Rotaxane Probes for the Detection of Hydrogen Peroxide by 129 Xe HyperCEST NMR Spectroscopy.

The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in 1 H MRI are impaired by the high abundance of protons in the body. 129 Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional 1 H MRI because of its ability to report specific chemical environments. To date, the scope of responsive 129 Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane-based 129 Xe hyperCEST NMR contrast agent that can be turned on in response to H2 O2 , which is upregulated in several disease states. Added H2 O2 was detected by 129 Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H2 O2 produced by HEK 293T cells activated with tumor necrosis factor.

Rotaxane probes for protease detection by 129Xe hyperCEST NMR.

We report a CB6 rotaxane for the 129Xe hyperCEST NMR detection of matrix metalloprotease 2 (MMP-2) activity. MMP-2 is overexpressed in cancer tissue, and hence is a cancer marker. A peptide containing an MMP-2 recognition sequence was incorporated into the rotaxane, synthesized via CB6-promoted click chemistry. Upon cleavage of the rotaxane by MMP-2, CB6 became accessible for 129Xe@CB6 interactions, leading to protease-responsive hyperCEST activation.

Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation.

Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA+ ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. We identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1-σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.

Targeted Molecular Imaging of Cancer Cells Using MS2-Based (129)Xe NMR.

We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo.

(129)Xe NMR Relaxation-Based Macromolecular Sensing.

We report a (129)Xe NMR relaxation-based sensing approach that exploits changes in the bulk xenon relaxation rate induced by slowed tumbling of a cryptophane-based sensor upon target binding. The amplification afforded by detection of the bulk dissolved xenon allows sensitive detection of targets. The sensor comprises a xenon-binding cryptophane cage, a target interaction element, and a metal chelating agent. Xenon associated with the target-bound cryptophane cage is rapidly relaxed and then detected after exchange with the bulk. Here we show that large macromolecular targets increase the rotational correlation time of xenon, increasing its relaxation rate. Upon binding of a biotin-containing sensor to avidin at 1.5 µM concentration, the free xenon T2 is reduced by a factor of 4.

Hyperpolarized xenon-based molecular sensors for label-free detection of analytes.

Nuclear magnetic resonance (NMR) can reveal the chemical constituents of a complex mixture without resorting to chemical modification, separation, or other perturbation. Recently, we and others have developed magnetic resonance agents that report on the presence of dilute analytes by proportionately altering the response of a more abundant or easily detected species, a form of amplification. One example of such a sensing medium is xenon gas, which is chemically inert and can be optically hyperpolarized, a process that enhances its NMR signal by up to 5 orders of magnitude. Here, we use a combinatorial synthetic approach to produce xenon magnetic resonance sensors that respond to small molecule analytes. The sensor responds to the ligand by producing a small chemical shift change in the Xe NMR spectrum. We demonstrate this technique for the dye, Rhodamine 6G, for which we have an independent optical assay to verify binding. We thus demonstrate that specific binding of a small molecule can produce a xenon chemical shift change, suggesting a general approach to the production of xenon sensors targeted to small molecule analytes for in vitro assays or molecular imaging in vivo.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1.

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI:http://dx.doi.org/10.7554/eLife.00813.001.

Heterogeneous seeding of a prion structure by a generic amyloid form of the fungal prion-forming domain HET-s(218-289).

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung ß-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked ß-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked ß-sheet fibrils nucleate the formation of the infectious ß-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious ß-solenoid fibrils) fibrillization. Several serial passages of stacked ß-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

Structure, function, and tethering of DNA-binding domains in σ54 transcriptional activators.

We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.

Conformational coupling across the plasma membrane in activation of the EGF receptor.

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Architecture and membrane interactions of the EGF receptor.

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR's intracellular module with the membrane are critical in maintaining this coupling.

Solution-state 2D NMR spectroscopy of plant cell walls enabled by a dimethylsulfoxide-d6/1-ethyl-3-methylimidazolium acetate solvent.

Lignocellulosic biomass is composed of the polysaccharides cellulose and hemicellulose and the polyphenol lignin. Many current methods for analyzing the structure of lignocelluloses involve a sequential extraction of the material and subsequent analysis of the resulting fractions, which is labor-intensive and time-consuming. The work presented here assesses the dissolution of whole lignocellulosic material, focusing on biomass derived from the perennial bioenergy grass Miscanthus. The solvent dimethylsulfoxide (DMSO)-d6 containing 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) was able to dissolve lignocellulosic material completely and gave high-resolution 2D heteronuclear single quantum coherence (HSQC) NMR spectra of the entire array of wall polymers. Extrapolated time-zero HSQC was applied using DMSO-d6/[Emim]OAc-d14 and enabled quantitative analysis of structural traits of lignocellulose components.

Structural mechanism of GAF-regulated σ(54) activators from Aquifex aeolicus.

The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ(54) family of σ subunits require the action of a 'transcriptional activator' protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ(54) directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ(54) activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ(54) activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ(54) activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ(54) activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states-an 'open', ATPase-inactive state; a 'closed', ATPase-inactive state; and a 'closed', ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ(54) activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ(54) activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.

A sterile alpha-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain.

NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.

Facile dimer synthesis for DNA-binding polyamide ligands.

Pyrrole-imidazole polyamide ligands are highly sequence specific synthetic DNA-binding ligands that bind with high affinity. To counter the synthetic difficulties associated with coupling the electron-rich heterocyclic acids to the electron-deficient nucleophilic imidazole amine, a novel approach is described for synthesis of Fmoc-protected dimers for solid-phase peptide synthesis (SPPS). This method produces the dimers in high yields, is broadly applicable to other heterocyclic-containing polyamides, and results in improved ligand yields and synthesis times.

Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.

ATP ground- and transition states of bacterial enhancer binding AAA+ ATPases support complex formation with their target protein, sigma54.

Transcription initiation by the sigma54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeF(x) (ground-state mimics), ADP-AlF(x) (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeF(x) state, and from the ADP-AlF(x) state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma54, with ADP-AlF(x) having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.