Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase.

Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

SAR optimization studies on modified salicylamides as a potential treatment for acute myeloid leukemia through inhibition of the CREB pathway.

Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

Long-Term Land Use Affects Phosphorus Speciation and the Composition of Phosphorus Cycling Genes in Agricultural Soils.

Agriculturally-driven land transformation is increasing globally. Improving phosphorus (P) use efficiency to sustain optimum productivity in diverse ecosystems, based on knowledge of soil P dynamics, is also globally important in light of potential shortages of rock phosphate to manufacture P fertilizer. We investigated P chemical speciation and P cycling with solution 31P nuclear magnetic resonance, P K-edge X-ray absorption near-edge structure spectroscopy, phosphatase activity assays, and shotgun metagenomics in soil samples from long-term agricultural fields containing four different land-use types (native and tame grasslands, annual croplands, and roadside ditches). Across these land use types, native and tame grasslands showed high accumulation of organic P, principally orthophosphate monoesters, and high acid phosphomonoesterase activity but the lowest abundance of P cycling genes. The proportion of inositol hexaphosphates (IHP), especially the neo-IHP stereoisomer that likely originates from microbes rather than plants, was significantly increased in native grasslands than croplands. Annual croplands had the largest variances of soil P composition, and the highest potential capacity for P cycling processes based on the abundance of genes coding for P cycling processes. In contrast, roadside soils had the highest soil Olsen-P concentrations, lowest organic P, and highest tricalcium phosphate concentrations, which were likely facilitated by the neutral pH and high exchangeable Ca of these soils. Redundancy analysis demonstrated that IHP by NMR, potential phosphatase activity, Olsen-P, and pH were important P chemistry predictors of the P cycling bacterial community and functional gene composition. Combining chemical and metagenomics results provides important insights into soil P processes and dynamics in different land-use ecosystems.

Characterizing the phosphorus forms extracted from soil by the Mehlich III soil test.

Phosphorus (P) can limit crop production in many soils, and soil testing is used to guide fertilizer recommendations. The Mehlich III (M3) soil test is widely used in North America, followed by colorimetric analysis for P, or by inductively coupled plasma-based spectrometry (ICP) for P and cations. However, differences have been observed in M3 P concentrations measured by these methods. Using 31P nuclear magnetic resonance (P-NMR) and mass spectrometry (MS), we characterized P forms in M3 extracts. In addition to the orthophosphate that would be detected during colorimetric analysis, several organic P forms were present in M3 extracts that would be unreactive colorimetrically but measured by ICP (molybdate unreactive P, MUP). Extraction of these P forms by M3 was confirmed by P-NMR and MS in NaOH-ethylenediaminetetraacetic acid extracts of whole soils and residues after M3 extraction. The most abundant P form in M3 extracts was myo-inositol hexaphosphate (myo-IHP, phytate), a compound that may not contribute to plant-available P if tightly sorbed in soil. Concentrations of myo-IHP and other organic P forms varied among soils, and even among treatment plots on the same soil. Extraction of myo-IHP in M3 appeared to be linked to cations, with substantially more myo-IHP extracted from soils fertilized with alum-treated poultry litter than untreated litter. These results suggest that ICP analysis may substantially over-estimate plant-available P in samples with high MUP concentrations, but there is no way at present to determine MUP concentrations without analysis by both colorimetry and ICP. This study also tested procedures that will improve future soil P-NMR studies, such as treatment of acid extracts, and demonstrated that techniques such as P-NMR and MS are complimentary, each yielding additional information that analysis by a single technique may not provide.

Long-term Changes in Grassland Soil Phosphorus with Fertilizer Application and Withdrawal.

Long-term phosphorus (P) applications can increase soil P concentrations in excess of agronomic optima, posing a risk to water quality. Once fertilization stops, however, it may take time for soil P concentrations to decline. Whereas P fertilization adds orthophosphate, little is known about changes in other soil P forms during P buildup and drawdown. This study examined changes in P pools (total P, Olsen P, Mehlich P, and water-extractable P) and P forms determined by P-nuclear magnetic resonance spectroscopy (P-NMR) in grazed grassland plots from Northern Ireland. Between 1994 and 1999, all plots received 8.3 kg P ha yr with variable rates of nitrogen (100-500 kg N ha yr). From 2000 to 2005, plots received 0, 20, 40, or 80 kg P ha yr and 250 kg N ha yr; from 2005 to 2010, no P fertilizer was applied to any plots. In 2005, soil P pool concentrations at the highest P fertilization rates were significantly elevated compared with those in 2000 but had decreased to 2000 concentrations by 2010. In soils receiving no P, soil P pool concentrations were significantly lower than those in 1994 only in 2010. There were few changes in P forms determined by P-NMR. Orthophosphate followed the same trend observed for the soil P pools; total organic P, total inositol phosphates, and total orthophosphate monoesters and diesters were highest in 2010 in the soil receiving no P fertilizer for 10 yr. For these soils, fertilizer application and cessation influenced inorganic P more than organic P.

The separate effects of lipids and proteins on brain MRI contrast revealed through tissue clearing.

Despite the widespread use of magnetic resonance imaging (MRI) of the brain, the relative contribution of different biological components (e.g. lipids and proteins) to structural MRI contrasts (e.g., T1, T2, T2*, proton density, diffusion) remains incompletely understood. This limitation can undermine the interpretation of clinical MRI and hinder the development of new contrast mechanisms. Here, we determine the respective contribution of lipids and proteins to MRI contrast by removing lipids and preserving proteins in mouse brains using CLARITY. We monitor the temporal dynamics of tissue clearance via NMR spectroscopy, protein assays and optical emission spectroscopy. MRI of cleared brain tissue showed: 1) minimal contrast on standard MRI sequences; 2) increased relaxation times; and 3) diffusion rates close to free water. We conclude that lipids, present in myelin and membranes, are a dominant source of MRI contrast in brain tissue.

Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis.

Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode "orphan" assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a "split, stuttering" module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.

Self-assembly and sequence length dependence on nanofibrils of polyglutamine peptides.

Huntington's disease (HD) is recognized as a currently incurable, inherited neurodegenerative disorder caused by the accumulation of misfolded polyglutamine (polyQ) peptide aggregates in neuronal cells. Yet, the mechanism by which newly formed polyQ chains interact and assemble into toxic oligomeric structures remains a critical, unresolved issue. In order to shed further light on the matter, our group elected to investigate the folding of polyQ peptides - examining glutamine repeat lengths ranging from 3 to 44 residues. To characterize these aggregates we employed a diverse array of technologies, including: nuclear magnetic resonance; circular dichroism; Fourier transform infrared spectroscopy; fluorescence resonance energy transfer (FRET), and atomic force microscopy. The data we obtained suggest that an increase in the number of glutamine repeats above 14 residues results in disordered loop structures, with different repeat lengths demonstrating unique folding characteristics. This differential folding manifests in the formation of distinct nano-sized fibrils, and on this basis, we postulate the idea of 14 polyQ repeats representing a critical loop length for neurotoxicity - a property that we hope may prove amenable to future therapeutic intervention. Furthermore, FRET measurements on aged assemblages indicate an increase in the end-to-end distance of the peptide with time, most probably due to the intermixing of individual peptide strands within the nanofibril. Further insight into this apparent time-dependent reorganization of aggregated polyQ peptides may influence future disease modeling of polyQ-related proteinopathies, in addition to directing novel clinical innovations.

Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.

CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.

Determination of Hydrogen Bond Structure in Water versus Aprotic Environments To Test the Relationship Between Length and Stability.

Hydrogen bonds profoundly influence the architecture and activity of biological macromolecules. Deep appreciation of hydrogen bond contributions to biomolecular function thus requires a detailed understanding of hydrogen bond structure and energetics and the relationship between these properties. Hydrogen bond formation energies (ΔGf) are enormously more favorable in aprotic solvents than in water, and two classes of contributing factors have been proposed to explain this energetic difference, focusing respectively on the isolated and hydrogen-bonded species: (I) water stabilizes the dissociated donor and acceptor groups much better than aprotic solvents, thereby reducing the driving force for hydrogen bond formation; and (II) water lengthens hydrogen bonds compared to aprotic environments, thereby decreasing the potential energy within the hydrogen bond. Each model has been proposed to provide a dominant contribution to ΔGf, but incisive tests that distinguish the importance of these contributions are lacking. Here we directly test the structural basis of model II. Neutron crystallography, NMR spectroscopy, and quantum mechanical calculations demonstrate that O-H···O hydrogen bonds in crystals, chloroform, acetone, and water have nearly identical lengths and very similar potential energy surfaces despite ΔGf differences >8 kcal/mol across these solvents. These results rule out a substantial contribution from solvent-dependent differences in hydrogen bond structure and potential energy after association (model II) and thus support the conclusion that differences in hydrogen bond ΔGf are predominantly determined by solvent interactions with the dissociated groups (model I). These findings advance our understanding of universal hydrogen-bonding interactions and have important implications for biology and engineering.

