Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

The TRiC/CCT Chaperonin and Its Role in Uncontrolled Proliferation.

The cell cycle is a sophisticated space-time regulated mechanism where a wide variety of protein modules and complexes associate functioning in a concerted manner to regulate and transfer the genetic material to daughter cells. CCT (chaperonin containing TCP-1, also known as TRiC) is a molecular machine that forms a high molecular weight complex (1000 KDa). CCT is emerging as a key molecule during mitosis due to its essential role in the folding of many important proteins involved in cell division (Cdh1, Plk1, p27, Cdc20, PP2a regulatory subunits, tubulin or actin) suggesting its involvement in uncontrolled proliferation. The assembly is formed by eight different subunits called CCTα, ß, γ, δ, ε, ζ, η and θ in mammals corresponding to CCT1-8 in yeast. CCT/TRiC is organized in a unique intra- and inter-ring arrangement. The chaperonin monomers share a common domain structure including an equatorial domain, which contains all the inter-ring contacts, most of the intra-ring contacts and the ATP binding site, whose binding and hydrolysis triggers the conformational changes that take place during the functional cycle. All chaperonins display an open substrate-receptive conformation, where the unfolded protein is recognized and trapped, and a closed conformation where the substrate is isolated from the bulk of the intracellular environment. In this chapter we discuss the complex set of intra- and inter-ring allosteric signals during chaperonin function.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.

Deep into the Aristolochia Flower: Expression of C, D, and E-Class Genes in Aristolochia fimbriata (Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.

Chaperonins: two rings for folding.

Chaperonins are ubiquitous chaperones found in Eubacteria, eukaryotic organelles (group I), Archaea and the eukaryotic cytosol (group II). They all share a common structure and a basic functional mechanism. Although a large amount of information has been gathered for the simpler group I, much less is known about group II chaperonins. Recent crystallographic and electron microscopy structures have provided new insights into the mechanism of these chaperonins and revealed important differences between group I and II chaperonins, mainly in the molecular rearrangements that take place during the functional cycle. These differences are evident for the most complex chaperonin, the eukaryotic cytosolic CCT, which highlights the uniqueness of this important molecular machine.

Molecular architecture of a multifunctional MCM complex.

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.

A Modified Schirmer Tear Test in Clinically Normal Horses: Measurement at 30 Seconds versus 60 Seconds.

This study sought to compare the Schirmer Tear Test (STT)-1 results at 30 (STT30) versus 60 (STT60) seconds in healthy horses. This study included a total of 56 healthy horses. STT-1 was performed in both eyes, right eye first, and the wetting lengths were measured in STT30 and STT60. To evaluate the reduction of the initial reflex phase, the wetting length velocity was measured during the first 30 seconds. The effects of eye, age, weight, sex, and ambient temperature and humidity on STT values were evaluated. Mean (standard deviation) STT30 and STT60 were 19.06 (3.88) and 24.26 (4.50) mm. There was a linear correlation between the STT 30 and STT60, expressed according to the following equation: STT60 = 2.20 + 1.18 × STT30 (P = .001). STT30 or STT60 values did not vary between the sexes or correlate with age, weight, ambient temperature, or humidity. In conclusion, STT30 allows for an accurate, reliable, and applicable diagnosis of tear production compared with the standard STT60 value. The proposed method is shorter and may be a suitable alternative to the 1-min test.

Expression of gynoecium patterning transcription factors in Aristolochia fimbriata (Aristolochiaceae) and their contribution to gynostemium development.

BACKGROUND: In Aristolochia (Aristolochiaceae) flowers, the congenital fusion of the anthers and the commissural, stigmatic lobes forms a gynostemium. Although the molecular bases associated to the apical-basal gynoecium patterning have been described in eudicots, comparative expression studies of the style and stigma regulatory genes have never been performed in early divergent angiosperms possessing a gynostemium. RESULTS: In this study, we assess the expression of five genes typically involved in gynoecium development in Aristolochia fimbriata. We found that all five genes (AfimCRC, AfimSPT, AfimNGA, AfimHEC1 and AfimHEC3) are expressed in the ovary, the placenta, the ovules and the transmitting tract. In addition, only AfimHEC3, AfimNGA and AfimSPT are temporarily expressed during the initiation of the stigma, while none of the genes studied is maintained during the elaboration of the stigmatic surfaces in the gynostemium. CONCLUSIONS: Expression patterns suggest that CRC, HEC, NGA and SPT homologs establish ovary and style identity in Aristolochia fimbriata. Only NGA,HEC3 and SPT genes may play a role in the early differentiation of the stigmatic lobes, but none of the genes studied seems to control late stigma differentiation in the gynostemium. The data gathered so far raises the possibility that such transient expression early on provides sufficient signal for late stigma differentiation or that unidentified late identity genes are controlling stigma development in the gynostemium. Our data does not rule out the possibility that stigmas could correspond to staminal filaments with convergent pollen-receptive surfaces.

