Neurotransmitter release is triggered by a calcium-induced rearrangement in the Synaptotagmin-1/SNARE complex primary interface.

The Ca2+ sensor synaptotagmin-1 triggers neurotransmitter release together with the neuronal SNARE complex formed by syntaxin-1, SNAP25 and synaptobrevin. Moreover, synaptotagmin-1 increases synaptic vesicle priming and impairs spontaneous vesicle release. The synaptotagmin-1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of synaptotagmin-1, and the mechanism underlying Ca2+-triggering of release is unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of synaptotagmin-1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of synaptotagmin-1 in vesicle priming and clamping of spontaneous release, and, importantly, show that Ca2+-triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with modeling and biophysical studies presented in the accompanying paper, our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast synaptic vesicle fusion.

Lincosamide Antibiotics: Structure, Activity, and Biosynthesis.

Lincosamides are naturally occurring antibiotics isolated from Streptomyces sp. Currently, lincomycin A and its semisynthetic analogue clindamycin are used as clinical drugs. Due to their unique structures and remarkable biological activities, derivatizations of lincosamides via semi-synthesis and biosynthetic studies have been reported. This review summarizes the structures and biological activities of lincosamides, and the recent studies of lincosamide biosynthetic enzymes.

Deep mutational scanning of proteins in mammalian cells.

Protein mutagenesis is essential for unveiling the molecular mechanisms underlying protein function in health, disease, and evolution. In the past decade, deep mutational scanning methods have evolved to support the functional analysis of nearly all possible single-amino acid changes in a protein of interest. While historically these methods were developed in lower organisms such as E. coli and yeast, recent technological advancements have resulted in the increased use of mammalian cells, particularly for studying proteins involved in human disease. These advancements will aid significantly in the classification and interpretation of variants of unknown significance, which are being discovered at large scale due to the current surge in the use of whole-genome sequencing in clinical contexts. Here, we explore the experimental aspects of deep mutational scanning studies in mammalian cells and report the different methods used in each step of the workflow, ultimately providing a useful guide toward the design of such studies.

Antibody sequence-based prediction of pH gradient elution in multimodal chromatography.

Multimodal chromatography has emerged as a promising technique for antibody purification, owing to its capacity to selectively capture and separate target molecules. However, the optimization of chromatography parameters remains a challenge due to the intricate nature of protein-ligand interactions. To tackle this issue, efficient predictive tools are essential for the development and optimization of multimodal chromatography processes. In this study, we introduce a methodology that predicts the elution behavior of antibodies in multimodal chromatography based on their amino acid sequences. We analyzed a total of 64 full-length antibodies, including IgG1, IgG4, and IgG-like multispecific formats, which were eluted using linear pH gradients from pH 9.0 to 4.0 on the anionic mixed-mode resin Capto adhere. Homology models were constructed, and 1312 antibody-specific physicochemical descriptors were calculated for each molecule. Our analysis identified six key structural features of the multimodal antibody interaction, which were correlated with the elution behavior, emphasizing the antibody variable region. The results show that our methodology can predict pH gradient elution for a diverse range of antibodies and antibody formats, with a test set R² of 0.898. The developed model can inform process development by predicting initial conditions for multimodal elution, thereby reducing trial and error during process optimization. Furthermore, the model holds the potential to enable an in silico manufacturability assessment by screening target antibodies that adhere to standardized purification conditions. In conclusion, this study highlights the feasibility of using structure-based prediction to enhance antibody purification in the biopharmaceutical industry. This approach can lead to more efficient and cost-effective process development while increasing process understanding.

Structural and functional characterisation of HepTH1-5 peptide as a potential hepcidin replacement.

