Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa.

Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and kcat/Km values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the KmGSH and I50 values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (kcat/KmCDNB) plot was used to estimate the pKa value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pKa value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pKa of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.

Thermal Dynamics of a Novel Radio-Frequency Device for Endoscopic Spine Surgery: An Experimental Model.

STUDY DESIGN: Experimental study. OBJECTIVE: In this study, the ambient temperature of a radiofrequency (RF) electrode tip was compared and analyzed in terms of products, mode, flow quantity, and flow rate. SUMMARY OF BACKGROUND DATA: Endoscopic spine surgery is a widely used operation for degenerative lumbar stenosis and herniated lumbar disc. To perform endoscopic spine surgery, dedicated instruments like a RF generator and electrode are essential. METHODS: An evaluation system capable of measuring temperature under equal conditions at a certain distance from the electrode tip was manufactured. The distance between the electrode tip and the temperature sensor was set to 1, 5, and 10 mm. The flow quantities of 0, 50, 100, and 150 mL/min and the flow rates of 0, 0.20, 0.53, and 0.80 m/s were compared and statistically analyzed. RESULTS: The temperatures measured in the experiments conducted on the four combinations of RF device showed similar values, and showed differences according to the characteristics of each mode of the RF. As the distance between the electrode tip and the temperature sensor increased, the temperature decreased, and as flow quantity or flow rate increased, the temperature decreased. The maximum temperatures differed significantly according to flow quantity, between flow quantities of 0 and 100 mL/min (P  = 0.03) and between 0 and 150 mL/min (P ≤ 0.01). The maximum temperatures also differed significantly between the flow rate of 0 m/s, and the flow rates of 0.20, 0.53, and 0.80 m/s, with P ≤ 0.01 in all three comparisons. CONCLUSION: This is the first study in which we made a customized RF temperature evaluation system and verified the temperature changes in various environments. When irrigation was performed, we could confirm that the maximum temperature was less than 60°C. Irrigation is considered essential in endoscopic spine surgery. LEVEL OF EVIDENCE: 3.

Structure and substrate specificity of ß-ketoacyl-acyl carrier protein synthase III from Acinetobacter baumannii.

Originally annotated as the initiator of fatty acid synthesis (FAS), ß-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.

Structure-activity relationship-based screening of antibiotics against Gram-negative Acinetobacter baumannii.

To discover potent antibiotics against the Gram-negative bacteria, we performed a structure-activity relationship (SAR) study of YKsa-6, which was the most potent inhibitor of Staphylococcus aureus ß-ketoacyl acyl carrier protein III in our previous study. We identified and selected 11 candidates, and finally screened two active compounds, YKab-4 (4-[(3-chloro-4-methylphenyl)aminoiminomethyl]benzene-1,3-diol) and YKab-6 (4-[[3-(trifluoromethyl)phenyl]aminoiminomethyl]phenol) as inhibitors of Acinetobacter baumannii KAS III (abKAS III). They showed potent antimicrobial activities at 2 or 8 µg/mL, specifically against Acinetobacter baumannii and a strong binding affinity for abKAS III. From the homology modeling, we defined the three-dimensional (3D) structure of abKAS III for the first time and found that it had an extra loop region compared with common Gram-negative bacteria derived KAS IIIs. The docking study revealed that the hydroxyl groups of inhibitors formed extensive hydrogen bonds and the complicated hydrophobic and cation-stacking interactions are important to binding with abKAS III. We confirmed that the hydrophobicity of these compounds might be the essential factor for their antimicrobial activities against Gram-negative bacteria as well as their structural rigidity, a cooperative feature for retaining the hydrophobic interactions between abKAS III and its inhibitors. This study may provide an insight developing strategies for potent antibiotics against A. baumannii.

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane.

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.

The Anti-Inflammatory Effect of Human Telomerase-Derived Peptide on P. gingivalis Lipopolysaccharide-Induced Inflammatory Cytokine Production and Its Mechanism in Human Dental Pulp Cells.