High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage.

Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells.

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.

Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the n-terminal domain unwinds DNA bidirectionally.

Bacillus subtilis bacteriophage SPP1 G40P hexameric replicative DNA helicase unidirectionally translocates with a 5'-->3' polarity while separating the DNA strands. A G40P mutant derivative lacking the N-terminal domain (containing amino acid residues 110-442 from G40P, G40PDeltaN109) was purified and characterized. G40PDeltaN109 showed an ATPase activity that was dependent on the presence of single-stranded (ss) DNA. Unlike G40P, G40PDeltaN109 was shown to bind with similar affinity both ssDNA arms of forked structures by nuclease protection assays. In a pH-dependent manner, G40PDeltaN109 unwound a branched double-arm substrate preferentially with a 3'-->5' polarity. Our results show that the linker region and the C-terminal domain of G40P are sufficient to render an enzyme capable of encircling the ssDNA tails of the forked DNA and to unwind DNA with both 5'-->3' and 3'-->5' polarity. The presence of the N-terminal domain, which does not play an essential role in helicase action, might be required indirectly for strand discrimination and polarity of translocation.

Quaternary polymorphism of replicative helicase G40P: structural mapping and domain rearrangement.

Quaternary polymorphism is a distinctive structural feature of the DnaB family of replicative DNA hexameric helicases. The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P) belongs to this family. Three different quaternary states have been described for G40P homohexamers, two of them with C(3) symmetry, and the other with C(6) symmetry. We present three-dimensional reconstructions of the different architectures of G40P hexamers and a variant lacking the N-terminal domain. Comparison of the G40P and the deletion mutant structures sheds new light on the functional roles of the N and C-terminal domains, at the same time that it allows the direct structural mapping of these domains. Based on this new information, hybrid EM/X-ray models are presented for all the different symmetries. These results suggest that quaternary polymorphism of hexameric helicases may be implicated in the translocation along the DNA.

Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P.

Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the tau subunit of both DNA polymerases III (Pol IIIs). The B.subtilis full-length protein and their mutant derivatives tau(373- 563) (lacking the N-terminal, domains I-III or amino acid residues 1-372) and tau(1-372) (lacking the C-terminal region or amino acids 373-563) have been purified. The tau protein forms tetramers, tau(373- 563) forms dimers, whereas tau(1-372), depending on the ionic strength, forms trimers or tetramers in solution. In the absence of single-stranded (ss) DNA and a nucleotide cofactor, tau interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry. G40P(109-442), lacking the N-terminal amino acid residues 1-108, interacts with the C-terminal moiety of tau. The data indicate that the interaction of G40P with the tau subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.

Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase, G40P, interacts with forked DNA.

SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5' tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3' tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5' tail, interacts with the duplex DNA at the ss-double-stranded DNA junction and excludes the 3' tail of the forked DNA.

Characterization of the NTPR and BD1 interacting domains of the human PICH-BEND3 complex.

Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH-BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58 Šand with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 Šresolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source.

Directly from the source: endogenous preparations of molecular machines.

Purification from a source enriched in large macromolecular machines with basic cellular function is still the method of choice in many cases. Such complexes occur in sufficiently high copy numbers in the cell and can be isolated using classical protein purification protocols. Although advanced DNA recombinant technologies and sophisticated overexpression strategies are available, many complexes like the ribosome, RNA polymerase II and membrane protein complexes involved in photosynthesis or in oxidative phosphorylation can only be purified from a rich source. Here, we review recent accomplishments and limitations in applying this strategy.