Backbone Resonance Assignments Research Articles

Backbone 1H, 15N and 13C resonance assignments for an E2 ubiquitin conjugating enzyme-UBE2T.

Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.

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.

Backbone 1H, 13C and 15N assignments of the apo-acyl carrier protein (ACP1) of Pseudomonas aeruginosa.

The N-acyl-L-homoserine lactone (AHL) quorum sensing regulates virulence in the opportunistic pathogen, Pseudomonas aeruginosa. The LasI and RhlI AHL synthases use acyl carrier protein substrates to synthesize, respectively, the 3-oxododecanoyl-L-homoserine lactone (3-oxoC12-HSL) and butyryl-L-homoserine lactone (C4-HSL) QS signals for this bacterium. Although P. aeruginosa genome contains three open reading frames to encode three acyl carrier proteins, namely the ACP1, ACP2 and ACP3, microarray and gene replacement studies show that only the ACP1 carrier protein is under quorum sensing regulation. In this study, we isotopically enriched one of the acyl carrier proteins, ACP1 from P. aeruginosa and describe the backbone resonance assignments for this protein to delineate the structural and molecular basis of ACP1 recognition in P. aeruginosa AHL quorum sensing signal synthesis.

Backbone 1H, 15N and 13C resonance assignments for dengue NS2B without the NS3 protease cofactor region in detergent micelles.

Dengue virus is an important human pathogen affecting people especially in tropical and subtropical regions. Its genome encodes seven non-structural proteins that are important for viral assembly and replication. Dengue NS2B is a membrane protein containing four transmembrane helices and involved in protein-protein interactions. Its transmembrane helices are critical for location of NS2B on the cell membrane while one cytoplasmic region composed of approximately 40 amino acids serves as a cofactor of viral NS3 protease by forming a tight complex with the N-terminal region of NS3. Here, we report the backbone resonance assignments for a dengue NS2B construct referred to as mini-NS2B containing only the transmembrane regions without NS3 cofactor region in detergent micelles. Mini-NS2B exhibits well-dispersed cross-peaks in the 1H-15N-HSQC spectrum and contains four helices in solution. The available mini-NS2B and its assignment will be useful for determining the structure of NS2B and identifying small molecules binding to the transmembrane regions.

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.

Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B.

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.

Complete non-proline backbone resonance assignments of the S. aureus neutrophil serine protease inhibitor, EapH1.

The S. aureus extracellular adherence protein (Eap) and its homologs, EapH1 and EapH2, serve roles in evasion of the human innate immune system. EapH1 binds with high-affinity and inhibits the neutrophil azurophilic granule proteases neutrophil elastase, cathepsin-G and proteinase-3. Previous structural studies using X-ray crystallography have shown that EapH1 binds to neutrophil elastase and cathepsin-G using a globally similar binding mode. However, whether the same holds true in solution is unknown and whether the inhibitor experiences dynamic changes following binding remains uncertain. To facilitate solution-phase structural and biochemical studies of EapH1 and its complexes with neutrophil granule proteases, we have characterized EapH1 by multidimensional NMR spectroscopy. Here we report a total of 100% of the non-proline backbone resonance assignments of EapH1 with BMRB accession number 50,304.

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.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

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, 13C, 15N backbone chemical shift assignments of the extended ARID domain in human AT-rich interactive domain protein 5a (Arid5a)

The family of AT-rich interactive domain (ARID) containing proteins -Arids- contains 15 members that have almost exclusively been described as DNA-binding proteins. Interestingly, a decade ago the family member Arid5a was found to bind and stabilize mRNAs of immune system key players and thereby account for driving inflammatory and autoimmune diseases. How exactly binding to DNA and RNA is coordinated by the Arid5a ARID domain remains unknown, mainly due to the lack of atom-resolved information on nucleic acid-binding. This in particular applies to the protein’s ARID domain, despite the comfortable size of its core unit for NMR-based investigations. Furthermore, the core domain of ARID domains is found to be extended by functionally relevant, often flexible stretches, but whether such elongations are present and crucial for the versatile Arid5a functions is unknown. We here provide a near-complete NMR backbone resonance assignment of the Arid5a ARID domain with N- and C-terminal extensions, which serves as a basis for further studies of its nucleic acid-binding preferences and targeted inhibition by means of NMR. Our data thus significantly contribute to unravelling mechanisms of Arid5a-mediated gene regulation and diseases.

