Merging NMR Data and Computation Facilitates Data-Centered Research.

The Biological Magnetic Resonance Data Bank (BMRB) has served the NMR structural biology community for 40 years, and has been instrumental in the development of many widely-used tools. It fosters the reuse of data resources in structural biology by embodying the FAIR data principles (Findable, Accessible, Inter-operable, and Re-usable). NMRbox is less than a decade old, but complements BMRB by providing NMR software and high-performance computing resources, facilitating the reuse of software resources. BMRB and NMRbox both facilitate reproducible research. NMRbox also fosters the development and deployment of complex meta-software. Combining BMRB and NMRbox helps speed and simplify workflows that utilize BMRB, and enables facile federation of BMRB with other data repositories. Utilization of BMRB and NMRbox in tandem will enable additional advances, such as machine learning, that are poised to become increasingly powerful.

A novel phosphoglycerol serine-glycine lipodipeptide of Porphyromonas gingivalis is a TLR2 ligand.

Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

Structural and functional analyses of the N-terminal domain of the A subunit of a Bacillus megaterium spore germinant receptor.

Germination of Bacillus spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of the Bacillus megaterium GerK3 GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.

Concomitant disorder and high-affinity zinc binding in the human zinc- and iron-regulated transport protein 4 intracellular loop.

The human zinc- and iron-regulated transport protein 4 (hZIP4) protein is the major plasma membrane protein responsible for the uptake of zinc in the body, and as such it plays a key role in cellular zinc homeostasis. hZIP4 plasma membrane levels are regulated through post-translational modification of its large, disordered, histidine-rich cytosolic loop (ICL2) in response to intracellular zinc concentrations. Here, structural characteristics of the isolated disordered loop region, both in the absence and presence of zinc, were investigated using nuclear magnetic resonance (NMR) spectroscopy. NMR chemical shifts, coupling constants and temperature coefficients of the apoprotein, are consistent with a random coil with minor propensities for transient polyproline Type II helices and ß-strand in regions implicated in post-translational modifications. The ICL2 protein remains disordered upon zinc binding, which induces exchange broadening. Paramagnetic relaxation enhancement experiments reveal that the histidine-rich region in the apoprotein makes transient tertiary contacts with predicted post-translational modification sites. The residue-specific data presented here strengthen the relationship between hZIP4 post-translational modifications, which impact its role in cellular zinc homeostasis, and zinc sensing by the intracellular loop. Furthermore, the zinc sensing mechanism employed by the ICL2 protein demonstrates that high-affinity interactions can occur in the presence of conformational disorder.

Nonuniform sampling in multidimensional NMR for improving spectral sensitivity.

The development of multidimensional NMR spectroscopy enabled an explosion of structural and dynamical investigations on proteins and other biomacromolecules. Practical limitations on data sampling, based on the Jeener paradigm of parametric sampling of indirect time domains, have long placed limits on resolution in the corresponding frequency dimensions. The emergence of nonuniform sampling (NUS) in indirect time dimensions circumvents those limitations, affording high resolution spectra from short data records collected in practically realized measurement times. In addition to substantially improved resolution, NUS can also be exploited to improve sensitivity, with gains comparable to those obtained using cryogenically cooled probes. We describe a general approach for acquiring and processing multidimensional NUS NMR data for improving sensitivity.

Practical Nonuniform Sampling and Non-Fourier Spectral Reconstruction for Multidimensional NMR.

A general approach to accelerating multidimensional NMR experiments via nonuniform sampling and maximum entropy spectral reconstruction was first demonstrated by Laue and colleagues in 1987. Following decades of continual improvements involving dozens of software packages for non-Fourier spectral analysis and many different schemes for nonuniform sampling, we still lack a clear consensus on best practices for sampling or spectral reconstruction, and programs for processing nonuniformly sampled data are not particularly user-friendly. Nevertheless, it is possible to discern conservative and general guidelines for nonuniform sampling and spectral reconstruction. Here, we describe a robust semi-automated workflow that employs these guidelines for simplifying the selection of a sampling schedule and the processing of the resulting nonuniformly sampled multidimensional NMR data. Our approach is based on NMRbox, a shared platform for NMR software that facilitates workflow development and execution, and enables rapid comparison of alternate approaches.

Robust and transferable quantification of NMR spectral quality using IROC analysis.

