Central Cleft Research Articles

The EV71 2A protease occupies the central cleft of SETD3 and disrupts SETD3-actin interaction

SETD3 is an essential host factor for the replication of a variety of enteroviruses that specifically interacts with viral protease 2A. However, the interaction between SETD3 and the 2A protease has not been fully characterized. Here, we use X-ray crystallography and cryo-electron microscopy to determine the structures of SETD3 complexed with the 2A protease of EV71 to 3.5 Å and 3.1 Å resolution, respectively. We find that the 2A protease occupies the V-shaped central cleft of SETD3 through two discrete sites. The relative positions of the two proteins vary in the crystal and cryo-EM structures, showing dynamic binding. A biolayer interferometry assay shows that the EV71 2A protease outcompetes actin for SETD3 binding. We identify key 2A residues involved in SETD3 binding and demonstrate that 2A’s ability to bind SETD3 correlates with EV71 production in cells. Coimmunoprecipitation experiments in EV71 infected and 2A expressing cells indicate that 2A interferes with the SETD3-actin complex, and the disruption of this complex reduces enterovirus replication. Together, these results reveal the molecular mechanism underlying the interplay between SETD3, actin, and viral 2A during virus replication.

Mammalian PIWI-piRNA-target complexes reveal features for broad and efficient target silencing.

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

Intrasellar Arachnoid Diverticulae as a Risk Factor for Intraoperative Cerebrospinal Fluid Leakage in Patients Undergoing Endoscopic Transsphenoidal Surgery.

Intrasellar arachnoid diverticulae can often be identified on preoperative imaging in patients undergoing endoscopic transsphenoidal surgery. The objective of this study was to characterize arachnoid diverticulae both qualitatively and quantitatively in a large institutional cohort of patients with pituitary tumors and to evaluate its association with intraoperative cerebrospinal fluid (CSF) leak. Preoperative imaging studies of 530 patients who underwent primary endoscopic transsphenoidal resection of pituitary tumors were examined both quantitatively and qualitatively for the presence of an intrasellar arachnoid diverticulum. A matched cohort analysis was performed to compare patients with a "significant" (>50% sellar depth) diverticulum with those with nonsignificant/no diverticulum. Morphologically, diverticulae were separately classified as Type 1 (ventral CSF cleft with no tumor/gland tissue between sellar face and infundibulum) or Type 2 (central CSF cleft with tumor/gland tissue between sellar face and infundibulum). Arachnoid diverticulae were noted in 40.2% of cases, and diverticulum depth was linearly correlated with tumor size and body mass index. A significant diverticulum was identified in 66 cases (12.5%) and was significantly associated with the functional tumor subtype ( P = .005) and intraoperative CSF leak ( P < .001). Type 1 clefts were associated with nonfunctional pathology ( P = .034) and the presence of suprasellar extension ( P = .035) and tended to be deeper than Type 2 clefts ( P < .001), with a higher incidence of intraoperative CSF leak ( P = .093). On logistic regression analysis, only the presence of a significant diverticulum was independently associated with intraoperative CSF leak (odds ratio 4.545; 95% CI 2.418-8.544; P < .001). The presence of an intrasellar arachnoid diverticulum should alert the surgeon to an elevated risk of intraoperative CSF leak during transsphenoidal surgery for pituitary tumors. A relatively limited surgical exposure tailored to the craniocaudal extent of the sellar pathology should be considered in these patients.

Age, growth, and feeding of boarfish, Capros aper (Linnaeus, 1758) in the Southwest of the Iberian Peninsula

ABSTRACT The boarfish (Capros aper) is a pelagic species that appears on numerous occasions as bycatch or discard in trawl fisheries in the Northeast Atlantic and Mediterranean. Existing information on this species and its occurrence as bycatch is incomplete for the Atlantic coasts. The objectives of this work were to study its growth, ageing, and diet in the southwest of the Iberian Peninsula. We found that the length–weight relationship was TW = 0.022*TL2.89 (R2 = 0.94). We observed age classes ranging from 1 to 5 years and that otoliths vary in shape and size according to the age of the individual, becoming more irregular in the lateral areas and increasing the size of the central cleft as the specimens grow older. The parameters of the von Bertalanffy growth equation were L∞ = 11.80 cm; k = 0.21 yr−1; t0 = −1.60 yr. Diet analysis indicated that copepods were the most abundant prey in the stomachs analysed, (IRI index value of 84.24%). This study serves to increase knowledge on the biology of the boarfish, which may be useful for future studies on the species, as well as for other members of the family and fisheries in which it is discarded.

Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering.

