Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus.

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Cryo-EM structure of the diapause chaperone artemin.

The protein artemin acts as both an RNA and protein chaperone and constitutes over 10% of all protein in Artemia cysts during diapause. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 Å resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage but in a different configuration than previously hypothesized with molecular modeling. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition.

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

De novo sequencing and native mass spectrometry revealed hetero-association of dirigent protein homologs and potential interacting proteins in Forsythia × intermedia.

The discovery of dirigent proteins (DPs) and their functions in plant phenol biochemistry was made over two decades ago with Forsythia × intermedia. Stereo-selective, DP-guided, monolignol-derived radical coupling in vitro was then reported to afford the optically active lignan, (+)-pinoresinol from coniferyl alcohol, provided one-electron oxidase/oxidant capacity was present. It later became evident that DPs have several distinct sub-families, presumably with different functions. Some known DPs require other essential enzymes/proteins (e.g. oxidases) for their functions. However, the lack of a fully sequenced genome for Forsythia × intermedia made it difficult to profile other components co-purified with the (+)-pinoresinol forming DP. Herein, we used an integrated bottom-up, top-down, and native mass spectrometry (MS) approach to de novo sequence the extracted proteins via adaptation of our initial report of DP solubilization and purification. Using publicly available transcriptome and genomic data from closely related species, we identified 14 proteins that were putatively associated with either DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for potential protein-protein interactions, none were found to form stable protein complexes with DPs in native MS under the specific experimental conditions we have explored. Interestingly, two new DP homologs were found and they formed hetero-trimers. Molecular dynamics simulations suggested that similar hetero-trimers were possible between Arabidopsis DP homologs with comparable sequence similarities. Nevertheless, our integrated mass spectrometry method development helped prepare for future investigations directed to the discovery of novel proteins and protein-protein interactions. These advantages can be highly beneficial for plant and microbial research where fully sequenced genomes may not be readily available.

Tunable Heteroassembly of a Plant Pseudoenzyme-Enzyme Complex.

Pseudoenzymes have emerged as key regulatory elements in all kingdoms of life despite being catalytically nonactive. Yet many factors defining why one protein is active while its homologue is inactive remain uncertain. For pseudoenzyme-enzyme pairs, the similarity of both subunits can often hinder conventional characterization approaches. In plants, a pseudoenzyme, PDX1.2, positively regulates vitamin B6 production by association with its active catalytic homologues such as PDX1.3 through an unknown assembly mechanism. Here we used an integrative experimental approach to learn that such pseudoenzyme-enzyme pair associations result in heterocomplexes of variable stoichiometry, which are unexpectedly tunable. We also present the atomic structure of the PDX1.2 pseudoenzyme as well as the population averaged PDX1.2-PDX1.3 pseudoenzyme-enzyme pair. Finally, we dissected hetero-dodecamers of each stoichiometry to understand the arrangement of monomers in the heterocomplexes and identified symmetry-imposed preferences in PDX1.2-PDX1.3 interactions. Our results provide a new model of pseudoenzyme-enzyme interactions and their native heterogeneity.

In Liquid Infrared Scattering Scanning Near-Field Optical Microscopy for Chemical and Biological Nanoimaging.

Imaging biological systems with simultaneous intrinsic chemical specificity and nanometer spatial resolution in their typical native liquid environment has remained a long-standing challenge. Here, we demonstrate a general approach of chemical nanoimaging in liquid based on infrared scattering scanning near-field optical microscopy (IR s-SNOM). It is enabled by combining AFM operation in a fluid cell with evanescent IR illumination via total internal reflection, which provides spatially confined excitation for minimized IR water absorption, reduced far-field background, and enhanced directional signal emission and sensitivity. We demonstrate in-liquid IR s-SNOM vibrational nanoimaging and conformational identification of catalase nanocrystals and spatio-spectral analysis of biomimetic peptoid sheets with monolayer sensitivity and chemical specificity at the few zeptomole level. This work establishes the principles of in-liquid and in situ IR s-SNOM spectroscopic chemical nanoimaging and its general applicability to biomolecular, cellular, catalytic, electrochemical, or other interfaces and nanosystems in liquids or solutions.

Tip-Enhanced Raman Nanospectroscopy of Smooth Spherical Gold Nanoparticles.

We record nanoscale-resolved chemical images of thiobenzonitrile (TBN)-functionalized smooth gold nanospheres on silicon via tip-enhanced Raman (TER) nanospectroscopy. The recorded images trace the nascence of the familiar doughnut-shaped scattering profile of nanoparticles on silicon at its origin (the particle surface), which appears as a horseshoe-shaped scattering pattern under our experimental conditions. The local optical field maps are in agreement with their simulated finite-difference time-domain analogues. Analysis of the recorded spectra with the aid of ab-initio-molecular-dynamics-based Raman spectral simulations further suggests that optical rectification and molecular charging take place throughout the course of atomic-force-microscopy-based TER nanoscale chemical imaging.

