Cerebrospinal fluid (CSF) is a biofluid in direct contact with the brain and as such constitutes a sample of choice in neurological disorder research, including neurodegenerative diseases such as Alzheimer or Parkinson. Human CSF has still been less studied using proteomic technologies compared to other biological fluids such as blood plasma or serum. In this work, a pool of “normal” human CSF samples was analysed using a shotgun proteomic workflow that combined removal of highly abundant proteins by immunoaffinity depletion and isoelectric focussing fractionation of tryptic peptides to alleviate the complexity of the biofluid. The resulting 24 fractions were analysed using liquid chromatography coupled to a high-resolution and high-accuracy timsTOF Pro mass spectrometer. This state-of-the-art mass spectrometry-based proteomic workflow allowed the identification of 3’174 proteins in CSF. The dataset reported herein completes the pool of the most comprehensive human CSF proteomes obtained so far. An overview of the identified proteins is provided based on gene ontology annotation. Mass and tandem mass spectra are made available as a possible starting point for further studies exploring the human CSF proteome. Specifications table•A comprehensive proteomic profile of “normal” human CSF, among the largest reported so far using LC-MS/MS, is provided•The data is useful for enhanced characterization and annotation of the human CSF proteome•The data is valuable for the proteomic community for spectral library generation and as a starting point for clinical studies focussing on CSF and neurological disorders•The data provides information for targeted protein/peptide assay development in human CSF1Supplementary Table S1. The human CSF sample analyzed in this report was previously analyzed with different LC-MS/MS intrumentations to assess throughput and robustness of an automated pipeline for biomarker discovery Binding and Catalytic activity represented 78% of the molecular functions. Cellular process was the most important biological process represented ; lastly, Cell and Cell part (21% each) were the major cellular components identified in this dataset.The dataset presented herein identified 3’174 proteins and their respective 25’227 peptides in “normal” CSF; protein and peptide lists are provided in semaphorin receptor activity”, “axon guidance receptor activity” or “semaphorin-plexin signaling pathway involved in neuron projection guidance” were particularly enriched in this dataset.A GO enrichment was also performed with Gorilla i.e., 2’390 proteins) were common to both datasets, as well as almost 14’000 peptides   .Fig. 2Co22.1vacuum centrifuge (Thermo Scientific). The dried samples were diluted in depletion Buffer A (Agilent Technologies) containing 9.65 µg/mL of β-lactoglobulin from bovine milk. Abundant CSF proteins were removed using MARS columns (Agilent Technologies) and HPLC systems (Thermo Scientific) equipped with an HTC-PAL  fraction collector. Buffer exchange was performed with Strata-X 33u polymeric reversed-phase (RP) (30 mg/1 mL) cartridges mounted on a 96-hole holder and a vacuum manifold, as previously described via the addition of 10 μL of trypsin/Lys-C at 0.25 μg/μL in 100 mM TEAB (incubation overnight at 37°C). TMT labeling was performed via the addition of 0.8 mg of TMT 6-plex reagent in 41 μL of CH3CN (incubation for 1 h at room temperature). After reaction, a volume of 8 μL of hydroxylamine 5% in H2O was added to each tube to react for 15 min. Samples from a given TMT 6-plex experiment were pooled together in a new tube. Pooled samples  were purified by solid phase extraction with Oasis HLB cartridges from Waters and Strata-X-C 33u polymeric strong cation cartridges from Phenomenex. All samples were resuspended in 200 μL of H2O/CH3CN/formic acid 96.9/3/0.1; 75 μL of the 16 resulting pooled samples were mixed together , dried, and dissolved in 3232.8 µL H2O with 345.6 µL glycerol 50% and 21.6 µL of IPG buffer pH 3-10 . The sample was separated in 24 fractions with isoelectric focusing according to a previously published protocol The sample preparation was performed previously ,5. Brief2.22O/CH3CN/formic acid (FA) 96.9/3/0.1%. A volume of 3 µL of each of the fractions were