Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer.

The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA-p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA-RelA homodimer is critical as it facilitates the formation of the more stable RelA-p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA-RelA homodimer, the kinetics of the RelA-p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA-RelA specific target gene regulation in physiology.

Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα.

The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.

Analysis of the RelA:CBP/p300 interaction reveals its involvement in NF-κB-driven transcription.

NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components--first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.

Backbone triple resonance assignments of the dimerization domain of NF-kappaB p52 subunit.

NF-kappaB is a family of inducible transcription factors playing an important role in immune response in vertebrates. All the five members of the family function as dimers in various combinations. Though all the family members recognize and bind to similar DNA elements to regulate the transcription of its target genes, the dimer composition can lead to differential transcriptional outcomes. Here we report the backbone resonance assignment of the 24.2 kDa homodimer of p52 subunit of the NF-kB family. The p52 subunit of NF-kB is a crucial player in the non-canonical NF-kB pathway and its dysregulation has shown detrimental effects in immune response leading to various inflammatory diseases and cancers. While the ß-strands predicted using the backbone chemical shifts in this study largely conform with the available crystal structure, the helical turns present in the crystal structure are not observed in our results.

Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites.

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which causes severe illness in humans and is responsible for epidemic outbreaks in Africa, Asia, North and South America, and Europe. Despite its increased global prevalence, no licensed vaccines are available to date for treating or preventing CHIKV infection. The envelope E2 protein is one of the promising subunit vaccine candidates against CHIKV. In this study, we describe successful cloning, expression, and purification of CHIKV E2 full-length (E2-FL) and truncated (E2-ΔC and E2-ΔNC) proteins in the Escherichia coli expression system. The recombinant E2 proteins were purified from inclusion bodies using Ni-NTA chromatography. Further, we describe a detailed refolding procedure for obtaining the CHIKV E2-FL protein in native conformation, which was confirmed using circular dichroism and Fourier transform infrared spectroscopy. BALB/c mice immunized with the three different E2 proteins exhibited increased E2-specific antibody titers compared to sham-immunized controls, suggesting induction of strong humoral immune response. On analyzing the E2-specific antibody response generated in immunized mice, the CHIKV E2-FL protein was observed to be the most immunogenic among the three different CHIKV E2 antigens used in the study. Our B-cell and T-cell epitope mapping results indicate that the presence of specific immunogenic peptides located in the N-terminal and C-terminal regions of the CHIKV E2-FL protein may contribute to its increased immunogenicity, compared to truncated CHIKV E2 proteins. In summary, our study provides a detailed protocol for expressing, purifying, and refolding of the CHIKV E2-FL protein and provides an understanding of its immunogenic epitopes, which can be exploited for the development of novel multiepitope-based anti-CHIKV vaccine strategies.

Insights into the NF-κB-DNA Interaction through NMR Spectroscopy.

Transcription factors bind specifically to their target elements in the genome, eliciting specific gene expression programs. The nuclear factor-κB (NF-κB) system is a family of proteins comprising inducible transcription activators, which play a critical role in inflammation and cancer. The NF-κB members function as dimers with each monomeric unit binding the κB-DNA. Despite the available structures of the various NF-κB dimers in complex with the DNA, the structural features of these dimers in the nucleic acid-free form are not well-characterized. Using solution NMR spectroscopy, we characterize the structural features of 73.1 kDa p50 subunit of the NF-κB homodimer in the DNA-free form and compare it with the κB DNA-bound form of the protein. The study further reveals that in the nucleic acid-free form, the two constituent domains of p50, the N-terminal and the dimerization domains, are structurally independent of each other. However, in a complex with the κB DNA, both the domains of p50 act as a single unit. The study also provides insights into the mechanism of κB DNA recognition by the p50 subunit of NF-κB.

Backbone resonance assignments of the dimeric domain of the p50 NF-kappaB subunit.

The Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a family of transcription factor recognizing a 9-11 base pair kappaB sites on the promoter/enhancer region of their target genes. The family comprises of five members forming dimers amongst themselves in various combinations. Here we report the backbone resonance assignments of the 24 kDa homodimer of the p50 subunit of NF-kappaB. This is the first step towards understanding the mechanism of dimer formation in solution. The secondary structure derived from the chemical shifts for the dimer is largely consistent with that observed in the available crystal structures of the protein in DNA-bound form.

Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein.

Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways.

Super-Resolved Nuclear Magnetic Resonance Spectroscopy.

We present a novel method that breaks the resolution barrier in nuclear magnetic resonance (NMR) spectroscopy, allowing one to accurately estimate the chemical shift values of highly overlapping or broadened peaks. This problem is routinely encountered in NMR when peaks have large linewidths due to rapidly decaying signals, hindering its application. We address this problem based on the notion of finite-rate-of-innovation (FRI) sampling, which is based on the premise that signals such as the NMR signal, can be accurately reconstructed using fewer measurements than that required by existing approaches. The FRI approach leads to super-resolution, beyond the limits of contemporary NMR techniques. Using this method, we could measure for the first time small changes in chemical shifts during the formation of a Gold nanorod-protein complex, facilitating the quantification of the strength of such interactions. The method thus opens up new possibilities for the application and acceleration of multidimensional NMR spectroscopy across a wide range of systems.

Structural basis for the observed differential magnetic anisotropic tensorial values in calcium binding proteins.

