Side Chain Resonance Assignments Research Articles

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 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.

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.

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.

Backbone and side chain NMR assignments and secondary structure calculation of the pheromone binding protein3 of Ostrinia nubilalis, an agricultural pest.

Ostrinia nubilalis, also known as European Corn Borer (ECB), is a serious pest in Europe and North America, as well as in Central Asia and Northern Africa. It damages a variety of agricultural crops such as corn, oats, buckwheat, millet, and soybeans. causing annually at least one billion dollars in loss. The Ostrinia nubilalis pheromone-binding protein3 (OnubPBP3), preferentially expressed in the male moth antenna, has been implicated in the detection of the female-secreted pheromone blend during the mating process. Understanding the structure of and function of OnubPBP3, including the mechanism of pheromone binding and its release at the dendritic olfactory neuron (ORN), is essential if integrated pest management through sensory inhibition is to be achieved. We report here the backbone and side-chain resonance assignments of OnubPBP3 at pH 6.5 using various triple resonance NMR experiments on a 13C, 15N-labeled protein sample. The secondary structure of OnubPBP3 consists of six α-helices and an unstructured C-terminus based on backbone chemical shifts.

1H, 13C and 15N backbone and side-chain resonance assignments of ∆∆BmSA1, the surface antigen of Babesia microti.

Human babesiosis is a vector-borne zoonotic infection caused mostly by the Apicomplexan parasite Babesia microti, distributed worldwide. The infection can result in severe symptoms such as hemolytic anemia, especially in immunodeficient patients. Also, asymptomatic patients continue transmission as unscreened blood donors, and represent a risk for Public Health. Early host-parasite interactions are mediated by BmSA1, the major surface antigen of Babesia microti, crucial for invasion and immune escape. Hence, a structural and functional characterization of the BmSA1 protein constitutes a first strategic milestone toward the development of innovative tools to control infection. Knowledge of the 3D structure of such an important antigen is crucial for the development of vaccines or new diagnostic tests. Here, we report the 1H, 15N and 13C NMR resonance assignment of ∆∆BmSA1, a truncated recombinant version of BmSA1 without the N-terminal signal peptide and the hydrophobic C-terminal GPI-anchor. Secondary structure prediction using CSI.3 and TALOS-N demonstrates a high content of alpha-helical structure. This preliminary study provides foundations for further structural characterization of BMSA1.

Resonance assignments of the PUB domain of the RNF31 protein.

E3 ubiquitin protein ligase RNF31 is present in human proteins and is involved in linear ubiquitin chain assembly complex (LUBAC) activity and cell growth. RNF31 is involved in ubiquitination, which is the post-translational modification of proteins. Ubiquitin molecules connect with amino acid residues of target proteins under the action of ubiquitin-activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, so as to achieve certain physiological functions. The abnormal expression of ubiquitination promotes the formation of cancer. In studies of breast cancer, RNF31 mRNA levels were found to be higher in cancer cells than in other tissues. The PUB domain of RNF31 is the binding site of the ubiquitin thioesterase otulin. Here, we report the backbone and side-chain resonance assignments of the PUB domain of RNF31 and study the backbone relaxation of the domain. These studies will contribute to further understanding of the structural and functional relationship of RNF31 protein, which may also be a target for drug research.

1H, 15N, and 13C 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.

Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

1H, 15N and 13C resonance assignments of a minimal CPSF73-CPSF100 C-terminal heterodimer.

The initial pre-mRNA transcript in eukaryotes is processed by a large multi-protein complex in order to correctly cleave the 3' end, and to subsequently add the polyadenosine tail. This cleavage and polyadenylation specificity factor (CPSF) is composed of separate subunits, with structural information available for both isolated subunits and also larger assembled complexes. Nevertheless, certain key components of CPSF still lack high-resolution atomic data. One such region is the heterodimer formed between the first and second C-terminal domains of the endonuclease CPSF73, with those from the catalytically inactive CPSF100. Here we report the backbone and sidechain resonance assignments of a minimal C-terminal heterodimer of CPSF73-CPSF100 derived from the parasite Encephalitozoon cuniculi. The assignment process used several amino-acid specific labeling strategies, and the chemical shift values allow for secondary structure prediction.

1H, 15N, 13C backbone and sidechain resonance assignments and secondary structure of mouse NOTCH1 EGF27.

NOTCH1 is a transmembrane receptor in metazoans that is linked to a variety of disorders. The receptor contains an extracellular domain (ECD) with 36 tandem epidermal growth factor-like (EGF) repeats. The ECD is responsible for intercellular signaling via protein-ligand interactions with neighboring cells. Each EGF repeat consists of approximately 40 amino acids and 3 conserved disulfide bonds. The Abruptex region (EGF24-29) is critical for NOTCH1 signaling and is known for its missense mutations. Certain EGF repeats are modified with the addition of O-linked glycans and many have calcium binding sites, which give each EGF repeat a unique function. It has been shown that the loss of the O-fucose site of EGF27 alters NOTCH1 activity. To investigate the role of glycosylation in the NOTCH1 signaling pathway, nuclear magnetic resonance spectroscopy has been employed to study the structures of EGF27 and its glycoforms. Here, we report the backbone and sidechain 1H, 15N, and 13C-resonance assignments of the unmodified EGF27 protein and the predicted secondary structure derived from the assigned chemical shifts.

