Activity of Tumor Necrosis Factor α Is Modulated by Dynamic Conformational Rearrangements.

The homotrimeric ligand tumor necrosis factor α (TNFα) is a key cytokine and immune regulator; however, when deregulated, it leads to several major chronic inflammatory diseases. Perturbation of the protein-protein interface has proven to be an efficient strategy to inactivate TNFα, but the atomic-resolution mechanism of its inactivation remains poorly understood. Here, we probe the solution structure and dynamics of active and inactive TNFα using NMR spectroscopy. The data reveal that TNFα undergoes motions on different time scales. Furthermore, by site-directed mutagenesis of residues at the trimerization interface and by targeting the interface with a low molecular weight inhibitor, we show that TNFα retains its overall structure and trimeric state. However, upon perturbation, TNFα exhibits increased conformational dynamics spanning from the trimerization interface to the regions mediating receptor binding. These findings provide novel insights into the inactivation mechanism of TNFα and the basis for strategies to target TNFα activity.

Computer vision-based automated peak picking applied to protein NMR spectra.

MOTIVATION: A detailed analysis of multidimensional NMR spectra of macromolecules requires the identification of individual resonances (peaks). This task can be tedious and time-consuming and often requires support by experienced users. Automated peak picking algorithms were introduced more than 25 years ago, but there are still major deficiencies/flaws that often prevent complete and error free peak picking of biological macromolecule spectra. The major challenges of automated peak picking algorithms is both the distinction of artifacts from real peaks particularly from those with irregular shapes and also picking peaks in spectral regions with overlapping resonances which are very hard to resolve by existing computer algorithms. In both of these cases a visual inspection approach could be more effective than a 'blind' algorithm. RESULTS: We present a novel approach using computer vision (CV) methodology which could be better adapted to the problem of peak recognition. After suitable 'training' we successfully applied the CV algorithm to spectra of medium-sized soluble proteins up to molecular weights of 26 kDa and to a 130 kDa complex of a tetrameric membrane protein in detergent micelles. Our CV approach outperforms commonly used programs. With suitable training datasets the application of the presented method can be extended to automated peak picking in multidimensional spectra of nucleic acids or carbohydrates and adapted to solid-state NMR spectra. AVAILABILITY AND IMPLEMENTATION: CV-Peak Picker is available upon request from the authors. CONTACT: gsw@mol.biol.ethz.ch; michal.walczak@mol.biol.ethz.ch; adam.gonczarek@pwr.edu.pl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Interactions of the acyl chain with the Saccharomyces cerevisiae acyl carrier protein.

Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability.

Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

Posttranslational modifications of intact proteins detected by NMR spectroscopy: application to glycosylation.

Posttranslational modifications (PTMs) are an integral part of the majority of proteins. The characterization of structure and function of PTMs can be very challenging especially for glycans. Existing methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications, the inability to identify linkage types and thus chemical structure. We present a direct, robust, and simple NMR spectroscopy method for the detection and identification of PTMs in proteins. No isotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approach is well suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.

Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10.

A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3 mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80 % of the resonances of the (13)C-(1)H moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.

Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments.

A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [(13)C, (15)N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1'C1'CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98 % assignment completeness for all (13)C-(1)H, (15)N1/9 and (31)P resonances of a stem-loop with 14 nucleotides.

The RING domain of the scaffold protein Ste5 adopts a molten globular character with high thermal and chemical stability.

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the ß and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M). Furthermore, it exhibits high thermal stability in native conditions. Binding of the Ste5 RING-H2 domain to the physiological Gß/γ (Ste4/Ste18) ligand is accompanied by a conformational transition into a better folded, more globular structure. This study reveals novel insights into the folding mechanism and recruitment of binding partners by the Ste5 RING-H2 domain. We speculate that many RING domains may share a similar mechanism of substrate recognition and molten-globule-like character.

4D experiments measured with APSY for automated backbone resonance assignments of large proteins.

Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40 kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a 'just-in-time' manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90 % of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4 kDa.

Structural analysis of the conserved ubiquitin-binding motifs (UBMs) of the translesion polymerase iota in complex with ubiquitin.

Ubiquitin-binding domains (UBDs) provide specificity to the ubiquitin system, which is also involved in translesion synthesis (TLS) in eukaryotic cells. Upon DNA damage, the UBDs (UBM domains) of polymerase iota (Pol ι) interact with ubiquitinated proliferating cell nuclear antigen to regulate the interchange between processive DNA polymerases and TLS. We report a biophysical analysis and solution structures of the two conserved UBM domains located in the C-terminal tail of murine Pol ι in complex with ubiquitin. The 35-amino acid core folds into a helix-turn-helix motif, which belongs to a novel domain fold. Similar to other UBDs, UBMs bind to ubiquitin on the hydrophobic surface delineated by Leu-8, Ile-44, and Val-70, however, slightly shifted toward the C terminus. In addition, UBMs also use electrostatic interactions to stabilize binding. NMR and fluorescence spectroscopy measurements revealed that UBMs bind monoubiquitin, and Lys-63- but not Lys-48-linked chains. Importantly, these biophysical data are supported by functional studies. Indeed, yeast cells expressing ubiquitin mutants specifically defective for UBM binding are viable but sensitive to DNA damaging conditions that require TLS for repair.

Sugar-to-base correlation in nucleic acids with a 5D APSY-HCNCH or two 3D APSY-HCN experiments.

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5 h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all (15)N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.

Automated projection spectroscopy and its applications.

This chapter presents the NMR technique APSY (automated projection spectroscopy) and its applications for sequence-specific resonance assignments of proteins. The result of an APSY experiment is a list of chemical shift correlations for an N-dimensional NMR spectrum (N≥3). This list is obtained in a fully automated way by the dedicated algorithm GAPRO (geometric analysis of projections) from a geometric analysis of experimentally recorded, low-dimensional projections. Because the positions of corresponding peaks in multiple projections are correlated, thermal noise and other uncorrelated artifacts are efficiently suppressed. We describe the theoretical background of the APSY method and discuss technical aspects that guide its optimal use. Further, applications of APSY-NMR spectroscopy for fully automated sequence-specific backbone and side chain assignments of proteins are described. We discuss the choice of suitable experiments for this purpose and show several examples. APSY is of particular interest for the assignment of soluble unfolded proteins, which is a time-consuming task by conventional means. With this class of proteins, APSY-NMR experiments with up to seven dimensions have been recorded. Sequence-specific assignments of protein side chains in turn are obtained from a 5D TOCSY-APSY-NMR experiment.

Automated projection spectroscopy (APSY) for the assignment of NMR resonances of biological macromolecules.

High resolution nuclear magnetic resonance (NMR) spectroscopy in solution is an established technique in structural biology. Detailed functional and structural studies of biological macromolecules by NMR require the assignment of the chemical shifts to specific nuclei. In biological applications, the necessary data is usually obtained from a number of two- and three-dimensional (2D and 3D) NMR experiments. Often, these data cannot be fully analyzed by automated computer programs due to insufficient separation and resolution of the signals in the available spectra. Then, complete resonance assignment requires manual interaction and can become a long and labor-intensive task. Automated projection spectroscopy (APSY) allows the substantial improvement of the resolution by providing spectral information from four and higher dimensional experiments without measuring the full spectrum, which would by far exceed any acceptable measuring time. APSY only measures a series of projections of the high-dimensional spectrum which can be obtained in a much shorter time. Peak picking of the projection spectra provides the basis for the calculation of the high-dimensional chemical shift correlation space by the algorithm GAPRO. The resulting high-dimensional peak lists are commonly artifact-free and of exceptional precision. Along with their high number of correlated nuclei they provide an ideal basis for reliable automated assignment. We will introduce the basic concepts of APSY, illustrate them with an application of a 6D APSY-seq-HNCOCANH experiment, and discuss some practical aspects.

Relaxation optimized double acquisition (RODA) as an alternative for virtual decoupling of NMR spectra.

We introduce an alternative way for spin-state selection, RODA, which yields higher sensitivity for spin systems exhibiting a TROSY effect. With RODA, the TROSY component of a doublet is recorded twice using a double acquisition scheme. RODA works by simple addition of consecutive NMR signals, and does not require any special processing. Thus, this pulse sequence element can seamlessly be integrated into existing experiments. We demonstrate the broad applicability of RODA with several systems exhibiting a TROSY effect on 15N-1H, 19F-13C or 1H-13C moieties. Further, we show that virtual decoupling with increased sensitivity is possible in a single double acquisition experiment in situations as encountered with dissolution DNP.

4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins.

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on (1)H(ß) and (13)C(δ) efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13 kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCH(aro) sequence are proposed.

Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5.

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell-cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gßγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell-cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress-triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell-cell fusion during mating.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

Automated NMR assignment of protein side chain resonances using automated projection spectroscopy (APSY).

This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.

Real-time imaging of the spatial distribution of rf-heating in NMR samples during broadband decoupling.

Continuous radio-frequency (rf) irradiation during decoupling and spin-lock periods in NMR pulse sequences may lead to undesired sample heating. Heat-sensitive samples can suffer damage from the sudden temperature rise which cannot be adequately compensated by the temperature control system. Moreover, as the heating is spatially inhomogeneous, higher temperature increases can arise locally than are indicated by the average increase detected by the temperature controller. In this work we present a technique that allows measurement of a real-time 2D-image of the temperature distribution inside an NMR sample during an experiment involving rf-heating. NMR imaging methods have previously been used to project the temperature distribution inside an NMR sample onto a single spatial axis or to acquire steady-state 2D- temperature distributions. The real-time 2D-temperature profiles obtained with our procedure provide much more detailed data. Our results show, that not only inhomogeneous heating but also inhomogeneous sample cooling contribute to the build-up of temperature gradients across the sample. The technique can be used to visualize rf-heating in order to protect sensitive samples and to experimentally test new coil geometries or to guide probehead design.

TROSY in NMR studies of the structure and function of large biological macromolecules.

Transverse relaxation-optimized spectroscopy (TROSY), in combination with various isotope-labeling techniques, has opened avenues to study biomolecules with molecular masses of up to 1000000Da by solution NMR. Important recent applications of TROSY include the structure determination of membrane proteins in detergent micelles, structural and functional studies of large proteins in both monomeric form and macromolecular complexes, and investigations of intermolecular interactions in large complexes. TROSY improves the measurement of residual dipolar couplings and the detection of scalar couplings across hydrogen bonds - techniques that promise to further enhance the determination of solution structures of large proteins and oligonucleotides.