Porphyromonas gingivalis is considered with inducing pulpal inflammation and has lipopolysaccharide (LPS) as an inflammatory stimulator. GV1001 peptide has anticancer and anti-inflammation activity due to inhibiting activation of signaling molecules after penetration into the various types of cells. Therefore, this study examined inhibitory effect of GV1001 on dental pulp cells (hDPCs) stimulated by P. gingivalis LPS. The intracellular distribution of GV1001 was analyzed by confocal microscopy. Real-time RT-PCR was performed to determine the expression levels of TNF-α and IL-6 cytokines. The role of signaling by MAP kinases (ERK and p38) was explored using Western blot analysis. The effect of GV1001 peptide on hDPCs viability was measured by MTT assay. GV1001 was predominantly located in hDPC cytoplasm. The peptide inhibited P. gingivalis LPS-induced TNF-α and IL-6 production in hDPCs without significant cytotoxicity. Furthermore, GV1001 treatment markedly inhibited the phosphorylation of MAP kinases (ERK and p38) in LPS-stimulated hDPCs. GV1001 may prevent P. gingivalis LPS-induced inflammation of apical tissue. Also, these findings provide mechanistic insight into how GV1001 peptide causes anti-inflammatory actions in LPS-stimulated pulpitis without significantly affecting cell viability.

Crystal structure of Legionella pneumophila type IV secretion system effector LegAS4.

The SET domain of LegAS4, a type IV secretion system effector of Legionella pneumophila, is a eukaryotic protein motif involved in histone methylation and epigenetic modulation. The SET domain of LegAS4 is involved in the modification of Lys4 of histone H3 (H3K4) in the nucleolus of the host cell, thereby enhancing heterochromatic rDNA transcription. Moreover, LegAS4 contains an ankyrin repeat domain of unknown function at its C-terminal region. Here, we report the crystal structure of LegAS4 in complex with S-adenosyl-l-methionine (SAM). Our data indicate that the ankyrin repeats interact extensively with the SET domain, especially with the SAM-binding amino acids, through conserved residues. Conserved surface analysis marks Glu159, Glu203, and Glu206 on the SET domain serve as candidate residues involved in interaction with the positively charged histone tail. Conserved surface residues on the ankyrin repeat domain surround a small pocket, which is suspected to serve as a binding site for an unknown ligand.

A new class of peptidomimetics targeting the polo-box domain of Polo-like kinase 1.

Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.

Effect from surface treatment of nickel-titanium rotary files on the fracture resistance.

This study was aimed to compare the cyclic fatigue resistance and torsional resistance of rotary instruments with and without surface treatment. G6 A2 (Group A2) with and G6 A2 without surface treatment after machining (Group AN) were compared in this study. ProTaper F2 (Group F2) which has similar dimension and shape was also used for comparison. To evaluate the torsional resistance, ultimate torsional strength and distortion angle until fracture were recorded, and the toughness was calculated. The cyclic fatigue resistance was compared by evaluating the number of cycles to failure in a simulated canal. Statistical analysis was performed by one-way analysis of variance and Tukey post hoc test (p = 0.05). After torsional and cyclic fatigue tests, all fracture fragments were observed under a scanning electron microscope. Group A2 showed higher cyclic fatigue resistance than the groups AN and F2 (p < 0.05). Although group A2 demonstrated lower ultimate torsional strength than the others, there were no significant differences in toughness among the groups. While obvious machining grooves were seen in groups AN and F2, group A2 showed smooth surface resulting from the surface treatment. The specimens of fracture fragments showed typical features of cyclic failure such as micro-cracks, overloaded fast fracture zone, and torsional fracture such as unwinding helix, circular abrasion marks and dimples. Under the conditions of this study, the surface treated instruments may improve cyclic fatigue resistance while maintaining the torsional resistances and mechanical properties.

