Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles.

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologues. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTC's m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent, and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

Three-layered control of mRNA poly(A) tail synthesis in Saccharomyces cerevisiae.

Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.

Down-regulation of Fra a 1.02 in strawberry fruits causes transcriptomic and metabolic changes compatible with an altered defense response.

The strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits.

The NAC transcription factor FaRIF controls fruit ripening in strawberry.

In contrast to climacteric fruits such as tomato, the knowledge on key regulatory genes controlling the ripening of strawberry, a nonclimacteric fruit, is still limited. NAC transcription factors (TFs) mediate different developmental processes in plants. Here, we identified and characterized Ripening Inducing Factor (FaRIF), a NAC TF that is highly expressed and induced in strawberry receptacles during ripening. Functional analyses based on stable transgenic lines aimed at silencing FaRIF by RNA interference, either from a constitutive promoter or the ripe receptacle-specific EXP2 promoter, as well as overexpression lines showed that FaRIF controls critical ripening-related processes such as fruit softening and pigment and sugar accumulation. Physiological, metabolome, and transcriptome analyses of receptacles of FaRIF-silenced and overexpression lines point to FaRIF as a key regulator of strawberry fruit ripening from early developmental stages, controlling abscisic acid biosynthesis and signaling, cell-wall degradation, and modification, the phenylpropanoid pathway, volatiles production, and the balance of the aerobic/anaerobic metabolism. FaRIF is therefore a target to be modified/edited to control the quality of strawberry fruits.

A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2 ß2 or (α2 ß2 )2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development.

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes.

Structural Bases for the Allergenicity of Fra a 1.02 in Strawberry Fruits.

Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients. These reactions can be due to the patient's previous sensitization to the major birch pollen allergen Bet v 1, by which IgE generated in response to Bet v 1 cross-reacts with the structurally related strawberry Fra a 1 protein family. Fra a 1.02 is the most expressed paralog in ripe strawberries and is highly allergenic. To better understand the molecular mechanisms regulating this allergic response, we have determined the three-dimensional structure of Fra a 1.02 and four site-directed mutants that were designed based on their positions in potential epitopes. Fra a 1.02 and mutants conform to the START fold. We show that the cross-reactivity of all the mutant variants to IgE from patients allergic to Bet v 1 was significantly reduced without altering the conserved structural fold, so that they could potentially be used as hypoallergenic Fra a 1 variants for the generation of vaccines against strawberry allergy in atopic patients.

Current developments in Coot for macromolecular model building of Electron Cryo-microscopy and Crystallographic Data.

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo-EM model building and refinement, as cryo-EM structures with resolution ranging 2.5-4 A are now routinely available. Model building into these maps can be time-consuming and requires experience in both biochemistry and building into low-resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman-McClure restraints, full-chain refinement, and Fourier-model based residue-type-specific Ramachandran restraints. Here, we present the current state-of-the-art in Coot usage.

Methods for merging data sets in electron cryo-microscopy.

Recent developments have resulted in electron cryo-microscopy (cryo-EM) becoming a useful tool for the structure determination of biological macromolecules. For samples containing inherent flexibility, heterogeneity or preferred orientation, the collection of extensive cryo-EM data using several conditions and microscopes is often required. In such a scenario, merging cryo-EM data sets is advantageous because it allows improved three-dimensional reconstructions to be obtained. Since data sets are not always collected with the same pixel size, merging data can be challenging. Here, two methods to combine cryo-EM data are described. Both involve the calculation of a rescaling factor from independent data sets. The effects of errors in the scaling factor on the results of data merging are also estimated. The methods described here provide a guideline for cryo-EM users who wish to combine data sets from the same type of microscope and detector.

Interpretation of medium resolution cryoEM maps of multi-protein complexes.