Lanthanide ions (Ln(3+)), which have ionic radii similar to those of Ca(2+), can displace the latter in a calcium binding protein, without affecting its tertiary structure. The paramagnetic Ln(3+) possesses large anisotropic magnetic susceptibilities and produce pseudocontact shifts (PCSs), which have r(-3) dependence. The PCS can be seen for spins as far as 45 A from the paramagnetic ion. They aid in structure refinement of proteins by providing long-range distance constraints. Besides, they can be used to determine the interdomain orientation in multidomain proteins. This is particularly important in the context of a calcium binding protein from Entamoeba histolytica (EhCaBP), which consists of two globular domains connected by a flexible linker region containing 8 residues. As a first step to obtain the interdomain orientation in EhCaBP, a suite of 2D and 3D heteronuclear experiments were recorded on EhCaBP by displacing calcium with Ce(3+), Ho(3+), Er(3+), Tm(3+), Dy(3+), and Yb(3+) ions in separate experiments, and the PCS of (1)H(N) and (15)N spins were measured. Such data have been used in the refinement of the individual domain structures of the protein in parallel with the calculation of the respective magnetic anisotropy tensorial values, which differ substantially (2.1-2.8 times) from what is found in other Ca(2+) binding loops. This study provides a structural basis for such variations in the magnetic anisotropy tensorial values.

NMR characterization of a 72 kDa transcription factor using differential isotopic labeling.

NF-κB is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF-κB have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF-κB monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple-resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA-binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full-length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue-specific labeling and comparison with previously-published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF-κB has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF-κB in the cell.

Structural basis for sequential displacement of Ca(2+) by Yb(3+) in a protozoan EF-hand calcium binding protein.

We have studied the displacement of Ca(2+)by the trivalent lanthanide ions (Yb(3+)) in a protozoan (Entamoeba histolytica) Ca(2+)-binding protein (EhCaBP), by NMR and thermodynamics. We have demonstrated, for the first time, how one can use in a combined fashion the utility of NMR and thermodynamics to have an insight to the relative binding specificities/affinity between Ca(2+) and Yb(3+). As revealed by the titration experiments, Yb(3+) displaces Ca(2+) from the four metal binding sites present in EhCaBP in a sequential manner. The study provides a structural origin for such a sequential Ca(2+) displacement by Yb(3+) in EhCaBP.

Magnesium promotes structural integrity and conformational switching action of a calcium sensor protein.

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.

Energetics of the native energy landscape of a two-domain calcium sensor protein: distinct folding features of the two domains.

The protein folding energy landscape allows a thorough understanding of the protein folding problem which in turn helps in understanding various aspects of biological functions. Characterizing the cooperative unfolding units and the intermediates along the folding funnel of a protein is a challenging task. In this paper, we investigated the native energy landscape of EhCaBP, a calcium sensor, belonging to the same EF-hand superfamily as calmodulin. EhCaBP is a two-domain EF-hand protein consisting of two EF-hands in each domain and binding to four Ca2+ cations. Native-state hydrogen exchange (HX) was used to assess the folding features of the landscape and also to throw light on the structure-folding function paradigm of calcium sensor proteins. HX measurements under the EX2 regime provided the thermodynamic information about the protein folding events under native conditions. HX studies revealed that the unfolding of EhCaBP is not a two-state process. Instead, it proceeds through cooperative units. The C-terminal domain exhibits less denaturant dependence than the N-terminal domain, suggesting that the former is dominated by local fluctuations. It is interesting to note that the N- and C-terminal domains of EhCaBP have distinct folding features. In fact, these observed differences can regulate the domain-dependent target recognition of two-domain Ca2+ sensor proteins.

Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state.

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.

Measurement of 1J(Ni,Calpha(i)), 1J(Ni,C'i-1), 2J(Ni,Calpha(i-1)), 2J(H(N)i,C'i-1) and 2J(H(N)i,Calpha(i)) values in 13C/15N-labeled proteins.

Use of partial or selective (13)C/(15)N labeling of specific amino acid residues in a given protein to measure the values of (1)J((15)N(i),(13)C(alpha) (i)), (2)J((1)H(N),(13)C(alpha) (i)), (2)J((15)N(i),(13)C(alpha) (i-1)), (1)J((15)N(i),(13)C'(i-1)) and (2)J((1)H(N),(13)C'(i-1)) is described. This was achieved by recording a sensitivity-enhanced 2D [(15)N-(1)H] HSQC experiment, without mixing the spin states of C(alpha) and C' during the course of entire experiment.

Energetics and mechanism of Ca2+ displacement by lanthanides in a calcium binding protein.

The displacement of Ca(2+) by trivalent lanthanide ions (Tm(3+)) in a protozoan (Entamoeba histolytica) Ca(2+) binding protein has been studied by NMR and isothermal calorimetry (ITC). The study provides a basis for understanding the behavior of lanthanides when used as a substitute for Ca(2+), the pattern of sequential binding, the structural changes involved, the range and magnitude of paramagnetic interaction, and the associated energetics and mechanism. The progressive Ca(2+) displacement from site III first, followed by displacement from site II, I, and IV, as observed during the NMR titration experiments, is interpreted in the light of ITC data to provide a deeper insight into the intradomain and, for the first time, interdomain cooperativity and information about the statistical phenomenon involved in it. A theoretical model governing Ca(2+) displacement is provided. The small structural changes involved in Ca(2+) displacement by a diamagnetic lanthanide (La(3+)) has also been monitored.