Exploring the binding nature of pyrrolidine pocket-dependent interactions in the polo-box domain of polo-like kinase 1.

BACKGROUND: Over the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1), which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1-3), respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups. CONCLUSION/SIGNIFICANCE: The progress made using our site-directed ligands validated this approach to specifically direct the ligand into the unique pyrrolidine-binding region, and it extends the applicability of the strategy for discovering selective protein-protein interaction inhibitors.

Effects of calcium phosphate endodontic sealers on the behavior of human periodontal ligament fibroblasts and MG63 osteoblast-like cells.

In regard to biological properties of endodontic sealers, there are many characteristics that should be considered. The aim of this study was to examine the biological effects of new calcium phosphate-based root canal sealers, CAPSEAL I and CAPSEAL II (CPS), on human periodontal fibroblast cells by examining the expression levels of inflammatory mediators and to compare the effects of CPS on the viability and osteogenic potential of human osteoblast MG63 cells compared to those of other commercially available calcium phosphate sealers [Apatite Root Sealer type I (ARS I) and Apatite Root Sealer III (ARS III); Sankin Kogyo, Tokyo, Japan] and a zinc oxide eugenol-based sealer (Pulp Canal Sealer EWT [PCS EWT]; Kerr, Detroit, MI). The levels of IL-6 in the new CPS group (CAPSEAL I, II) were higher than those in the control and all experimental groups at all time points after 2 h. TGF-ß1 and FGF-1 levels decreased at 72 h compared to the levels in the control, in cells treated with every sealers except ARS I. The new CPS sealers showed low cytotoxicity. Reverse transcription polymerase chain reaction showed that CAPSEAL I, II, and Apatite Root Sealer type III induced expression of early stage markers of differentiation (alkaline phosphatase and osteopontin) at 7 days. Also, new CPS showed higher mineralized nodule formation at 28 days. These results suggest that CAPSEAL I and II facilitate the periapical dentoalveolar and alveolar healing by controlling cellular mediators from PDL cells and osteoblast differentiation of precursor cells.

Expression, purification, crystallization and preliminary X-ray analysis of the human NORE1 SARAH domain.

NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366-413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7 Šresolution and belonged to space group P6(1)22, with unit-cell parameters a = b = 73.041, c = 66.092 Å, α = ß = 90, γ = 120°.

Protective effects of luteolin against apoptotic liver damage induced by D-galactosamine/lipopolysaccharide in mice.

In this study, the protective effects of luteolin (1, a major component of Cirsium japonicum) were examined against d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice received an intraperitoneal injection of 1 (25, 50, 100, and 200 mg·kg(-1)) 1 h before treatment with GalN (700 mg·kg(-1))/LPS (10 µg·kg(-1)). Treatment with GalN/LPS resulted in increased mortality and serum aminotransferase activity. These increases were attenuated by pretreatment with 1. Treatment with GalN/LPS induced an increase in the serum level of tumor necrosis factor-α (TNF-α) and protein expression of TNF-α receptor-associated death domain, and these increases were prevented by 1. In addition, 1 attenuated apoptosis induced by GalN/LPS treatment, which was analyzed using a caspase-3 and -8 activity assay, as well as by proapoptotic BH3-only protein and cytochrome c protein expression, and by a terminal deoxynuleotidyl transferase-mediated dUTP nick end-labeling method. After GalN/LPS injection, nuclear phosphorylated c-Jun levels showed a significant increase, which were attenuated by 1. The present findings suggest that luteolin ameliorates D-GalN/LPS-induced liver injury and that this protection is likely due to inhibition of the extrinsic and intrinsic apoptotic pathways.

Anti-inflammatory and analgesic effects of human placenta extract.