Backbone and side-chain resonance assignments of the NISTmAb-scFv and antigen-binding study

Monoclonal antibodies (mAbs) therapeutics are the largest and fastest growing class of biologic drugs, amongst which, the vast majority are immunoglobulin G1 (IgG1). Their antigen binding abilities are used for the treatment of immunologic diseases, cancer therapy, reversal of drug effects, and targeting viruses and bacteria. The high importance of therapeutic mAbs and their derivatives has called for the generation of well-characterized standards for method development and calibration. One such standard, the NISTmAb RM 8621 based on the antibody motavizumab, has been developed by the National Institute of Standards and Technologies (NIST) in the US. Here, we present the resonance assignment of the single chain variable fragment, NISTmAb-scFv, that was engineered by linking the variable domains of the heavy and light chains of the NISTmAb. Also, addition of a peptide, corresponding to the target antigen of motavizumab, to samples of NISTmAb-scFv has induced chemical shift perturbations on residues lining the antigen binding interface thereby indicating proper folding of the NISTmAb-scFv.

Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the β1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain’s secondary structure elements.

Resonance assignments of the ORC2-WH domain of the human ORC protein

ORC2 is a small subunit of the origin recognition complex (ORC), which is important for gene replication. The ORC2 WH domain recognizes dsDNA sequences with its flexible β-sheet hairpins as anchors. Here, we report near-complete NMR backbone and side chain resonance assignments of the WH domain and study the backbone relaxation of the WH domain. These studies will contribute to further understanding of the structure-function relationship of the ORC protein.

1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the 1H, 13C and 15N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (SΔ60), which lacks the 1-60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that SΔ60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds.

ILV methyl NMR resonance assignments of the 81kDa E. coli β-clamp.

The ring-shaped E. coli β-clamp protein is an 81kDa head-to-tail homodimer, which serves as a processivity factor anchoring the replicative polymerase to DNA, thereby increasing replication processivity and speed. In addition, it facilitates numerous protein transactions that take place on DNA during replication, repair, and damage response. We used a structure-based approach to obtain nearly complete Ile, Leu and Val side-chain methyl NMR resonance assignments of the wild-type β-clamp and its stabilized T45R/S107R variant based on site directed mutagenesis and the analysis of methyl-methyl NOESY data. The obtained assignments will facilitate future studies of the β-clamp interactions and dynamics.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

Backbone and side chain resonance assignment of the intrinsically disordered human DBNDD1 protein

The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein.

NMR resonance assignments of mouse lipocalin-type prostaglandin D synthase/prostaglandin J2 complex

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to produce PGD2, an endogenous somenogen, in the brains of various mammalians. We recently reported that various other PGs also bind to L-PGDS, suggesting that it could serve as an extracellular carrier for PGs. Although the solution and crystal structure of L-PGDS has been determined, as has the structure of L-PGDS complexed PGH2 analog, a structural analysis of L-PGDS complexed with other PGs is needed in order to understand the mechanism responsible for the PG trapping. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of the L-PGDS/PGJ2 complex and the binding site for PGJ2 on L-PGDS.

Backbone and sidechain 1H, 15N and 13C resonance assignments of the free and RNA-bound tandem zinc finger domain of the tristetraprolin family member from Selaginella moellendorffii.

Members of the tristetraprolin (TTP) family of RNA binding proteins (RBPs) regulate the metabolism of a variety of mRNA targets. In mammals, these proteins modulate many physiological processes, including immune cell activation, hematopoiesis, and embryonic development. Regulation of mRNA stability by these proteins requires that the tandem zinc finger (TZF) domain binds initially and directly to target mRNAs, ultimately leading to their deadenylation and decay. Proteins of this type throughout eukarya possess a highly conserved TZF domain, suggesting that they are all capable of high-affinity RNA binding. However, the mechanism of TTP-mediated mRNA decay is largely undefined. Given the vital role that these TTP family proteins play in maintaining RNA homeostasis throughout eukaryotes, we focused here on the first, key step in this process: recognition and binding of the TZF domain to target RNA. For these studies, we chose a primitive plant, the spikemoss Selaginella moellendorffii, which last shared a common ancestor with humans more than a billion years ago. Here we report the near complete backbone and side chain resonance assignments of the spikemoss TZF domain, including: (1) the assignment of the RNA-TZF domain complex, representing one of only two data sets currently available for the entire TTP family of proteins; and (2) the first NMR resonance assignments of the entire TZF domain, in the RNA-free form. This work will serve as the basis for further NMR structural investigations aimed at gaining insights into the process of RNA recognition and the mechanisms of TTP-mediated mRNA decay.