Hepcidin is a principal regulator of iron homeostasis and its dysregulation has been recognised as a causative factor in cancers and iron disorders. The strategy of manipulating the presence of hepcidin peptide has been used for cancer treatment. However, this has demonstrated poor efficiency and has been short-lived in patients. Many studies reported using minihepcidin therapy as an alternative way to treat hepcidin dysregulation, but this was only applied to non-cancer patients. Highly conserved fish hepcidin protein, HepTH1-5, was investigated to determine its potential use in developing a hepcidin replacement for human hepcidin (Hepc25) and as a therapeutic agent by targeting the tumour suppressor protein, p53, through structure-function analysis. The authors found that HepTH1-5 is stably bound to ferroportin, compared to Hepc25, by triggering the ferroportin internalisation via Lys42 and Lys270 ubiquitination, in a similar manner to the Hepc25 activity. Moreover, the residues Ile24 and Gly24, along with copper and zinc ligands, interacted with similar residues, Lys24 and Asp1 of Hepc25, respectively, showing that those molecules are crucial to the hepcidin replacement strategy. HepTH1-5 interacts with p53 and activates its function through phosphorylation. This finding shows that HepTH1-5 might be involved in the apoptosis signalling pathway upon a DNA damage response. This study will be very helpful for understanding the mechanism of the hepcidin replacement and providing insights into the HepTH1-5 peptide as a new target for hepcidin and cancer therapeutics.Communicated by Ramaswamy H. Sarma.

Boosting the Full Potential of PyMOL with Structural Biology Plugins.

Over the past few decades, the number of available structural bioinformatics pipelines, libraries, plugins, web resources and software has increased exponentially and become accessible to the broad realm of life scientists. This expansion has shaped the field as a tangled network of methods, algorithms and user interfaces. In recent years PyMOL, widely used software for biomolecules visualization and analysis, has started to play a key role in providing an open platform for the successful implementation of expert knowledge into an easy-to-use molecular graphics tool. This review outlines the plugins and features that make PyMOL an eligible environment for supporting structural bioinformatics analyses.

Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport.

Structure-function analysis is a powerful strategy to characterize the contribution of specific residues to the biogenesis and function of a protein. This approach requires the characterization of strains that express mutant alleles in the absence of the wild-type protein. When studying nonessential bacterial genes, collections of mutants can be easily constructed by introducing plasmid-encoded alleles of interest into a strain that already lacks the wild-type gene. However, this high-throughput approach is not applicable to studying essential genes since their respective null strains are not viable. While there are several tools currently available to modify essential genes, they can be greatly limited by the amount of effort it takes to build and analyze each mutant strain. Here, we describe a high-throughput system for the rapid structure-function analysis of essential genes involved in lipopolysaccharide transport in Escherichia coli. This method, which can be applied to study any essential gene, relies on the initial construction of a single bacterial strain that can be used to generate and functionally characterize multiple plasmid-encoded alleles in under 24 h. We will discuss the advantages and possible shortcomings of our protocol in comparison to other commonly used methods.

Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry.

Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance.

Synthesis of Polyoxygenated Tropolones and their Antiviral Activity against Hepatitis B Virus and Herpes Simplex Virus-1.

Polyoxygenated tropolones possess a broad range of biological activity, and as a result are promising lead structures or fragments for drug development. However, structure-function studies and subsequent optimization have been challenging, in part due to the limited number of readily available tropolones and the obstacles to their synthesis. Oxidopyrylium [5+2] cycloaddition can effectively generate a diverse array of seven-membered ring carbocycles, and as a result can provide a highly general strategy for tropolone synthesis. Here, we describe the use of 3-hydroxy-4-pyrone-based oxidopyrylium cycloaddition chemistry in the synthesis of functionalized 3,7-dimethoxytropolones, 3,7-dihydroxytropolones, and isomeric 3-hydroxy-7-methoxytropolones through complementary benzyl alcohol-incorporating procedures. The antiviral activity of these molecules against herpes simplex virus-1 and hepatitis B virus is also described, highlighting the value of this approach and providing new structure-function insights relevant to their antiviral activity.

Na+ Sensitivity of the KAT2-Like Channel Is a Common Feature of Cucurbits and Depends on the S5-P-S6 Segment.