In this study, we investigated the effects of human placenta extract (HPE, Laennec inj.) on pro-inflammatory cytokines and mediators secreted from lipopolysaccharide-stimulated RAW264.7 macrophages. We found that HPE significantly inhibited the production of nitric oxide, tumour necrosis factor-α and cyclooxygenase-2. We studied the anti-inflammatory and analgesic potential of HPE in murine models of inflammation/inflammatory pain. Rats were assigned to six groups and were administered either saline or HPE (0.33, 1, 3 and 6 mL kg⁻¹) intraperitoneally. Diclofenac was used as a positive control. HPE attenuated the swelling of the rat's hind paw. The vascular permeability induced by acetic acid was significantly reduced by HPE. HPE reduced the formation of granuloma in carrageenan air pouch and hind paw oedema in complete Freund's adjuvant-induced chronic arthritis in rats. HPE attenuated writhing episodes. An increase in hot-plate latency was observed in mice receiving HPE. HPE also increased the pain threshold in the Randall-Selitto test. In the tail-flick assay, HPE prolonged the reaction time of rats to radiant heat stimulation. These results suggest that HPE has potent anti-inflammatory and anti-nociceptive activities.

Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats.

AIM OF THE STUDY: GCSB-5 (traditional name: Chungpa-Juhn), an herbal medicine composed of 6 crude herbs (Saposhnikovia divaricata Schiskin, Achyranthis bidentata Blume, Acanthopanax sessiliflorum Seem, Cibotium baromets J. Smith, Glycine max Meriill, and Eucommia ulmoides Oliver), has been widely used in Asia for treatment of neuropathic and inflammatory diseases. This study investigated the protective effect of GCSB-5 against peripheral nerve injury in vitro and in vivo. MATERIALS AND METHODS: After left sciatic nerve transection, rats received oral administration of GCSB-5 (30, 100, 300, and 600 mg/kg), or saline (vehicle), respectively, once daily for 8 weeks. Motor functional recovery and axonal nerve regeneration were evaluated by measurement of sciatic functional index (SFI), sensory regeneration distance, and gastrocnemius muscle mass ratio. The myelinated axon number was counted by morphometric analysis. In the in vitro study, the effects of GCSB-5 on H(2)O(2)-induced oxidative damage in SH-SY5Y cells were investigated by measurement of cell viability, production of reactive oxygen species (ROS), lipid peroxidation, release of lactate dehydrogenease (LDH), and cellular glutathione contents. Neurite outgrowth was also determined. RESULTS: After 8 weeks of nerve transection, SFI, regeneration distance, and gastrocnemius muscle mass ratio and myelinated axon number showed a significant decrease and these decreases were attenuated by GCSB-5. GCSB-5 significantly inhibited H(2)O(2)-induced cell death and oxidative stress, as evidenced by decreases in production of ROS and lipid peroxidation and release of LDH, and by increase in total GSH content. CONCLUSIONS: The neuroprotective effect afforded by GCSB-5 is due in part to reduced oxidative stress.

Mesiobuccal root canal anatomy of Korean maxillary first and second molars by cone-beam computed tomography.

OBJECTIVE: The aim of this study was to investigate the types of canal configurations and the incidence of a second mesiobuccal (MB2) canal in Korean maxillary molar mesiobuccal (MB) roots by analyzing cone-beam computed tomographic (CBCT) images. STUDY DESIGN: Three-dimensional CBCT images of 458 maxillary first molars and 467 second molars from 276 Korean patients were analyzed to determine the incidence of an MB2 canal, the types of canal configurations, and the correlations between the incidence of an MB2 canal and age, gender, and tooth position. RESULTS: The incidence of 2-canaled MB roots was 71.8% in first molars and 42.2% in second molars, with the most common configurations being Weine types III and II. The frequency of an MB2 canal decreased with age in both molars (P < .05). CONCLUSIONS: Types III and II canal configurations were the most prevalent in the 2-canaled MB roots of Korean maxillary molars.

Periapical bone regeneration after endodontic microsurgery with three different root-end filling materials: amalgam, SuperEBA, and mineral trioxide aggregate.