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1.

CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).

The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

The dynamic process of ß(2)-adrenergic receptor activation.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.

Complementary Phosphorus Speciation in Agricultural Soils by Sequential Fractionation, Solution P Nuclear Magnetic Resonance, and Phosphorus K-edge X-ray Absorption Near-Edge Structure Spectroscopy.

Ultisols in China need phosphorus (P) fertilization to sustain crop production but are prone to P loss in runoff. Balancing P inputs and loss requires detailed information about soil P forms because P speciation influences P cycling. Analytical methods vary in the information they provide on P speciation; thus, we used sequential fractionation (SF), solution P nuclear magnetic resonance (P-NMR), and P K-edge X-ray absorption near-edge structure (XANES) spectroscopy to investigate organic P (P) and inorganic P (P) species in Chinese Ultisols managed for different crops and with different fertilizer inputs in the first study to combine these techniques to characterize soil P. Sequential fractionation showed that moderately labile NaOH-P was the largest P pool in these soils, P varied from 20 to 47%, and residual P ranged from 9 to 31%. Deoxyribonucleic acid (1-5%) and -inositol hexakisphosphate (-IHP, 4-10%) were the major P forms from P-NMR. Orthophosphate diesters determined by NMR were significantly correlated with labile NaHCO-P in SF ( > 0.981; < 0.001). Soil P was shown to be predominantly associated with iron and soluble calcium (Ca) by XANES. Furthermore, XANES identified hydroxyapatite in the soil receiving the highest rates of Ca-phosphate fertilizer, which had the highest HCl-P pool by SF, and also identified IHP (7%) in the soil with the highest proportion of -IHP from P-NMR. These results strongly suggest that a combined use of SF, solution P-NMR, and P K-edge XANES spectroscopy will provide the comprehensive information about soil P species needed for effective soil P management.

Small-molecule displacement of a cryptic degron causes conditional protein degradation.

The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

Ligand-switchable substrates for a ubiquitin-proteasome system.

Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.

Rationally designed turn promoting mutation in the amyloid-ß peptide sequence stabilizes oligomers in solution.

Enhanced production of a 42-residue beta amyloid peptide (Aß(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aß(42) peptide (Aß(42)Nle35p37) that appear to organize Aß(42) into stable oligomers. 2D NMR on the Aß(42)Nle35p37 peptide revealed the occurrence of two ß-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aß(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase.

The assembly-line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6-deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein-protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF3-S-ACP, was developed as a ¹9F NMR spectroscopic probe of protein-protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.

Phosphorus forms and chemistry in the soil profile under long-term conservation tillage: a phosphorus-31 nuclear magnetic resonance study.

In many regions, conservation tillage has replaced conventional tilling practices to reduce soil erosion, improve water conservation, and increase soil organic matter. However, tillage can have marked effects on soil properties, specifically nutrient redistribution or stratification in the soil profile. The objective of this research was to examine soil phosphorus (P) forms and concentrations in a long-term study comparing conservation tillage (direct drilling, "No Till") and conventional tillage (moldboard plowing to 20 cm depth, "Till") established on a fine sandy loam (Orthic Humo-Ferric Podzol) in Prince Edward Island, Canada. No significant differences in total carbon (C), total nitrogen (N), total P, or total organic P concentrations were detected between the tillage systems at any depth in the 0- to 60-cm depth range analyzed. However, analysis with phosphorus-31 nuclear magnetic resonance spectroscopy showed differences in P forms in the plow layer. In particular, the concentration of orthophosphate was significantly higher under No Till than Till at 5 to 10 cm, but the reverse was true at 10 to 20 cm. Mehlich 3-extractable P was also significantly higher in No Till at 5 to 10 cm and significantly higher in Till at 20 to 30 cm. This P stratification appears to be caused by a lack of mixing of applied fertilizer in No Till because the same trends were observed for pH and Mehlich 3-extractable Ca (significantly higher in the Till treatment at 20 to 30 cm), reflecting mixing of applied lime. The P saturation ratio was significantly higher under No Till at 0 to 5 cm and exceeded the recommended limits, suggesting that P stratification under No Till had increased the potential for P loss in runoff from these sites.