNA-binding glycine-rich protein that regulates key processes in A. thaliana. AtGRP2 is a nucleo-cytoplasmic protein with preferential expression in developing tissues, such as meristems, carpels, anthers, and embryos. AtGRP2 knockdown leads to an early flowering phenotype. In addition, AtGRP2-silenced plants exhibit a reduced number of stamens and abnormal development of embryos and seeds, suggesting its involvement in plant development. AtGRP2 expression is highly induced by cold and abiotic stresses, such as high salinity. Moreover, AtGRP2 promotes double-stranded DNA/RNA denaturation, indicating its role as an RNA chaperone during cold acclimation. AtGRP2 is composed of an N-terminal cold shock domain (CSD) followed by a C-terminal flexible region containing two CCHC-type zinc fingers interspersed with glycine-rich sequences. Despite its functional relevance in flowering time regulation and cold adaptation, the molecular mechanisms employed by AtGRP2 are largely unknown. To date, there is no structural information regarding AtGRP2 in the literature. Here, we report the 1H, 15N, and 13C backbone and side chain resonance assignments, as well as the chemical shift-derived secondary structure propensities, of the N-terminal cold shock domain of AtGRP2, encompassing residues 1-90. These data provide a framework for AtGRP2-CSD three-dimensional structure, dynamics, and RNA binding specificity investigation, which will shed light on its mechanism of action.

Self-assembly of dengue virus empty capsid-like particles in solution.

Nucleocapsid (NC) assembly is an essential step of the virus replication cycle. It ensures genome protection and transmission among hosts. Flaviviruses are human viruses for which envelope structure is well known, whereas no information on NC organization is available. Here we designed a dengue virus capsid protein (DENVC) mutant in which a highly positive spot conferred by arginine 85 in α4-helix was replaced by a cysteine residue, simultaneously removing the positive charge and restricting the intermolecular motion through the formation of a disulfide cross-link. We showed that the mutant self-assembles into capsid-like particles (CLP) in solution without nucleic acids. Using biophysical techniques, we investigated capsid assembly thermodynamics, showing that an efficient assembly is related to an increased DENVC stability due to α4/α4' motion restriction. To our knowledge, this is the first time that flaviviruses' empty capsid assembly is obtained in solution, revealing the R85C mutant as a powerful tool to understand the NC assembly mechanism.

The 1H, 15N and 13C resonance assignments of dengue virus capsid protein with the deletion of the intrinsically disordered N-terminal region.

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.

IgA quantification as a good predictor of the neutralizing antibodies levels after vaccination against SARS-CoV-2.

Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions.

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M ß-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried ß-sheet. The two mutations located in this ß-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M ß-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

Deciphering the Path of S-nitrosation of Human Thioredoxin: Evidence of an Internal NO Transfer and Implication for the Cellular Responses to NO.

Nitric oxide (NO) is a free radical with a signaling capacity. Its cellular functions are achieved mainly through S-nitrosation where thioredoxin (hTrx) is pivotal in the S-transnitrosation to specific cellular targets. In this study, we use NMR spectroscopy and mass spectrometry to follow the mechanism of S-(trans)nitrosation of hTrx. We describe a site-specific path for S-nitrosation by measuring the reactivity of each of the 5 cysteines of hTrx using cysteine mutants. We showed the interdependence of the three cysteines in the nitrosative site. C73 is the most reactive and is responsible for all S-transnitrosation to other cellular targets. We observed NO internal transfers leading to C62 S-nitrosation, which serves as a storage site for NO. C69-SNO only forms under nitrosative stress, leading to hTrx nuclear translocation.

On the caveats of a multiplex test for SARS-CoV-2 to detect seroconversion after infection or vaccination.

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.

Characterization of the RNA-dependent RNA polymerase from Chikungunya virus and discovery of a novel ligand as a potential drug candidate.

Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an acute febrile and arthritogenic illness with no effective treatments available. The development of effective therapeutic strategies could be significantly accelerated with detailed knowledge of the molecular components behind CHIKV replication. However, drug discovery is hindered by our incomplete understanding of their main components. The RNA-dependent RNA-polymerase (nsP4-CHIKV) is considered the key enzyme of the CHIKV replication complex and a suitable target for antiviral therapy. Herein, the nsP4-CHIKV was extensively characterized through experimental and computational biophysical methods. In the search for new molecules against CHIKV, a compound designated LabMol-309 was identified as a strong ligand of the nsp4-CHIKV and mapped to bind to its active site. The antiviral activity of LabMol-309 was evaluated in cellular-based assays using a CHIKV replicon system and a reporter virus. In conclusion, this study highlights the biophysical features of nsP4-CHIKV and identifies a new compound as a promising antiviral agent against CHIKV infection.

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

15N, 13C, and 1H resonance assignments of Jarastatin: a disintegrin of Bothrops jararaca.

Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play a fundamental role in the regulation of many physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of recombinant disintegrin Jarastatin (rJast), which was further validated by secondary structure prediction using the TALOS-N server. Taken together, these data are essential to perform NMR-based experiments, including structure determination, backbone dynamics, mapping ligand sites and enabling a deeper understanding of the effect of hydrophobic surface clusters, which are important elements to stabilize some 3D proteins structure/folding.