Protein structural biology using cell-free platform from wheat germ.

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.

Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp.

The atomic structure of the infectious, protease-resistant, ß-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the ß2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.

Identification and Characterization of a Class of MALAT1-like Genomic Loci.

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript (MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant long noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Thus, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures.

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.

A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage.

Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this âˆ¼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.

Enhanced Raman scattering from aromatic dithiols electrosprayed into plasmonic nanojunctions.

We describe surface enhanced Raman spectroscopy (SERS) experiments in which molecular coverage is systematically varied from 3.8 × 10(5) to 3.8 × 10(2) to 0.38 molecules per µm(2) using electrospray deposition of ethanolic 4,4'-dimercaptostilbene (DMS) solutions. The plasmonic SERS substrate used herein consists of a well-characterized 2-dimensional (2D) array of silver nanospheres (see El-Khoury et al., J. Chem. Phys., 2014, 141, 214308), previously shown to feature uniform topography and plasmonic response, as well as intense SERS activity. When compared to their ensemble averaged analogues, the spatially and temporally averaged spectra of a single molecule exhibit several unique features including: (i) distinct relative intensities of the observable Raman-active vibrational states, (ii) more pronounced SERS backgrounds, and (iii) broader Raman lines indicative of faster vibrational dephasing. The first observation may be understood on the basis of an intuitive physical picture in which the removal of averaging over multiple molecules exposes the tensorial nature of Raman scattering. When an oriented single molecule gives rise to the recorded SERS spectra, the relative orientation of the molecule with respect to vector components of the local electric field determines the relative intensities of the observable vibrational states. Using a single molecule SERS framework, described herein, we derive a unique molecular orientation in which a single DMS molecule is isolated at a nanojunction formed between two silver nanospheres in the 2D array. The DMS molecule is found lying nearly flat with respect to the metal. The derived orientation of a single molecule at a plasmonic nanojunction is consistent with observations (ii) and (iii). In particular, a careful inspection of the temporal spectral variations along the recorded single molecule SERS time sequences reveals that the time-averaged SERS backgrounds arise from individual molecular events, marked by broadened SERS signatures. We assign the broadened spectra along the SERS time sequence--which sum up to a SERS background in the averaged spectra--to instances in which the π-framework of the DMS molecule is parallel to the metal at a classical plasmonic nanojunction. This also accounts for Raman line broadening as a result of fast vibrational dephasing, and driven by molecular reorientation at a plasmonic nanojunction. Furthermore, we report on the molecular orientation dependence of single molecule SERS enhancement factors. We find that in the case of a single DMS molecule isolated at a plasmonic nanojunction, molecular orientation may affect the derived single molecule SERS enhancement factor by up to 5 orders of magnitude. Taking both chemical effects as well as molecular orientation into account, we were able to estimate a single molecule enhancement factor of ∼10(10) in our measurements.

Tackling structures of long noncoding RNAs.

RNAs are important catalytic machines and regulators at every level of gene expression. A new class of RNAs has emerged called long non-coding RNAs, providing new insights into evolution, development and disease. Long non-coding RNAs (lncRNAs) predominantly found in higher eukaryotes, have been implicated in the regulation of transcription factors, chromatin-remodeling, hormone receptors and many other processes. The structural versatility of RNA allows it to perform various functions, ranging from precise protein recognition to catalysis and metabolite sensing. While major housekeeping RNA molecules have long been the focus of structural studies, lncRNAs remain the least characterized class, both structurally and functionally. Here, we review common methodologies used to tackle RNA structure, emphasizing their potential application to lncRNAs. When considering the complexity of lncRNAs and lack of knowledge of their structure, chemical probing appears to be an indispensable tool, with few restrictions in terms of size, quantity and heterogeneity of the RNA molecule. Probing is not constrained to in vitro analysis and can be adapted to high-throughput sequencing platforms. Significant efforts have been applied to develop new in vivo chemical probing reagents, new library construction protocols for sequencing platforms and improved RNA prediction software based on the experimental evidence.

3S: shotgun secondary structure determination of long non-coding RNAs.