Inhibition of Shaker K+ channel activity by external Na+ was previously reported in the melon (Cucumis melo L.) inwardly rectifying K+ channel MIRK and was hypothesized to contribute to salt tolerance. In this study, two inward Shaker K+ channels, CsKAT2 from cucumber (Cucumis sativus) and ClKAT2 from watermelon (Citrullus lanatus), were identified and characterized in Xenopus oocytes. Both channels were inwardly rectifying K+ channels with higher permeability to potassium than other monovalent cations and more active when external pH was acidic. Similarly to MIRK, their activity displayed an inhibition by external Na+, thus suggesting a common feature in Cucurbitaceae (Cucumis spp., Citrullus spp.). CsKAT2 and ClKAT2 are highly expressed in guard cells. After 24 h of plant treatment with 100 mM NaCl, the three KAT2-like genes were significantly downregulated in leaves and guard cells. Reciprocal chimeras were obtained between MIRK and Na+-insensitive AtKAT2 cDNAs. The chimera where the MIRK S5-P-S6 segment was replaced by that from AtKAT2 no longer showed Na+ sensitivity, while the inverse chimera gained Na+ sensitivity. These results provide evidence that the molecular basis of the channel blockage by Na+ is located in the S5-P-S6 region. Comparison of the electrostatic property in the S5-P-S6 region in AtKAT2 and MIRK revealed four key amino acid residues potentially governing Na+ sensitivity.

Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica.

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.

Structure-Function Analysis of Two Interacting Vaccinia Proteins That Are Critical for Viral Morphogenesis: L2 and A30.5.

An enduring mystery in poxvirology is the mechanism by which virion morphogenesis is accomplished. A30.5 and L2 are two small regulatory proteins that are essential for this process. Previous studies have shown that vaccinia A30.5 and L2 localize to the ER and interact during infection, but how they facilitate morphogenesis is unknown. To interrogate the relationship between A30.5 and L2, we generated inducible complementing cell lines (CV1-HA-L2; CV1-3xFLAG-A30.5) and deletion viruses (vΔL2; vΔA30.5). Loss of either protein resulted in a block in morphogenesis and a significant (>100-fold) decrease in infectious viral yield. Structure-function analysis of L2 and A30.5, using transient complementation assays, identified key functional regions in both proteins. A clustered charge-to-alanine L2 mutant (L2-RRD) failed to rescue a vΔL2 infection and exhibits a significantly retarded apparent molecular weight in vivo (but not in vitro), suggestive of an aberrant posttranslational modification. Furthermore, an A30.5 mutant with a disrupted putative N-terminal α-helix failed to rescue a vΔA30.5 infection. Using our complementing cell lines, we determined that the stability of A30.5 is dependent on L2 and that wild-type L2 and A30.5 coimmunoprecipitate in the absence of other viral proteins. Further examination of this interaction, using wild-type and mutant forms of L2 or A30.5, revealed that the inability of mutant alleles to rescue the respective deletion viruses is tightly correlated with a failure of L2 to stabilize and interact with A30.5. L2 appears to function as a chaperone-like protein for A30.5, ensuring that they work together as a complex during viral membrane biogenesis. IMPORTANCE Vaccinia virus is a large, enveloped DNA virus that was successfully used as the vaccine against smallpox. Vaccinia continues to be an invaluable biomedical research tool in basic research and in gene therapy vector and vaccine development. Although this virus has been studied extensively, the complex process of virion assembly, termed morphogenesis, still puzzles the field. Our work aims to better understand how two small viral proteins that are essential for viral assembly, L2 and A30.5, function during early morphogenesis. We show that A30.5 requires L2 for stability and that these proteins interact in the absence of other viral proteins. We identify regions in each protein required for their function and show that mutations in these regions disrupt the interaction between L2 and A30.5 and fail to restore virus viability.

Inhibitory Effects of Periplocin on Lymphoma Cells: A Network Pharmacology Approach and Experimental Validation.

