Structural and enzymatic characterization of the sialidase SiaPG from Porphyromonas gingivalis.

The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Šresolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Structural basis for the tethered peptide activation of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1-6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7-12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical-bulge-ß-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved 'toggle switch' W6.53 and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49 and the P6.47/V6.47φφG6.50 motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gs coupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gs coupling.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2.

Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Determination of Five Major 8-Prenylflavones in Leaves of Epimedium by Solid-Phase Extraction Coupled with Capillary Electrophoresis.

A simple, accurate and reproducible method which is based on the capillary electrophoresis, coupled with solid-phase extraction, has been developed for simultaneous determination of multiple 8-prenylflavones from Chinese Herba Epimedii. In this study, the author has mainly illustrated the experimental process and research results of five major components including epimedin C, icariin, diphylloside A, epimedoside A and icarisoside A that have been extracted and identified from Herba Epimedii for the first time. Experimental conditions have been optimized to achieve the best separation efficiency for the following factors: the buffer pH, buffer concentration and applied voltage. The experiment can be conducted through two separable stages: the first stage is to obtain the crude extracts through the solid-phase extraction; and the second stage is to further separate five major components by using the capillary electrophoresis. The separation of the five components and the analysis of the experiment are relatively fast and can be completed within 20 min. The concentration ranges of the construction of standard curves of five major 8-prenylflavones are 32.0-395.0, 23.4-292.0, 42.1-526.0, 18.8-233.5 and 29.7-371.0 µg mL(-1) respectively, which have showed acceptable linearity with a correlation coefficient, r ≥ 0.999. The coefficient varies within 2.0% for both intra- and inter-days tests. The recoveries of five components range from 92.3 to 104.1%. The relative standard deviations of recoveries of five components range from 1.2 and 2.8%. This new method will facilitate the extraction and expedite the determination of medical components from Herba Epimedii.

[Prenatal restraint stress decreases neurogranin expression in rat offspring hippocampus].

Neurogranin, a neuron-specific postsynaptic protein, has been considered to play an important role in synaptic plasticity and learning and memory. The present study aimed to investigate the effects of prenatal restraint stress on neurogranin expression in rat offspring hippocampus. Pregnant rats were given a restraint stress (3 times a day for 7 d, 45 min each time) at the late stage of gestation except that in the control group. The offspring rats were divided into four groups: female control group, male control group, female stress group and male stress group. Expression of neurogranin was determined by immunohistochemistry and Western blot. The results showed that neurogranin-positive immunostaining was detected in all areas of the hippocampus. The staining density was stronger in the CA1 and CA3 regions than that in the dentate gyrus (DG) region. Western blot assay showed that neurogranin protein level in female and male prenatal stressed offspring was significantly lower than that in the controls (P<0.01). Neurogranin level was significantly lower in the female stress group than that in the male stress group, whereas there was no significant gender difference in the control group. Immunohistochemical data further confirmed these results. The present study provides evidence that prenatal restraint stress induces gender-dependent decrease in neurogranin expression in the offspring hippocampus. The prenatal restraint stress-induced decrease in neurogranin expression in the hippocampus might be associated with the deficit in spatial learning and memory reported previously.

Investigation of the acid-base properties of mononitro-calix[4]arene with chemometric methods.

The acid-base properties of mononitro-calix[4]arene was studied with chemometric methods by measurement of its UV absorbance under different pH. The chemometric method-iterative target transformation factor (ITTFA) was employed to resolve the acid-base fraction curves. Combining with other chemometric methods-principal component analysis (PCA) and evolving factor analysis (EFA), the proton dissociation behavior of the derivative was investigated in detail. The pK(a) values of the derivative were determined and the fraction curves and pure absorbing spectra of each absorbing component were obtained.

[Gender-dependent difference of NF-kappaB expression in the hippocampus of prenatally stressed offspring rats].

In this study, immunohistochemistry and Western blot were used to determine whether the expression of NF-kappaB in the hippocampus of prenatally stressed offspring rats is gender-dependent. The results were as follows: In the female offspring rats, the expressions of p65 in the hippocampal dentate gyrus in mid-term stress (MS) and late-term stress (LS) groups were significantly less than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly more than that in the control group (P<0.01). A significant difference was also present between MS and LS groups (P<0.01). In the male offspring rats, the expressions of p65 in the hippocampal dentate gyrus in MS and LS groups were evidently more than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly less than that in the control group (P<0.05, P<0.01). There was also a significant difference in p65 expression between MS and LS groups (P<0.01). In addition, in the control group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly more than that of male ones (P<0.01). However, in LS group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly less than that of male ones (P<0.01). Moreover, there was no significant difference in p65 expression between female and male offspring rats in MS group. In the control group the gender difference in the expression of p50 was only observed in hippocampal CA1 (P<0.01). The expressions of p50 in all regions of hippocampus of female offspring rats were significantly more than that of male ones in LS group (P<0.01). There was no significant difference in p50 expression between female and male offspring rats in MS group. The results of Western blot were similar to those of immunohistochemical study. These results indicate that prenatal stress in different gestational periods significantly affects the expressions of p65 and p50 in hippocampus, and this effect is gender-dependent. This may be one of the mechanisms underlying the gender difference in the ability of learning and memory of the prenatally stressed offspring rats.

[Effects of prenatal stress on kinetic properties of high-voltage-activated Ca2+ channel in freshly isolated offspring rat hippocampal CA3 pyramidal neurons].

OBJECTIVE: To investigate the effects of prenatal stress (PNS) on kinetic properties of high-voltage-activated (HVA) Ca(2+) channel in freshly isolated offspring rat hippocampal CA3 pyramidal neurons. METHODS: The pregnant rats were exposed to restraint stress, and the hippocampal CA3 pyramidal neurons were freshly isolated from the offspring rats to record voltage-gate Ca(2+) channel currents in these neurons using whole-cell configuration of patch-clamp technique. RESULTS: PNS significantly increased HVA calcium current density and integral current but did not alter the steady-state activation and steady-state inactivation properties of HVA Ca(2+) channel in the offspring rat CA3 neurons. The maximal HVA calcium current density was -40.89-/+0.31 pA/pF in the control group (n=10) and -49.44-/+0.37 pA/pF in PNS group (n=8, P<0.01). The maximal integral current of the HVA Ca(2+) channel was 106.81-/+4.20 nA*ms in the control group (n=10) and 133.49-/+2.59 nA*ms in the PNS group (n=8, P<0.01). CONCLUSION: Maternal exposure to stress during the critical phase of pregnancy may result in long-lasting effects on the ion channels of the hippocampal neurons in the offspring rats.

[The effects of prenatal stress on Ca2+ channel and K+ channel of hippocampal CA3 pyramidal neurons in rat offspring].

To investigate the effects of prenatal stress (PNS) on high-voltage-activated (HVA) Ca2+ channel and delayed rectifier potassium currents (I(KD)) of freshly isolated rat offspring hippocampal CA3 pyramidal neurons, the pregnant rats of PNS group were exposed to restraint stress and patch clamp technique of whole-cell mode was employed to record HVA Ca2+ and K+ channel currents in hippocampal CA3 pyramidal neurons. It was observed that PNS increased HVA calcium peak current amplitude. The maximal HVA calcium peak current amplitudes were -576.52 +/- 7.03 pA in the control group and -702.05 +/- 6.82 pA in the PNS group respectively (P < 0.01). The conductance-voltage relationship of HVA Ca2+ channel was not changed. Current-voltage relationship and conductance-voltage relationship of I(KD) in offspring CA3 neurons were also not affected by PNS. The data suggested that exposure of pregnant animals to a period of stress could impose lasting effects on the offspring hippocampus CA3 neurons Ca2+ channel during a critical phase of fetal development. The mechanism may include two ways. One was that high CORT led to enhance level of expression for the HVA Ca2+ channel subunit mRNAs and increased HVA calcium currents. Another was that PNS caused an increase in the production of ROS in hippocampal CA3 region and ROS caused an increase in the phosphorylation of HVA Ca2+ channel of offspring hippocampal CA3 neurons.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells.

AIM: To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS: Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS: The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca2+ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION: The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Developmental expression of FXPRLamide neuropeptides in peptidergic neurosecretory cells of diapause- and nondiapause-destined individuals of the cotton bollworm, Helicoverpa armigera.

The diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) gene encodes five neuropeptides, DH, PBAN, alpha-SGNP, beta-SGNP, and gamma-SGNP (subesophageal ganglion neuropeptide). All share the C-terminal pentapeptide FXPRLamide sequence and are produced in the subesophageal ganglion (SG). Expression of the DH-PBAN gene in the central nervous system of embryonic, larval, pupal, and adult Helicoverpa armigera (Har) was studied using in situ hybridization, whole-mount immunocytochemistry, and competitive ELISA. Both Har-DH-PBAN mRNA and protein are localized in the mandibular, maxillary, and labial cell clusters of the SG and a pair of ventral midline neurons of each thoracic ganglion. The FXPRLamide titers in hemolymph are significantly higher in diapause-destined larvae during the fifth and sixth instar than in similar nondiapause-destined individuals. In contrast, the FXPRLamide titers in diapause-destined pupae are significantly lower than in nondiapause-destined pupae. The results from immunocytochemistry and in situ hybridization are consistent with changes of FXPRLamide titers as measured by ELISA. These data suggest that the expression of DH-PBAN might be correlated with diapause induction at the larval stage of diapause-destined individuals and continuous development at pupal stage of nondiapause-destined individuals. Thus, the DH-PBAN gene may play an important regulatory role in aspects of insect development besides diapause termination and pheromone biosynthesis. The transport pathways of FXPRLamide neuropeptides suggest that humoral route is involved in their regulation of development.

[Resolution for the kinetic process of ultrasound-assisted electro-catalytic degradation of benzoic acid].

The two-way kinetic-spectral data can be acquired by monitoring the process of the ultrasound-assisted electro-catalytic degradation of benzoic acid. The product of absorbing intermediate is confirmed by FSMWEFA(Fix-sized Moving Window Evolving Factor Analysis). The real kinetic spectra and absorption spectra, which are hard to measure directly, are resolved by ITTFA (Iterative Target Transformation Factor Analysis) combined with the isoabsorptive points between components. The kinetic model of benzoic acid degradation is also studied.

Studies on the Relationship Between the Biological Activities and the Circular Dichroism of South Anhui Dienagkistrodon Acutus Hemorrhagins.

The solution conformations of three hemorrhagic toxins, designated as AaH I, AaH III and AaH IV, from South Anhui Dienagkistrodon acutus have been studied by CD spectra. The secondary structure of AaH I consisted of 25.8% alpha-helix, 12.7% beta-sheet and 26.8% beta-turns, together with 34.7% random coil. For AaH III, the secondary structure contents were 23.9%, 20.6%, 23.7% and 31.8%, and for AaH IV they were 18.2%, 31.0%, 17.2% and 33.8%, respectively. When pH was lower than 4.0 or higher than 11.0, the alpha-helix decreased but beta-sheet increased, meanwhile, the caseinolytic activities of the three toxins decreased. The activities could be inhibited by EDTA, which indicated that all the three toxins were all metalloproteinases. EDTA, Cu(2+), Zn(2+), Ca(2+) and Mg(2+) could change the secondary structures and play an important role in caseinolytic activities.