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.

Structural insights into the multifunctionality of rabies virus P3 protein.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

15N-Detected TROSY NMR experiments to study large disordered proteins in high-field magnets.

Intrinsically disordered regions (IDRs) of proteins are critical in the regulation of biological processes but difficult to study structurally. Nuclear magnetic resonance (NMR) is uniquely equipped to provide structural information on IDRs at atomic resolution; however, existing NMR methods often pose a challenge for large molecular weight IDRs. Resonance assignment of IDRs using 15ND-detection was previously demonstrated and shown to overcome some of these limitations. Here, we improve the methodology by overcoming the need for deuterated buffers and provide better sensitivity and resolution at higher magnetic fields and physiological salt concentrations using transverse relaxation optimized spectroscopy (TROSY). Finally, large disordered regions with low sequence complexity can be assigned efficiently using these new methods as demonstrated by achieving near complete assignment of the 398-residue N-terminal IDR of the transcription factor NFAT1 harboring 18% prolines.

Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells.

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5'-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.

Local Deuteration Enables NMR Observation of Methyl Groups in Proteins from Eukaryotic and Cell-Free Expression Systems.

Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13 C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of "local" deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.

1H, 15N, and 13C backbone resonance assignments of the C4b-binding region from the S. aureus extracellular adherence protein.

The Extracellular Adherence Protein (Eap) from Staphylococcus aureus is a potent inhibitor of the classical and lectin pathways of the complement system. Previous studies have shown that Eap binds with nanomolar affinity to complement component C4b and prevents C4b binding the pro-protease, C2, thereby inhibiting formation of the pro-C3 convertase shared by the classical and lectin pathways (Woehl et al. in J Immunol 193:6161-6171, 2014). The C4b-binding and complement-inhibitory properties of Eap from S. aureus strain Mu50 lie within the two C terminal-most Eap domains (i.e. Eap34) (Woehl et al. J Immunol 193:6161-6171, 2014). Interestingly, Eap34 binds C4b with an apparent KD that is nearly 100-fold tighter than that of either Eap3 or Eap4 alone (Woehl et al. in Protein Sci 26:1595-1608, 2017). This suggests that linking these two domains into a single molecule is a significant determinant of Eap function. To better understand this property at the structural level, we undertook a solution NMR study of the ~ 23 kDa Eap34 protein. In this communication, we report that greater than 98% of the total non-proline backbone residues have been assigned. These data have been deposited in the BMRB database under the accession number 50210.

Rapid nuclear magnetic resonance data acquisition with improved resolution and sensitivity for high-throughput metabolomic analysis.

Nuclear magnetic resonance (NMR)-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution, and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy, and fast-pulsing methods for identification and assignments of metabolites.

Pre-T cell receptors topologically sample self-ligands during thymocyte ß-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αßTCRs. Using x-ray crystallography, we show how a preTCR applies the concave ß-sheet surface of its single variable domain (Vß) to "horizontally" grab the protruding MHC α2-helix. By contrast, αßTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVß module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3ß reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αßTCR canonical docking mode. "Horizontal" docking precludes germline CDR1ß- and CDR2ß-MHC binding to broaden ß-chain repertoire diversification before αßTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

Nearest-neighbor NMR spectroscopy: categorizing spectral peaks by their adjacent nuclei.

Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. However, resonance assignment remains challenging. The problem is to combine existing structural informational with sparse distance restraints and search for the most compatible assignment among the permutations. Prior classification of peaks as either from isoleucine, leucine, or valine reduces the search space by many orders of magnitude. However, this is hindered by overlapped leucine and valine frequencies. In contrast, the nearest-neighbor nuclei, coupled to the methyl carbons, resonate in distinct frequency bands. Here, we develop a framework to imprint additional information about passively coupled resonances onto the observed peaks. This depends on simultaneously orchestrating closely spaced bands of resonances along different magnetization trajectories, using principles from control theory. For methyl-NMR, the method is implemented as a modification to the standard fingerprint spectrum (the 2D-HMQC). The amino acid type is immediately apparent in the fingerprint spectrum. There is no additional relaxation loss or an increase in experimental time. The method is validated on biologically relevant proteins. The idea of generating new spectral information using passive, adjacent resonances is applicable to other contexts in NMR spectroscopy.

Correction to: The role of NMR in leveraging dynamics and entropy in drug design.

Unfortunately, in the original publication, Fig. 5 was published incorrectly. The correct version is given below.

The role of NMR in leveraging dynamics and entropy in drug design.