Virtually all living cells are covered with glycans. Their structures are primarily controlled by the specificities of glycosyltransferases (GTs). GTs typically adopt one of the three folds, namely, GT-A, GT-B, and GT-C. However, what defines their specificities remain poorly understood. Here, we developed a genetic glycoengineering platform by reprogramming the capsular polysaccharide pathways in Streptococcus pneumoniae to interrogate GT specificity and manipulate glycan structures. Our findings suggest that the central cleft of GT-B enzymes is important for determining acceptor specificity. The constraint of the glycoengineering platform was partially alleviated when the specificity of the precursor transporter was reduced, indicating that the transporter contributes to the overall fidelity of glycan synthesis. We also modified the pneumococcal capsule to produce several medically important mammalian glycans, as well as demonstrated the importance of regiochemistry in a glycosidic linkage on binding lung epithelial cells. Our work provided mechanistic insights into GT specificity and an approach for investigating glycan functions.

Chimeric deletion mutation of rpoC2 underlies the leaf-patterning of Clivia miniata var. variegata.

The deletion mutated rpoC2 leads to yellow stripes of Clivia miniata var. variegata by down regulating the transcription of 28 chloroplast genes and disturbing chloroplast biogenesis and thylakoid membrane development. Clivia miniata var. variegata (Cmvv) is a common mutant of Clivia miniata but its genetic basis is unclear. Here, we found that a 425bp deletion mutation of chloroplast rpoC2 underlies the yellow stripes (YSs) of Cmvv. Both RNA polymerase PEP and NEP coexist in seed-plant chloroplasts and the β″ subunit of PEP is encoded by rpoC2. The rpoC2 mutation changed the discontinuous cleft domain required to form the PEP central cleft for DNA binding from 1103 to 59aa. RNA-Seq revealed that 28 chloroplast genes (cpDEGs) were all down-regulated in YSs, of which, four involved in chloroplast protein translation and 21 of photosynthesis system (PS)I, PSII, cytochrome b6/f complex and ATP synthase are crucial for chloroplast biogenesis/development. The accuracy and reliability of RNA-Seq was verified by qRT-PCR. Moreover, the chlorophyll (Chl) a/b content, ratio of Chla/Chlb and photosynthetic rate (Pn) of YS decreased significantly. Meanwhile, chloroplasts of the YS mesophyll cells were smaller, irregular in shape, contain almost no thylakoid membrane, and even proplastid was found in YS. These findings indicate that the rpoC2 mutation down-regulated expression of the 28 cpDEGs, which disturb chloroplast biogenesis and its thylakoid membrane development. Thus, there are not enough PSI and II components to bind Chl, so that the corresponding areas of the leaf are yellow and show a low Pn. In this study, the molecular mechanism of three phenotypes of F1 (Cmvv ♀ × C. miniata ♂) was revealed, which lays a foundation for the breeding of variegated plants.

Experimental and computational evaluation of dipeptidyl peptidase III inhibitors based on quinazolinone-Schiff’s bases

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that sequentially hydrolyzes biologically active peptides by cleaving dipeptides from their N-termini. Although its fundamental role is not been fully elucidated, human DPP III (hDPP III) has been recognized in several pathophysiological processes of interest for drug development. In this article 27 quinazolinone-Schiff’s bases were studied for their inhibitory activity against hDPP III combining an in vitro experiment with a computational approach. The biochemical assay showed that most compounds exhibited inhibitory activity at the 100 μM concentration. The best QSAR model included descriptors from the following 2D descriptor groups: information content indices, 2D autocorrelations, and edge adjacency indices. Five compounds were found to be the most potent inhibitors with IC50 values below 10 µM, while molecular docking predicted that these compounds bind to the central enzyme cleft and interact with residues of the substrate binding subsites. Molecular dynamics simulations of the most potent inhibitor (IC50=0.96 µM) provided valuable information explaining the role of PHE109, ARG319, GLU327, GLU329, and ILE386 in the mechanism of the inhibitor binding and stabilization. This is the first study that gives insight into quinazolinone-Schiff’s bases binding to this metalloenzyme. Communicated by Ramaswamy H. Sarma

The molecular mechanism of microRNA duplex selectivity of Arabidopsis ARGONAUTE10.

Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3′ region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3′ region mismatches are ignored and mismatches opposite the guide 5′ end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting.

Early results of geometric ring annuloplasty for bicuspid aortic valve repair during aortic aneurysm surgery.