Non-Fourier methods are increasingly utilized in NMR spectroscopy because of their ability to handle nonuniformly-sampled data. However, non-Fourier methods present unique challenges due to their nonlinearity, which can produce nonrandom noise and render conventional metrics for spectral quality such as signal-to-noise ratio unreliable. The lack of robust and transferable metrics (i.e. applicable to methods exhibiting different nonlinearities) has hampered comparison of non-Fourier methods and nonuniform sampling schemes, preventing the identification of best practices. We describe a novel method, in situ receiver operating characteristic analysis (IROC), for characterizing spectral quality based on the Receiver Operating Characteristic curve. IROC utilizes synthetic signals added to empirical data as "ground truth", and provides several robust scalar-valued metrics for spectral quality. This approach avoids problems posed by nonlinear spectral estimates, and provides a versatile quantitative means of characterizing many aspects of spectral quality. We demonstrate applications to parameter optimization in Fourier and non-Fourier spectral estimation, critical comparison of different methods for spectrum analysis, and optimization of nonuniform sampling schemes. The approach will accelerate the discovery of optimal approaches to nonuniform sampling experiment design and non-Fourier spectrum analysis for multidimensional NMR.

NMRbox: A Resource for Biomolecular NMR Computation.

Advances in computation have been enabling many recent advances in biomolecular applications of NMR. Due to the wide diversity of applications of NMR, the number and variety of software packages for processing and analyzing NMR data is quite large, with labs relying on dozens, if not hundreds of software packages. Discovery, acquisition, installation, and maintenance of all these packages is a burdensome task. Because the majority of software packages originate in academic labs, persistence of the software is compromised when developers graduate, funding ceases, or investigators turn to other projects. To simplify access to and use of biomolecular NMR software, foster persistence, and enhance reproducibility of computational workflows, we have developed NMRbox, a shared resource for NMR software and computation. NMRbox employs virtualization to provide a comprehensive software environment preconfigured with hundreds of software packages, available as a downloadable virtual machine or as a Platform-as-a-Service supported by a dedicated compute cloud. Ongoing development includes a metadata harvester to regularize, annotate, and preserve workflows and facilitate and enhance data depositions to BioMagResBank, and tools for Bayesian inference to enhance the robustness and extensibility of computational analyses. In addition to facilitating use and preservation of the rich and dynamic software environment for biomolecular NMR, NMRbox fosters the development and deployment of a new class of metasoftware packages. NMRbox is freely available to not-for-profit users.

Nuclear Magnetic Resonance Structures of GCN4p Are Largely Conserved When Ion Pairs Are Disrupted at Acidic pH but Show a Relaxation of the Coiled Coil Superhelix.

To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cß) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of 1H-15N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, 13C rotating frame T1 relaxation (T1ρ) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

Correction: Sparse sampling methods in multidimensional NMR.

Correction for 'Sparse sampling methods in multidimensional NMR' by Mehdi Mobli et al., Phys. Chem. Chem. Phys., 2012, 14, 10835-10843.

Nonuniform sampling of hypercomplex multidimensional NMR experiments: Dimensionality, quadrature phase and randomization.

Nonuniform sampling (NUS) in multidimensional NMR permits the exploration of higher dimensional experiments and longer evolution times than the Nyquist Theorem practically allows for uniformly sampled experiments. However, the spectra of NUS data include sampling-induced artifacts and may be subject to distortions imposed by sparse data reconstruction techniques, issues not encountered with the discrete Fourier transform (DFT) applied to uniformly sampled data. The characterization of these NUS-induced artifacts allows for more informed sample schedule design and improved spectral quality. The DFT-Convolution Theorem, via the point-spread function (PSF) for a given sampling scheme, provides a useful framework for exploring the nature of NUS sampling artifacts. In this work, we analyze the PSFs for a set of specially constructed NUS schemes to quantify the interplay between randomization and dimensionality for reducing artifacts relative to uniformly undersampled controls. In particular, we find a synergistic relationship between the indirect time dimensions and the "quadrature phase dimension" (i.e. the hypercomplex components collected for quadrature detection). The quadrature phase dimension provides additional degrees of freedom that enable partial-component NUS (collecting a subset of quadrature components) to further reduce sampling-induced aliases relative to traditional full-component NUS (collecting all quadrature components). The efficacy of artifact reduction is exponentially related to the dimensionality of the sample space. Our results quantify the utility of partial-component NUS as an additional means for introducing decoherence into sampling schemes and reducing sampling artifacts in high dimensional experiments.

NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein.

CUS-3 is a P22-like tailed dsDNA bacteriophage that infects Escherichia coli serotype K1. The CUS-3 coat protein, which forms the icosahedral capsid, has a conserved HK97-fold but with a non-conserved accessory domain known as the insertion domain (I-domain). Sequence alignment of the coat proteins from CUS-3 and P22 shows higher sequence similarity for the I-domains (35 %) than for the HK97-cores, suggesting the I-domains play important functional roles. The I-domain of the P22 coat protein, which has an NMR structure comprised of a six-stranded ß-barrel, has been shown to govern the assembly, stability and size of the resulting capsid particles. Here, we report the (1)H, (15)N, and (13)C assignments for the I-domain from the coat protein of bacteriophage CUS-3. The secondary structure and dynamics of the CUS-3 I-domain, predicted from the assigned NMR chemical shifts, agree with those of the P22 I-domain, suggesting the CUS-3 and P22 I-domains may have similar structures and functions in capsid assembly.