INTRODUCTION: The purpose of this study was to determine the bone regeneration potential to different root-end filling materials by evaluating the distance between the materials and newly regenerated bone after root-end surgery. MATERIAL AND METHODS: Periapical lesions were induced in premolars and molars of five female beagle dogs. The teeth were treated endodontically after the development of the lesions. After 1 week, the teeth underwent root-end surgery using modern microsurgical techniques. Three different root-end filing materials were used: amalgam (Tytin; Kerr Mfg Co, Romulus, MI), SuperEBA (Bosworth, Skokie, IL), and mineral trioxide aggregates (MTA; Dentsply, York, PA). After 4 months, the dogs were sacrificed, and the jaws were prepared for histological sectioning. The distances from the root-end filling materials to the regenerated bone were determined by the evaluation of microradiographic images of the sections with imaging software (Sigma Scan/Image; Jandel Scientific Software, San Rafael, CA). The results were statistically analyzed with analysis of variance using Sigma Stat software (Jandel Scientific Software, San Rafael, CA). RESULTS: The mean distances from the newly regenerated bone were 0.397 +/- 0.278 mm in the MTA group, 0.756 +/- 0.581 mm in the SuperEBA group, and 1.290 +/- 0.386 mm in the amalgam group. There was a statistically significant difference between the amalgam and MTA groups (p < 0.05). No significant differences existed for amalgam versus SuperEBA and SuperEBA versus MTA. CONCLUSION: MTA showed the most favorable periapical tissue response. The distance from MTA to the regenerated bone was similar to the normal average periodontal ligament thickness in dogs.

Palmatine attenuates D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice.

Palmatine is an isoquinoline alkaloid from Coptis chinensis, an herbal medicine used to treat various inflammatory diseases such as gastritis, edema and dermatitis. The present study examined the cytoprotective properties of palmatine on d(+)-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were intraperitoneally given GalN (700 mg/kg)/LPS (10 microg/kg). Palmatine (25, 50, 100, and 200mg/kg) was administered 1h before GalN/LPS. GalN/LPS increased the mortality and serum aminotransferase activities. These increases were attenuated by palmatine. GalN/LPS increased hepatic lipid peroxidation and decreased the contents of reduced glutathione. Palmatine did not affect the lipid peroxidation and glutathione content. GalN/LPS increased the circulating levels of tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6) and IL-10. Palmatine prevented the increase of serum TNF-alpha and augmented that of serum IL-10. GalN/LPS treatment also increased the levels of TNF-alpha, IL-6 and IL-10 mRNA expression in liver tissue. Palmatine decreased the TNF-alpha mRNA expression and increased the IL-10 mRNA expression. Palmatine attenuated the apoptosis of hepatocytes, as evidenced by the TUNEL method and capase-3 analysis. Our data suggest that palmatine alleviates GalN/LPS-induced liver injury by modulating the cytokine response and inhibiting apoptosis.

X-ray crystal analysis of a TASP: structural insights of a cavitein dimer.

Cavitein Q4 is a template assembled synthetic protein designed for X-ray crystallographic analysis. It is based on a previous monomeric helical bundle cavitein (N1GG) that consists of four identical parallel helical peptides. Crystals that were grown in the presence of bromide ions were used to solve the initial phases via single-wavelength anomalous dispersion (SAD). A 1.4 A resolution data set was then refined starting with the SAD phases to provide the crystal structure of cavitein Q4. The crystal structure revealed cavitein Q4 as an asymmetric dimer, although the cavitein appears to be largely monomeric in solution. A comparative analysis is carried out to discern any intrinsic differences between Q4 and its parent cavitein N1GG. We present herein the first X-ray crystal structure of a TASP system and relate this structure to the solution data for both Q4 and its parent N1GG.

Structure, binding, and activity of Syd, a SecY-interacting protein.

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners.