Backbone Resonance Assignments Research Articles

1H, 15N and 13C backbone resonance assignment of the N-terminal region of Zika virus NS4B protein in detergent micelles.

Zika virus has raised global concerns due to its link to microcephaly and Guillain-Barré syndrome in adults. One of viral nonstructural proteins-NS4B, an integral membrane protein, plays crucial roles in viral replication by interacting with both viral and host proteins, rendering it an attractive drug target for antiviral development. We purified the N-terminal region of ZIKV NS4B (NS4B NTD) and reconstituted it into detergent micelles. Here, we report the assignments of the backbone resonances of NS4B NTD in detergent micelles. The available assignment is useful for understanding its structure and ligand binding to provide useful information for developing NS4B inhibitors.

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

Chemical shift assignments of PA2072 CHASE4 domain.

Diverse extracellular sensor domains enable cells to regulate their behavior, physiological processes, and interspecies interactions in response to environmental stimuli. These sensing mechanisms facilitate the ultimate adaptation of organisms to their surrounding conditions. Pseudomonas aeruginosa (PAO1) is a clinically significant opportunistic pathogen in hospital infection. The CHASE4 domain, a putative extracellular sensing module, is found in the N-terminus of GGDEF-EAL-containing PA2072, a transmembrane receptor from P. aeruginosa. However, the signal identification and sensing mechanism of monomeric PA2072 CHASE4 remains largely unknown. Here, we report backbone and side chain resonance assignments of PA2072 CHASE4 as a basis for studying the structural mechanism of CHASE4-mediated signal recognition.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

The backbone NMR resonance assignments of the stabilized E. coli β clamp.

The 81 kDa E. coli β clamp is a ring-shaped head-to-tail homodimer that encircles DNA and plays a central role in bacterial DNA replication by serving as a processivity factor for DNA polymerases and a binding platform for other DNA replication and repair proteins. Here we report the backbone 1H, 15N, and 13C NMR resonance assignments of the stabilized T45R/S107R β clamp variant obtained using standard TROSY-based triple-resonance experiments (BMRB 52548). The backbone assignments were aided by 13C and 15N edited NOESY experiments, allowing us to utilize our previously reported assignments of the β clamp ILV side-chain methyl groups (BMRB 51430, 51431). The backbone assignments of the T45R/S107R β clamp variant were transferred to the wild-type β clamp using a minimal set of TROSY-based 15N edited NOESY, NHCO and NHCA experiments (BMRB 52549). The reported backbone and previous ILV side-chain resonance assignments will enable NMR studies of the β clamp interactions and dynamics using amide and methyl groups as probes.

NMR 1H, 13C, 15N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

1H, 13C and 15N backbone resonance assignment of the calcium-activated EndoU endoribonuclease.

The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with 13C and 15N labeling. A nearly complete assignment of backbone 1H, 15N, and 13C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases.

Solution NMR backbone resonance assignment of the full-length resistance-related calcium-binding protein Sorcin.

Sorcin is a penta-EF hand calcium-binding protein that confers multidrug resistance in cancer cells. It regulates cellular Ca2+ homeostasis by interacting with calcium channels such as Ryanodine receptor 2 and Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in a calcium-dependent manner. The crystal structure of the Sorcin has been determined in both calcium-free and calcium-bound states to understand calcium-binding induced conformational change. However, due to its flexibility, most of the N-terminal domain is invisible in these crystal structures. Here we report the 1H, 13C, and 15N backbone resonance assignments of full-length Sorcin in the calcium-free state using solution NMR. The protein secondary structure was predicted based on the assigned backbone chemical shifts using TALOS+ and CSI 3.0. Our backbone resonance assignment of the full-length Sorcin provides a foundation for future NMR spectroscopic studies to uncover the mechanism of Ca2+ sensing by Sorcin.

1H, 15N and 13C resonance assignments of eggcase silk protein 3.

Spider silk is a high-performance biomaterial known for its outstanding combination of strength and flexibility. Among the six distinct types of spider silk, eggcase silk stands out as it is exclusively produced from the tubuliform gland, playing a specialized role in offspring protection. In the spider species Latrodectus hesperus, eggcase silk is spun from a large spidroin complex, including the major silk component tubuliform spidroin 1 (TuSp1) and at least six different minor silk components. One of these minor components is eggcase protein 3 (ECP3), a small silk protein of 11.8kDa that lacks the typical spidroin architecture. ECP3 shows very limited homology to all known spidroins. In this study, we report nearly complete backbone and side-chain resonance assignments of ECP3 as a basis for studying the structural mechanisms involved in eggcase silk formation.

Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab

Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.

1H, 13C and 15N backbone resonance assignment of Cel45A from Phanerochaete chrysosporium

A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1H, 13C and 15N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.

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 24.2kDa 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 β-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.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

1H, 13C and 15N backbone and side-chain resonance assignments of the human oncogenic protein NCYM.

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1H, 15N and 13C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners.

Backbone 1H, 13C, and 15N chemical shift assignments for human SERF2.

Human small EDRK-rich factor protein SERF2 is a cellular driver of protein amyloid formation, a process that has been linked to neurodegenerative diseases including Alzheimer's and Parkinson's disease. SERF2 is a 59 amino acid protein, highly charged, and well conserved whose structure and physiological function is unclear. SERF family proteins including human SERF2 have shown a tendency to form fuzzy complexes with misfolded proteins such as α-Synuclein which has been linked to Parkinson's disease. SERF family proteins have been recently identified to bind nucleic acids, but the binding mechanism(s) remain enigmatic. Here, using multidimensional solution NMR, we report the 1H, 15N, and 13C chemical shift assignments (~ 86% of backbone resonance assignments) for human SERF2. TALOS-N predicted secondary structure of SERF2 showed three very short helices (3-4 residues long) in the N-terminal region of the protein and a long helix in the C-terminal region spanning residues 37-46 which is consistent with the helical content indicated by circular dichroism spectroscopy. Paramagnetic relaxation enhancement NMR analysis revealed that a short C-terminal region E53-K55 is in the proximity of the N-terminus. Having the backbone assignment of SERF2 allowed us to probe its interaction with α-Synuclein and to identify the residues in SERF2 binding interfaces that likely promote α-Synuclein aggregation.

1H, 13C and 15N backbone resonance assignments of hepatocyte nuclear factor-1-beta (HNF1β) POUS and POUHD.

Hepatocyte nuclear factor 1β (HNF1β) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1β plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1β POUS and POUHD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1β cause diseases.

Chemical shift assignments of the ACID domain of MED25, a subunit of the mediator complex in Arabidopsis thaliana.

Mediator complex is a key component that bridges various transcription activators and RNA polymerase during eukaryotic transcription initiation. The Arabidopsis thaliana Med25 (aMed25), a subunit of the Mediator complex, plays important roles in regulating hormone signaling, biotic and abiotic stress responses and plant development by interacting with a variety of transcription factors through its activator-interacting domain (ACID). However, the recognition mechanism of aMed25-ACID for various transcription factors remains unknown. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of aMED25-ACID (residues 551-681). TALOS-N analysis revealed that aMED25-ACID structure is comprised of three α-helices and seven β-strands, which lacks the C-terminal α-helix existing in the human MED25-ACID. This study lays a foundation for further research on the structure-function relationship of aMED25-ACID.

NMR 1H, 13C, 15N backbone resonance assignments of wild-type human K-Ras and its oncogenic mutants G12D and G12C bound to GTP.

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants.

Chemical shift assignments of wildtype human leptin.

Leptin is an adipose tissue-expressed 16-kDa hormone encoded by the ob/ob gene. It serves a crucial role in regulating diverse physiological processes, including body weight control, energy homeostasis regulation, promotion of cell proliferation, and more. Emerging research has also revealed potential implications of leptin in various aging-related diseases, suggesting multifaceted physiological roles of leptin. Structural investigation of wild-type leptin in apo form is of particular importance to understand its conformational plasticity for receptor interaction and recognition. Here, we report backbone and side-chain resonance assignments of wild-type human leptin as a basis for structural and functional studies on leptin-mediated signaling.