Long non-coding RNAs (lncRNAs) have emerged as an important class of RNAs playing key roles in development, disease and epigenetics. Knowledge of lncRNA structure may be critical in understanding function for many lncRNA systems. Due to the enormous number of possible folds for these sequences, secondary structure determination presents a significant challenge, both experimentally and computationally. Here, we present a new strategy capable of determining the RNA secondary structure in the wet lab without significant reliance on computational predictions. First, we chemically probe the entire lncRNA. Next, using a shotgun approach, we divide the RNA into overlapping fragments and probe these fragments. We then compare probing profiles of fragments with the profiles of the full RNA and identify similarities. Sequence regions with profiles that are similar in the fragment and full-length transcript possess only base pairing partners within the fragment. Thus, by experimentally folding smaller and smaller fragments of the full RNA and probing these chemically, we are able to isolate modular sub-domains, dramatically reducing the number of possible folds. The method also eliminates the possibility of pseudoknots within a modular sub-domain. The 3S technique is ideally suited for lncRNAs because it is designed for long RNA sequences. The 3S-determined secondary structure of a specific lncRNA in one species (e.g., human) enables searches for instances of the same lncRNA in other species.

Rise of the RNA machines: exploring the structure of long non-coding RNAs.

Novel, profound and unexpected roles of long non-coding RNAs (lncRNAs) are emerging in critical aspects of gene regulation. Thousands of lncRNAs have been recently discovered in a wide range of mammalian systems, related to development, epigenetics, cancer, brain function and hereditary disease. The structural biology of these lncRNAs presents a brave new RNA world, which may contain a diverse zoo of new architectures and mechanisms. While structural studies of lncRNAs are in their infancy, we describe existing structural data for lncRNAs, as well as crystallographic studies of other RNA machines and their implications for lncRNAs. We also discuss the importance of dynamics in RNA machine mechanism. Determining commonalities between lncRNA systems will help elucidate the evolution and mechanistic role of lncRNAs in disease, creating a structural framework necessary to pursue lncRNA-based therapeutics.

The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch.

Riboswitch operation involves the complex interplay between the aptamer domain and the expression platform. During transcription, these two domains compete against each other for shared sequence. In this study, we explore the cooperative effects of ligand binding and Magnesium interactions in the SAM-I riboswitch in the context of aptamer collapse and anti-terminator formation. Overall, our studies show the apo-aptamer acts as (i) a pre-organized aptamer competent to bind ligand and undergo structural collapse and (ii) a conformation that is more accessible to anti-terminator formation. We show that both Mg(2+) ions and SAM are required for a collapse transition to occur. We then use competition between the aptamer and expression platform for shared sequence to characterize the stability of the collapsed aptamer. We find that SAM and Mg(2+) interactions in the aptamer are highly cooperative in maintaining switch polarity (i.e. aptamer 'off-state' versus anti-terminator 'on-state'). We further show that the aptamer off-state is preferentially stabilized by Mg(2+) and similar divalent ions. Furthermore, the functional switching assay was used to select for phosphorothioate interference, and identifies potential magnesium chelation sites while characterizing their coordinated role with SAM in aptamer stabilization. In addition, we find that Mg(2+) interactions with the apo-aptamer are required for the full formation of the anti-terminator structure, and that higher concentrations of Mg(2+) (>4 mM) shift the equilibrium toward the anti-terminator on-state even in the presence of SAM.

Sizing up long non-coding RNAs: do lncRNAs have secondary and tertiary structure?

Long noncoding RNAs (lncRNAs) play a key role in many important areas of epigenetics, stem cell biology, cancer, signaling and brain function. This emerging class of RNAs constitutes a large fraction of the transcriptome, with thousands of new lncRNAs reported each year. The molecular mechanisms of these RNAs are not well understood. Currently, very little structural data exist. We review the available lncRNA sequence and secondary structure data. Since almost no tertiary information is available for lncRNAs, we review crystallographic structures for other RNA systems and discuss the possibilities for lncRNAs in the context of existing constraints.

Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

Engineering cooperative tecto-RNA complexes having programmable stoichiometries.

High affinity and specificity RNA-RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop-receptor interaction motifs. These interactions can be fused using flexible four-way junction motifs to create divalent, self-assembling scaffolding units ('tecto-RNA') that have favorable properties for nanomedicine and other applications. We describe the design and directed assembly of tecto-RNA units ranging from closed, cooperatively assembling ring-shaped complexes of programmable stoichiometries (dimers, trimers and tetramers) to open multimeric structures. The novelty of this work is that tuning of the stoichiometries of self-assembled complexes is achieved by precise positioning of the interaction motifs in the monomer units rather than changing their binding specificities. Structure-probing and transmission electron microscopy studies as well as thermodynamic analysis support formation of closed cooperative complexes that are highly resistant to nuclease digestion. The present designs provide two helical arms per RNA monomer for further functionalization aims.