1H, 15N, 13C backbone and Cβ resonance assignments for UBQLN1 UBA and UBAA domains

UBQLN1 functions in autophagy and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), and a flexible central region which functions as a chaperone to prevent protein aggregation. Here, we report the 1H, 15N, and 13C resonance assignments for the backbone (NH, N, C’, Cα, and Hα) and sidechain Cβ atoms of the UBQLN1 UBA and an N-terminally adjacent segment called the UBA-adjacent domain (UBAA). We find a subset of the resonances corresponding to the UBAA to have concentration-dependent chemical shifts, likely due to self-association. We also find the backbone amide nitrogen of T572 to be shifted upfield relative to the average value for a threonine amide nitrogen, a phenomenon likely caused by T572 Hγ1 engagement in a hydrogen bond with adjacent backbone carbonyl atoms. The assignments described in this manuscript can be used to study the protein dynamics of the UBQLN1 UBA and UBAA as well as the interaction of these domains with other proteins.

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.

Expression of 2H, 13C, 15N-labeled NIST-Fab fragment in the methylotrophic yeast Komagataella phaffii for nuclear magnetic resonance studies

Labeling of proteins with deuterium is an essential tool in overcoming size limitations in the application of nuclear magnetic resonance (NMR) spectroscopy to proteins larger than 30 kilodaltons (kDa). A non-originator antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, so called yNIST-Fab, is a ~ 50 kDa protein, with 5 native disulfide linkages, that can be expressed in properly folded form in methylotrophic Komagataella phaffii (formerly Pichia pastoris). Further, the K. phaffii host can support the production of perdeuterated yNIST-Fab which is necessary to obtain well-resolved TROSY-based tripleresonance NMR spectra for chemical shift assignment of the peptide backbone resonances. Here, we examined growth conditions and effects of media composition to maximize biomass generation and expression yield of the 2H, 13C, 15N-enriched NIST-Fab fragment. Triple-labeled yNIST-Fab with ~93% deuteration reduced the 1HN, 15N and 13C-linewidths in the NMR spectra, allowing sequential NMR assignment of backbone resonance a key step toward sequence-specific structural and dynamic studies of Fab fragments and intact antibodies.

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.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein.

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215-300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.

1H, 13C, 15N backbone resonance assignment of apo and ADP-ribose bound forms of the macro domain of Hepatitis E virus through solution NMR spectroscopy

The genome of Hepatitis E virus (HEV) is 7.2 kilobases long and has three open reading frames. The largest one is ORF1, encoding a non-structural protein involved in the replication process, and whose processing is ill-defined. The ORF1 protein is a multi-modular protein which includes a macro domain (MD). MDs are evolutionarily conserved structures throughout all kingdoms of life. MDs participate in the recognition and removal of ADP-ribosylation, and specifically viral MDs have been identified as erasers of ADP-ribose moieties interpreting them as important players at escaping the early stages of host-immune response. A detailed structural analysis of the apo and bound to ADP-ribose state of the native HEV MD would provide the structural information to understand how HEV MD is implicated in virus-host interplay and how it interacts with its intracellular partner during viral replication. In the present study we present the high yield expression of the native macro domain of HEV and its analysis by solution NMR spectroscopy. The HEV MD is folded in solution and we present a nearly complete backbone and sidechains assignment for apo and bound states. In addition, a secondary structure prediction by TALOS + analysis was performed. The results indicated that HEV MD has a α/β/α topology very similar to that of most viral macro domains.

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.