Nonuniform sampling and maximum entropy reconstruction in multidimensional NMR.

NMR spectroscopy is one of the most powerful and versatile analytic tools available to chemists. The discrete Fourier transform (DFT) played a seminal role in the development of modern NMR, including the multidimensional methods that are essential for characterizing complex biomolecules. However, it suffers from well-known limitations: chiefly the difficulty in obtaining high-resolution spectral estimates from short data records. Because the time required to perform an experiment is proportional to the number of data samples, this problem imposes a sampling burden for multidimensional NMR experiments. At high magnetic field, where spectral dispersion is greatest, the problem becomes particularly acute. Consequently multidimensional NMR experiments that rely on the DFT must either sacrifice resolution in order to be completed in reasonable time or use inordinate amounts of time to achieve the potential resolution afforded by high-field magnets. Maximum entropy (MaxEnt) reconstruction is a non-Fourier method of spectrum analysis that can provide high-resolution spectral estimates from short data records. It can also be used with nonuniformly sampled data sets. Since resolution is substantially determined by the largest evolution time sampled, nonuniform sampling enables high resolution while avoiding the need to uniformly sample at large numbers of evolution times. The Nyquist sampling theorem does not apply to nonuniformly sampled data, and artifacts that occur with the use of nonuniform sampling can be viewed as frequency-aliased signals. Strategies for suppressing nonuniform sampling artifacts include the careful design of the sampling scheme and special methods for computing the spectrum. Researchers now routinely report that they can complete an N-dimensional NMR experiment 3(N-1) times faster (a 3D experiment in one ninth of the time). As a result, high-resolution three- and four-dimensional experiments that were prohibitively time consuming are now practical. Conversely, tailored sampling in the indirect dimensions has led to improved sensitivity. Further advances in nonuniform sampling strategies could enable further reductions in sampling requirements for high resolution NMR spectra, and the combination of these strategies with robust non-Fourier methods of spectrum analysis (such as MaxEnt) represent a profound change in the way researchers conduct multidimensional experiments. The potential benefits will enable more advanced applications of multidimensional NMR spectroscopy to study biological macromolecules, metabolomics, natural products, dynamic systems, and other areas where resolution, sensitivity, or experiment time are limiting. Just as the development of multidimensional NMR methods presaged multidimensional methods in other areas of spectroscopy, we anticipate that nonuniform sampling approaches will find applications in other forms of spectroscopy.

NMR assignments for the cis and trans forms of the hemolysin II C-terminal domain.

Pathogenic bacteria secrete pore-forming toxins (PFTs) to selectively defend against immune cells and to break through cellular barriers in the host. Understanding how PFTs attack cell membranes is not only essential for therapeutic intervention but for designing agents to deliver drugs to specific human cell subtypes, for example in anti-cancer or anti-viral therapies. Many toxins contain accessory domains that help recognize specific molecular epitopes on the membranes of target cells, including proteins, carbohydrates, and lipids. Here we report NMR assignments for the 94-residue 10 kDa C-terminal accessory domain of Bacillus cereus hemolysin II, HlyIIC, that has no known structural or functional homologues. The HlyIIC domain exists in a dynamic equilibrium due to cis/trans isomerization of its Gly86-Pro87 peptide bond. The cis and trans forms are about equally populated and are in slow exchange on the NMR timescale, giving rise to separate signals for approximately half of the residues in the domain. Assignments for the cis and trans forms were achieved with the aid of a P87M mutant that stabilizes the trans form, and separate sequential walks for the two forms in 3D NMR spectra of the wild-type HlyIIC. Based on backbone chemical shifts, the domain has a α1-α2-ß1-ß2-ß3-ß4-α3-ß5 order of secondary structure elements. The last strand in the trans form and in the P87M mutant is shortened near Pro87 compared to the cis form. Both cis/trans isomerization and the P87M mutation cause large chemical shift changes throughout HlyIIC, suggesting that the proline is important in stabilizing the structure of the domain. The NMR assignments pave the way for solving the structures of the multiple conformational forms of HlyIIC and establishing their mechanism of interconversion.

Serine lipids of Porphyromonas gingivalis are human and mouse Toll-like receptor 2 ligands.

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2(-/-)) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2(-/-) mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Crystal structure of the ubiquitin-like small archaeal modifier protein 2 from Haloferax volcanii.

The discovery of ubiquitin-like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating-like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well-folded compact conformation in the presence of 2.5M of NaCl. Its 2.3-Å-resolution crystal structure reveals a characteristic α/ß central core domain and a unique ß-hinge motif. This motif anchors an unusual C-terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this ß-hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin-like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic ß-grasp fold in a salt-independent manner. These results provide insights into the structure-function relationship of sampylating proteins of fundamental importance in post-translational protein modification and environmental cues in Archaea.