ObjectivesGeometric ring annuloplasty has shown promise during bicuspid aortic valve repair for aortic insufficiency. This study examined early outcomes of bicuspid aortic valve repair associated with proximal aortic aneurysm replacement.MethodsFrom September 2017 to November, 2021, 127 patients underwent bicuspid aortic valve repair with concomitant proximal aneurysm reconstruction. Patient age was 50.6 ± 12.7 years (mean ± standard deviation), male gender was 83%, New York Heart Association Class was 2 (1-2) (median [interquartile range]), and preoperative aortic insufficiency grade was 3 (2-4). Ascending aortic diameter was 50 (46-54) mm, and all patients had ascending aortic replacement. Forty patients had sinus diameters greater than 45 mm, prompting remodeling root procedures. A total of 105 patients had Sievers type 1 valves, 3 patients had type 0, and 7 patients had type 2. A total of 118 patients had primarily right/left fusion, 8 patients had right/nonfusion, and 1 patient had left/nonfusion. Leaflet reconstruction used central leaflet plication and cleft closure, with limited ultrasonic decalcification in 31 patients.ResultsRing size was 23 (21-23) mm, and 26 of 40 root procedures were selective nonfused sinus replacements. Aortic clamp time was 139 (112-170) minutes, and bypass time was 178 (138-217) minutes. Postrepair aortic insufficiency grade was 0 (0-0) (P < .0001), and mean valve gradient was 10 (7-14) mm Hg. No early and 1 late mortality occurred. Four patients required reoperation for bleeding, and 4 patients required pacemakers. At a mean follow-up of 20 months (maximal 93), there were no valve-related complications, 5 late repair failures prompting valve replacement, and 1 death due to Coronavirus Disease 2019.ConclusionsGeometric ring annuloplasty for bicuspid aortic valve repair with proximal aortic aneurysm reconstruction is safe and associated with good early outcomes. Further experience and follow-up will help inform long-term durability.

Revival of generalized favus

Revival of generalized favus

Structure of an inactive RNA polymerase II dimer.

Eukaryotic gene transcription is carried out by three RNA polymerases: Pol I, Pol II and Pol III. Although it has long been known that Pol I can form homodimers, it is unclear whether and how the two other RNA polymerases dimerize. Here we present the cryo-electron microscopy (cryo-EM) structure of a mammalian Pol II dimer at 3.5 Å resolution. The structure differs from the Pol I dimer and reveals that one Pol II copy uses its RPB4-RPB7 stalk to penetrate the active centre cleft of the other copy, and vice versa, giving rise to a molecular handshake. The polymerase clamp domain is displaced and mobile, and the RPB7 oligonucleotide-binding fold mimics the DNA–RNA hybrid that occupies the cleft during active transcription. The Pol II dimer is incompatible with nucleic acid binding as required for transcription and may represent an inactive storage form of the polymerase.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Conserved strategies of RNA polymerase I hibernation and activation

RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms ‘activation by cleft contraction’ and ‘hibernation by dimerization’ are unique to the regulation of this enzyme, but structure-function analysis is limited to baker’s yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the ‘connector’ domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.

Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

Morpho–anatomical and ultrastructural analysis of extrafloral nectaries in Inga edulis (Vell.) Mart. (Leguminosae)

The presence of extrafloral nectaries (EFNs) between leaflets is an usual feature in Inga edulis (Vell.) Mart. (Leguminosae). Extrafloral nectaries are secretory structures involved in production of nectar and which serve in the protection of plants against herbivores through association with ants. This study aimed to characterize the EFNs of I. edulis at different developmental stages and describe their morphology, histochemistry and ultrastructure. Leaf fragments, containing secretory structures, were processed according to standard methods for light, scanning and transmission electron microscopy. The EFNs were classified into three stages based on morphology: pre‐secretory, secretory and post‐secretory. The pre‐secretory stage occurs in young leaves, whereas secretory and post‐secretory stages occur in developed and senescent leaves, respectively. The EFNs possess a concave surface and a central cleft in which nectar is accumulated and which was not observed in pre‐secretory EFNs. Histochemical tests identified the presence of sugars, proteins, phenolic compounds, mucilage and lipids at all developmental stages of the EFNs. Calcium crystals were identified in all tissues and stages of the EFNs. The secretory cells of the EFNs exhibit a granular cytoplasm, small vacuoles, prominent nuclei, smooth endoplasmic reticulum and mitochondria. Post‐secretory stage EFNs exhibited intense cytoplasmic degradation and the presence of microorganisms. The performance of EFNs of I. edulis appear to follow the leaf development.

Identification of Pelomyxa palustris Endosymbionts

Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.

DNA recognition by an RNA-guided bacterial Argonaute.

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.

Crystal structure of RNA polymerase II from Komagataella pastoris

RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, the crystal structure of Pol II from the methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While the structure is highly homologous to that of Pol II from the budding yeast Saccharomyces cerevisiae, the stalk and clamp modules of the K. pastoris Pol II displayed large inward rotations, closing the central cleft to a greater extent than in the known S. cerevisiae Pol II structures. The conformational differences reflect the inherent flexibilities of the stalk and the clamp, as additional low-resolution structures of K. pastoris Pol II in different crystal forms revealed diverse stalk and clamp orientations. Comparisons with other eukaryotic/archaeal RNA polymerase structures in the Protein Data Bank revealed the distributions of the stalk and clamp orientations. The conformational plasticity should be essential for transcriptional functions and binding various regulatory factors.

Structural Basis of RNA Polymerase I Transcription Initiation

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.