Nuclear magnetic resonance (NMR) spectroscopy has contributed to structure-based drug development (SBDD) in a unique way compared to the other biophysical methods. The potency of a ligand binding to a protein is dictated by the binding free energy, which is an intricate interplay between entropy and enthalpy. In addition to providing the atomic resolution structural information, NMR can help to identify protein-ligand interactions that potentially contribute to the enthalpic component of the free energy. NMR can also illuminate dynamic aspects of the interaction, which correspond to the entropic term of the free energy. The ability of NMR to access both terms in the free energy equation stems from the suite of experiments developed to shed light on various aspects that contribute to both entropy and enthalpy, deepening our understanding of the biological function of macromolecules and assisting to target them in physiological conditions. Here we provide a brief account of the contribution of NMR to SBDD, highlighting hallmark examples and discussing the challenges that demand further method development. In the era of integrated biology, the unique ability of NMR to directly ascertain structural and dynamical aspects of macromolecule and monitor changes in these properties upon engaging a ligand can be combined with computational and other structural and biophysical methods to provide a more complete picture of the energetics of drug engagement with the target. Such efforts can be used to engineer better drugs.

Fast NMR Methods for Identification of Resonances and Metabolic Pathways.

High-throughput analysis of NMR data in metabolomics involves both rapid data acquisition and analysis. We describe here a data collection and analysis protocol, which enables fast multidimensional NMR data acquisition and automated analysis of NMR spectra to rapidly identify the metabolites and assign them to active metabolic pathways in the system.

Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins.

The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-resolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Here, we summarize recent technical landmarks in solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic resolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.

Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Backbone resonance assignments of innate immune evasion protein EapH2 from the S. aureus.

Staphylococcus aureus is a ubiquitous and persistent pathogen of humans and livestock. The bacterium disrupts the host's innate immune system's ability to recognize and clear bacteria with optimal efficiency by expressing a wide variety of virulence proteins. Two single domain protein homologs (EapH1, EapH2) of the extracellular adherence protein (Eap) have been reported. Eap is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement and Neutrophil Serine Proteases (NSPs). EapH1 and EapH2 are also inhibitors of NSPs (Stapels et al., Proc Natl Acad Sci 111:13187-13192, 2014), but lack the ability to inhibit the classical, and lectin pathways of the complement activation system (Woehl et al., J Immunol 193:6161-6171, 2014). We continue the characterization of Eap domains, here with the experiments on EapH2, we acquired a series of 2D and 3D NMR spectra of EapH2 in solution. We completed 99% of expected non-proline backbone 1H, 15N, and 13C resonance assignments of EapH2 and predicted secondary structure via the TALOS-N server. The assignment data have been deposited in the BMRB data bank under Accession Number 27540.

Optimal control theory enables homonuclear decoupling without Bloch-Siegert shifts in NMR spectroscopy.

The Bloch-Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch-Siegert shifts. This approach enables accurate observation of the resonance frequencies of decoupled nuclear spins. We apply this method to increase the resolution of the HNCA experiment. We also observe a doubling in sensitivity for a 30 kDa protein. We demonstrate the use of band-selective Cß decoupling to produce amino acid-specific line shapes, which are valuable for assigning resonances to the protein sequence. Finally, we assign the backbone of a 30 kDa protein, Human Carbonic Anhydrase II, using only HNCA experiments acquired with band-selective decoupling schemes, and instrument time of one week.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins.

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart.

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.

Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment.

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαß) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.

Evaluation of Effect of Glucosamine-Chondroitin Sulfate, Tramadol, and Sodium Hyaluronic Acid on Expression of Cytokine Levels in Internal Derangement of Temporomandibular Joint.

AIM: Evaluation of the effect of glucosamine-chondroitin combination, tramadol, and sodium hyaluronic acid in temporomandibular joint (TMJ) disorders and its impact on the expression of various cytokines such as IL-6, IL-1ß, TNF-α, and PGE2. MATERIALS AND METHODS: The present study was conducted on 60 patients (males-30, females-30) suffering from internal derangement such as disc displacement with reduction of TMJ. The patients were divided into three groups of 20 each. Group I received a combination of 1.5g of glucosamine and 1.2 g of chondroitin sulfate per day and group II received 50 mg tramadol HCL peroral. Group III received sodium hyaluronate 10 mg/mL, 2 mL injection syringe on each joint. Pain (VAS) scale and maximum mouth opening (MMO) was measured. The level of IL-6, IL-1ß, TNF-α, and PGE2 levels were measured using Enzyme-linked immuno sorbent assay (ELISA). RESULTS: There was an improvement in maximum mouth opening in all three groups (p < 0.05). There was a reduction in pain in all groups. IL- 1ß, TNF-α, and PGE2 leve ls showed reduction while IL-6 showed an increase in value in group II and III. CONCLUSION: The efficacy of glucosamine chondroitin sulfate , tramadol and hyaluronic acid in TMJ disorders has been found to be effective. CLINICAL SIGNIFICANCE: IL-6, IL-1ß, TNF-α, and PGE2 levels indicate the risk of TMJ disorders. Thus earlier assessment of their levels helps in diagnosis, and better management may be done.