PURPOSE: Lymphoma is considered to be one of the most pressing health problems worldwide owing to its high incidence and mortality. Previous studies have shown that periplocin, a naturally occurring compound, inhibits growth and induces apoptosis in several cancers. However, the effects of periplocin on lymphoma and the underlying mechanisms of action remain unclear. METHODS: The PharmMapper database was used to predict the potential targets of periplocin. The GeneCard database was used to identify lymphoma-related genes. A few intersecting genes were obtained, and the protein-protein interaction network was visualized using STRING Gene ontology analysis. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using R project. MTS assay, flow cytometry, real-time quantitative polymerase chain reaction (qPCR), and Western blotting were used to verify whether periplocin possesses anti-lymphoma activity. RESULTS: A total of 216 intersecting genes were identified. Numerous cancer-related signaling pathways were visualized using Cytoscape software, with the PI3K-Akt signaling pathway being the highest-ranked pathway related to cell proliferation, apoptosis, and cell cycle progression. HuT 78 and Jurkat cell lines were used to verify the predictions. Periplocin significantly inhibited their proliferation in a dose- and time-dependent manner, but had no effect on the viability of peripheral blood lymphocytes. Flow cytometry revealed that treatment with periplocin increased the apoptotic rate and ratio of HuT 78 and Jurkat cells in the G2/M phase. CDK1 and cyclin B1 complex formation is a key gatekeeper to mitotic division in the G2/M phase. Western blot analysis revealed that periplocin significantly decreased the protein levels of CDK1 and cyclin B1; however, real-time qPCR revealed no effect on gene expression. CONCLUSION: Periplocin showed anti-tumor effects in lymphoma cells through multiple targets and signaling pathways, and could be a novel therapeutic agent for the treatment of lymphoma.

Plant SUMO E3 Ligases: Function, Structural Organization, and Connection With DNA.

Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.

Reprogramming Substrate and Catalytic Promiscuity of Tryptophan Prenyltransferases.

Prenylation is a process widely prevalent in primary and secondary metabolism, contributing to functionality and chemical diversity in natural systems. Due to their high regio- and chemoselectivities, prenyltransferases are also valuable tools for creation of new compounds by chemoenzymatic synthesis and synthetic biology. Over the last ten years, biochemical and structural investigations shed light on the mechanism and key residues that control the catalytic process, but to date crucial information on how certain prenyltransferases control regioselectivity and chemoselectivity is still lacking. Here, we advance a general understanding of the enzyme family by contributing the first structure of a tryptophan C5-prenyltransferase 5-DMATS. Additinally, the structure of a bacterial tryptophan C6-prenyltransferase 6-DMATS was solved. Analysis and comparison of both substrate-bound complexes led to the identification of key residues for catalysis. Next, site-directed mutagenesis was successfully implemented to not only modify the prenyl donor specificity but also to redirect the prenylation, thereby switching the regioselectivity of 6-DMATS to that of 5-DMATS. The general strategy of structure-guided protein engineering should be applicable to other related prenyltransferases, thus enabling the production of novel prenylated compounds.

Identifying Cancer-Relevant Mutations in the DLC START Domain Using Evolutionary and Structure-Function Analyses.

Rho GTPase signaling promotes proliferation, invasion, and metastasis in a broad spectrum of cancers. Rho GTPase activity is regulated by the deleted in liver cancer (DLC) family of bona fide tumor suppressors which directly inactivate Rho GTPases by stimulating GTP hydrolysis. In addition to a RhoGAP domain, DLC proteins contain a StAR-related lipid transfer (START) domain. START domains in other organisms bind hydrophobic small molecules and can regulate interacting partners or co-occurring domains through a variety of mechanisms. In the case of DLC proteins, their START domain appears to contribute to tumor suppressive activity. However, the nature of this START-directed mechanism, as well as the identities of relevant functional residues, remain virtually unknown. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset and evolutionary and structure-function analyses, we identify several conserved residues likely to be required for START-directed regulation of DLC-1 and DLC-2 tumor-suppressive capabilities. This pan-cancer analysis shows that conserved residues of both START domains are highly overrepresented in cancer cells from a wide range tissues. Interestingly, in DLC-1 and DLC-2, three of these residues form multiple interactions at the tertiary structural level. Furthermore, mutation of any of these residues is predicted to disrupt interactions and thus destabilize the START domain. As such, these mutations would not have emerged from traditional hotspot scans of COSMIC. We propose that evolutionary and structure-function analyses are an underutilized strategy which could be used to unmask cancer-relevant mutations within COSMIC. Our data also suggest DLC-1 and DLC-2 as high-priority candidates for development of novel therapeutics that target their START domain.