Electron cryo-microscopy (cryoEM) is used to determine structures of biological molecules, including multi-protein complexes. Maps at better than 3.0Å resolution are relatively straightforward to interpret since atomic models of proteins and nucleic acids can be built directly. Still, these resolutions are often difficult to achieve, and map quality frequently varies within a structure. This results in data that are challenging to interpret, especially when crystal structures or suitable homology models are not available. Recent advances in mass spectrometry techniques, computational methods and model building tools facilitate subunit/domain fitting into maps, elucidation of protein contacts, and de novo generation of atomic models. Here, we review techniques for map interpretation and provide examples from recent studies of multi-protein complexes.

Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex.

Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

The APT complex is involved in non-coding RNA transcription and is distinct from CPF.

The 3'-ends of eukaryotic pre-mRNAs are processed in the nucleus by a large multiprotein complex, the cleavage and polyadenylation factor (CPF). CPF cleaves RNA, adds a poly(A) tail and signals transcription termination. CPF harbors four enzymatic activities essential for these processes, but how these are coordinated remains poorly understood. Several subunits of CPF, including two protein phosphatases, are also found in the related 'associated with Pta1' (APT) complex, but the relationship between CPF and APT is unclear. Here, we show that the APT complex is physically distinct from CPF. The 21 kDa Syc1 protein is associated only with APT, and not with CPF, and is therefore the defining subunit of APT. Using ChIP-seq, PAR-CLIP and RNA-seq, we show that Syc1/APT has distinct, but possibly overlapping, functions from those of CPF. Syc1/APT plays a more important role in sn/snoRNA production whereas CPF processes the 3'-ends of protein-coding pre-mRNAs. These results define distinct protein machineries for synthesis of mature eukaryotic protein-coding and non-coding RNAs.

Architecture of eukaryotic mRNA 3'-end processing machinery.

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3' ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four ß propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3'-end processing.

Central role of FaGAMYB in the transition of the strawberry receptacle from development to ripening.

The receptacle of the strawberry (Fragaria × ananassa) fruit accounts for the main properties of the ripe fruit for human consumption. As it ripens, it undergoes changes similar to other fruits in sugar : acid ratio, volatile production and cell wall softening. However, the main regulators of this process have not yet been reported. The white stage marks the initiation of the ripening process, and we had previously reported a peak of expression for a FaGAMYB gene. Transient silencing of FaGAMYB using RNAi and further determination of changes in global gene expression by RNAseq, and composition of primary and secondary metabolites have been used to investigate the role played by this gene during the development of the receptacle. Down-regulation of FaGAMYB caused an arrest in the ripening of the receptacle and inhibited colour formation. Consistent with this, several transcription factors associated with the regulation of flavonoid biosynthetic pathway showed altered expression. FaGAMYB silencing also caused a reduction of ABA biosynthesis and sucrose content. Interestingly, exogenous ABA application to the RNAI-transformed receptacle reversed most defects caused by FaGAMYB down-regulation. The study assigns a key regulatory role to FaGAMYB in the initiation of strawberry receptacle ripening and acting upstream of the known regulator ABA.

The strawberry pathogenesis-related 10 (PR-10) Fra a proteins control flavonoid biosynthesis by binding to metabolic intermediates.

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low µm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

Purification, crystallization and preliminary X-ray analysis of the strawberry allergens Fra a 1E and Fra a 3 in the presence of catechin.

The strawberry Fra a proteins belong to the pathogenesis-related PR-10 protein family and share a common fold with the Bet v 1 major pollen allergen and the START/PYR/PYL proteins, which are characterized by the presence of a central cavity and are often involved in the binding of a variety of natural compounds. The Fra a proteins play a key role in the control of flavonoid biosynthesis in strawberries and are essential for pigment formation in fruits. In order to understand Fra a protein function, full-length Fra a 1E and Fra a 3 cDNAs were cloned and expressed in Escherichia coli, and the proteins were purified to homogeneity using metal-affinity chromatography. Diffraction-quality crystals of Fra a 1E and of Fra a 3 in the presence of (+)-catechin were obtained by the sitting-drop vapour-diffusion method. X-ray diffraction data from single crystals of Fra a 1E and Fra a 3 were processed to 2.2 and 3.0 Šresolution in space groups P212121 and P2221, with unit-cell parameters a = 70.02, b = 74.42, c = 84.04 Šand a = 137.91, b = 206.61, c = 174.7 Šfor Fra a 1E and Fra a 3, respectively.

Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB.

• We have reported previously that the gibberellin (GA) content in strawberry receptacle is high, peaking at specific stages, pointing to a role of this hormone in fruit development. In Arabidopsis, miR159 levels are dependent on GA concentration. This prompted us to investigate the role of two members of the miR159 family and their putative strawberry target gene, GAMYB, in relation to changes in GA content during the course of fruit development. • The highest expression level of the two Fa-MIR159 genes was in the fruit's receptacle tissue, with dramatic changes observed throughout development. The lowest levels of total mature miR159 (a and b) were observed during the white stage of receptacle development, which was concurrent with the highest expression of Fa-GAMYB. A functional interaction between miR159 and Fa-GAMYB has been demonstrated in receptacle tissue. • The application of bioactive GA (i.e. GA(3) ) to strawberry plants caused the down-regulated expression of Fa-MIR159a, but the expression of Fa-MIR159b was not affected significantly. Clear discrepancies between Fa-MIR159b and mature Fa-miR159b levels were indicative of post-transcriptional regulation of Fa-MIR159b gene expression. • We propose that Fa-miR159a and Fa-miR159b interact with Fa-GAMYB during the course of strawberry receptacle development, and that they act in a cooperative fashion to respond, in part, to changes in GA endogenous levels.

Template-assisted generation of nanocavities within plasma polymer films.

The generation of nanosized cavities within thin film layers is of interest for a number of fundamental and applied reasons. One challenge is to make such systems sufficiently robust mechanically. Plasma polymer (pp) films possess excellent mechanical stability if deposition conditions are selected such as to achieve a sufficient density of cross-linking and resistance to extraction of polymeric material by solvents. In this study, gold nanoparticles of 15 and 70 nm diameter were used as sacrificial templates to generate nanocavities in pp films of various thickness values in the tens of nanometers range. A first pp layer was deposited onto substrates using n-heptylamine (HA) to a thickness of 20 nm. Carboxy-thiolated gold nanoparticles were electrostatically bound onto the surface amine groups of the n-heptylamine plasma polymer (HApp) layer. A second HApp layer was then coated to various thicknesses onto the nanoparticle/HApp surface. The template particles embedded thus in-between the two HApp layers were then dissolved using aqueous KCN solution; monitoring of the plasmon resonance band of the gold nanoparticles enabled verification of template stripping and measurement of the kinetics of stripping. AFM topography images showed little change on extraction of the template nanoparticles, indicating that the plasma polymer layer maintained structural integrity upon template extraction and subsequent drying, and thereby prevented collapse of the empty nanocavities. The concept of template stripping to generate controlled size free volume in thin plasma polymer layers is thus shown to produce robust structures.

Solvent-induced porosity in ultrathin amine plasma polymer coatings.

Plasma polymers deposited from n-heptylamine onto silicon wafers have been found to form a porous microstructure when immersed in water and other solvents, with pores of dimensions and densities that vary considerably between coatings deposited under different plasma conditions. This solvent-induced pore formation was found to correlate with the observed percentage of extractable material. With low radio frequency (rf) power inputs, the resultant softer coatings possess considerably more extractable material than coatings deposited at higher applied power levels. The porosity is thus proposed to result from the formation of voids created by the extraction of soluble low-molecular-weight polymeric material, which produces shrinkage stress that the coating, firmly attached to the substrate, cannot relieve by macroscopic contraction. The microscopic contraction of plasma polymer volume creates voids that appear to span the entire film thickness. The effect of aging plasma polymers in air was also investigated. For films deposited at low power it led to reduced extraction of soluble material and different pore morphology, whereas for films deposited at higher rf power levels, the extracted amounts and pore formation were the same for aged coatings. It was also found that the density of surface amine groups was lower for films deposited under the two lowest power settings, in contrast to the commonly held belief that the use of minimal applied rf power aids retention of functional groups. These porous plasma polymer coatings with surface groups suitable for further interfacial chemical immobilization reactions may be useful for various membrane and biotechnology applications.