NMR mapping of PCNA interaction with translesion synthesis DNA polymerase Rev1 mediated by Rev1-BRCT domain.

Rev1 is a Y-family translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap filling. In the process of TLS, high-fidelity replicative DNA polymerases stalled by DNA damage are replaced by error-prone TLS enzymes responsible for the majority of mutagenesis in eukaryotic cells. The polymerase exchange that gains low-fidelity TLS polymerases access to DNA is mediated by their interactions with proliferating cell nuclear antigen (PCNA). Rev1 stands alone from other Y-family TLS enzymes since it lacks the consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. Here we report solution NMR structure of an 11-kDa BRCA1 C-terminus (BRCT) domain from Saccharomyces cerevisiae Rev1 and demonstrate with the use of transverse relaxation optimized spectroscopy (TROSY) NMR methods that Rev1-BRCT domain directly interacts with an 87-kDa PCNA in solution. The domain adopts α/ß fold (ß1-α1-ß2-ß3-α2-ß4-α3-α4) typical for BRCT domain superfamily. PCNA-binding interface of the Rev1-BRCT domain comprises conserved residues of the outer surface of the α1-helix and the α1-ß1, ß2-ß3 and ß3-α2 loops. On the other hand, Rev1-BRCT binds to the inter-domain region of PCNA that overlaps with the binding site for the PIP-box motif. Furthermore, Rev1-BRCT domain bound to PCNA can be displaced by increasing amounts of the PIP-box peptide from TLS DNA polymerase polη, suggesting that Rev1-BRCT and polη PIP-box interactions with the same PCNA monomer are mutually exclusive. These results provide structural insights into PCNA recognition by TLS DNA polymerases that help better understand TLS regulation in eukaryotes.

Formalism for hypercomplex multidimensional NMR employing partial-component subsampling.

Multidimensional NMR spectroscopy typically employs phase-sensitive detection, which results in hypercomplex data (and spectra) when utilized in more than one dimension. Nonuniform sampling approaches have become commonplace in multidimensional NMR, enabling dramatic reductions in experiment time, increases in sensitivity and/or increases in resolution. In order to utilize nonuniform sampling optimally, it is necessary to characterize the relationship between the spectrum of a uniformly sampled data set and the spectrum of a subsampled data set. In this work we construct an algebra of hypercomplex numbers suitable for representing multidimensional NMR data along with partial-component nonuniform sampling (i.e. the hypercomplex components of data points are subsampled). This formalism leads to a modified DFT-Convolution relationship involving a partial-component, hypercomplex point-spread function set. The framework presented here is essential for the continued development and appropriate characterization of partial-component nonuniform sampling.

(1)H, (13)C, and (15)N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum.

Phosphoethanolamine methyltransferases (PMTs also known as PEAMTs) catalyze the three-step s-adenosyl-methionione-dependent methylation of phosphoethanolamine to form phosphocholine. These enzymes play an important function in the synthesis of phosphatidylcholine, the major phospholipid in the membranes of lower and higher eukaryotes, as well as in the production of the compatible solute and osmoprotectant glycine betaine in plants. Genetic studies in plants, Caenhorhabditis elegans and Plasmodium falciparum have demonstrated that disruption of PMT activity results in severe defects in important cellular processes such as development, replication, survival and sexual maturation and differentiation. Here we report chemical shift assignments for PfPMT, the PMT from Plasmodium falciparum. X-ray crystal structures have been recently reported for complexes of PfPMT, but the structure of the apoenzyme remains unknown. The solution structure of the apoenzyme will help to elucidate important details of the mechanism of substrate binding by PfPMT, as residues comprising the substrate binding site are inaccessible to solvent in the conformation evident in the available crystal structures. In addition to enabling determination of the solution structure of the apoenzyme, the assignments will facilitate additional investigations into the interaction of PfPMT with its substrates and inhibitors.

Secondary structure, a missing component of sequence-based minimotif definitions.

Minimotifs are short contiguous segments of proteins that have a known biological function. The hundreds of thousands of minimotifs discovered thus far are an important part of the theoretical understanding of the specificity of protein-protein interactions, posttranslational modifications, and signal transduction that occur in cells. However, a longstanding problem is that the different abstractions of the sequence definitions do not accurately capture the specificity, despite decades of effort by many labs. We present evidence that structure is an essential component of minimotif specificity, yet is not used in minimotif definitions. Our analysis of several known minimotifs as case studies, analysis of occurrences of minimotifs in structured and disordered regions of proteins, and review of the literature support a new model for minimotif definitions that includes sequence, structure, and function.