New variant (Val597Ile) in transmembrane region of the TSH receptor with human chorionic gonadotropin hypersensitivity in familial gestational hyperthyroidism.

OBJECTIVES: Only two mutations at the lysine 183 amino acid in the extracellular N-terminal domain of human TSH receptor (hTSHR) have been associated with hypersensitivity to hCG and familial gestational hyperthyroidism. DESIGN: Describe a new variant of the TSHR gene with hCG hypersensitivity found in two women of the same family diagnosed with gestational hyperthyroidism. PATIENTS: A 38-year-old woman was seen during the first trimester of her second pregnancy for thyrotoxicosis with increased fT3 and fT4 concentrations and low TSH levels without anti-TSH receptor antibody. Thyrotoxicosis improved spontaneously during the 2nd trimester and persisted at the 3rd trimester. Similar clinical symptoms (weight loss, nausea, vomiting) were also reported during the first trimester of her first pregnancy and the first pregnancy of her mother. RESULTS: DNA sequencing of the hTSHR gene of this woman and her mother identifies a heterozygous variant changing valine to isoleucine residue at codon 597 in the transmembrane domain (TMD) of this receptor. In vitro functional studies of this variant showed increased constitutive activity in regard to the basal level of cAMP and IP3 production and to the low cell-surface expression, while response to TSH was reduced compared to that of the wild-type receptor. The Val597Ile variant presented a dose-dependent increase in cAMP response to hGC and human luteinizing hormone (hLH). Simulation of the protein dynamics showed a high structural impact of the Val597Ile variant on helices 3 (TMH3) and 5 (TMH5) of the transmembrane domain participating to constitutive activity and hCG sensitivity. CONCLUSION: We describe a new variant in the transmembrane region of the hTSHR gene with increased constitutive activity and hCG hypersensitivity in familial gestational hyperthyroidism.

The neuronal transcription factor Myt1L interacts via a conserved motif with the PAH1 domain of Sin3 to recruit the Sin3L/Rpd3L histone deacetylase complex.

The Sin3L/Rpd3L histone deacetylase (HDAC) complex is one of six major HDAC complexes in the nucleus, and its recruitment by promoter-bound transcription factors is an important step in many gene transcription regulatory pathways. Here, we investigate how the Myt1L zinc finger transcription factor, important for neuronal differentiation and the maintenance of neuronal identity, recruits this complex at the molecular level. We show that Myt1L, through a highly conserved segment shared with its paralogs, interacts directly and specifically with the Sin3 PAH1 domain, binding principally to the canonical hydrophobic cleft found in paired amphipathic helix domain (PAH) domains. Our findings are relevant not only for other members of the Myt family but also for enhancing our understanding of the rules of protein-protein interactions involving Sin3 PAH domains.

Unveiling the Synaptic Function and Structure Using Paired Recordings From Synaptically Coupled Neurons.

Synaptic transmission between neurons is the basic mechanism for information processing in cortical microcircuits. To date, paired recording from synaptically coupled neurons is the most widely used method which allows a detailed functional characterization of unitary synaptic transmission at the cellular and synaptic level in combination with a structural characterization of both pre- and postsynaptic neurons at the light and electron microscopic level. In this review, we will summarize the many applications of paired recordings to investigate synaptic function and structure. Paired recordings have been used to study the detailed electrophysiological and anatomical properties of synaptically coupled cell pairs within a synaptic microcircuit; this is critical in order to understand the connectivity rules and dynamic properties of synaptic transmission. Paired recordings can also be adopted for quantal analysis of an identified synaptic connection and to study the regulation of synaptic transmission by neuromodulators such as acetylcholine, the monoamines, neuropeptides, and adenosine etc. Taken together, paired recordings from synaptically coupled neurons will remain a very useful approach for a detailed characterization of synaptic transmission not only in the rodent brain but also that of other species including humans.

Dissecting the Regulatory Strategies of NF-κB RelA Target Genes in the Inflammatory Response Reveals Differential Transactivation Logics.

Nuclear factor κB (NF-κB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-κB RelA core response.