then diluted with 7 µL of H2O/FA 99.9/0.1% and only 2 µL of each diluted fraction were injected for separation on a 75 µm × 250 mm Aurora 2 C18 column (Ion Opticks). A typical RP gradient  was run on a nanoflow LC system  at a flow rate of 400 nL/min. Column temperature was controlled at 50°C. The LC run lasted for 120 min . The column was coupled online to a timsTOF Pro with a CaptiveSpray ion source . The temperature of the ion transfer capillary was set at 180°C. Ions were accumulated for 123 ms, and mobility separation was achieved by ramping the entrance potential from −160 V to −20 V within 123 ms.The purified 24 fractions were dissolved in 50 µL Hm/z), respectively. To enable the parallel accumulation-serial fragmentation (PASEF) method, precursor m/z and mobility information was first derived from full scan TIMS-MS experiments (with a mass range of m/z 100-1700). Singly charged precursors were excluded by their position in the m/z-ion mobility plane and precursors that reached a ‘target value’ of 20,000 a.u. were dynamically excluded for 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th form/z ≥ 700, for fragmentation, and the collision energies varied between 31 and 52 eV depending on precursor mass and charge. TIMS, MS operation and PASEF were controlled and synchronized using the control instrument software OtofControl 5.1 . LC-MS/MS data were acquired using the PASEF method with a total cycle time of 1.23 s, including 1 TIMS MS scan and 10 PASEF MS/MS scans. The 10 PASEF scans (123 ms each) contained on average 12 MS/MS scans per PASEF scan. Ion mobility resolved mass spectra, nested ion mobility versus m/z distributions, as well as summed fragment ion intensities were extracted from the raw data file with DataAnalysis 5.1 .The acquisition of mass and tandem mass spectra was done with average resolution of 60,000 and 50,000 full width at half maximum (mass range 100-1700 2.3Protein identification was performed against the human UniProtKB/Swiss-Prot database (2020/02 release) comprising 20’367 protein sequences in total. Mascot (version 2.4.6 from Matrix Sciences) was used as search engine. Variable amino acid modifications were: oxidized methionine, deamidated asparagine/glutamine, and 6-plex TMT-labeled peptide amino terminus; 6-plex TMT-labeled lysine was set as fixed modifications as well as carbamidomethylation of cysteine. Trypsin was selected as the proteolytic enzyme, with a maximum of two potential missed cleavages. Peptide and fragment ion tolerance were set to 15 ppm and 0.05 Da, respectively. All Mascot result files were loaded into Scaffold Q+S 4.8.4 (Proteome Software) to be further searched with X! Tandem ). The FDR in Scaffold was set up to 1% at protein and peptide level, with a one unique peptide criterion to report protein identification.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article. C. Macron, A. Núñez Galindo, M. Affolter and L. Dayon are employees of the Société des Produits Nestlé SA.


The CYP2E1 assay showed a good linearity (R2 > 0.999), good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80–120%), and low detection (4.972 µM and 1.997 µM) and quantitation limit values (15.068 µM and 6.052 µM), for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Salicylic acid acts as a mixed inhibitor (competitive and non-competitive inhibition), with Ki (inhibition constant) = 83.56 ± 2.730 µM and concentration of inhibitor causing 50% inhibition of original enzyme activity (IC50) exceeding 100 µM (IC50 = 167.12 ± 5.460 µM) for CYP2E1 enzyme activity. Salicylic acid in rats would have both low and high potential to cause toxicity and drug interactions with other drugs that are substrates for CYP2E1.Inhibition of cytochrome P450 (CYP) alters the pharmacokinetic parameters of the drug and causes drug–drug interactions. Salicylic acid been used for the treatment of colorectal cancer (CRC) and chemoprevention in recent decades. Thus, the aim of this study was to examine the in vitro inhibitory effect of salicylic acid on CYP2E1 activity in rat liver microsomes (RLMs) using high-performance liquid chromatography (HPLC). High-performance liquid chromatography analysis of a CYP2E1 assay was developed on a reversed phase C Members of the cytochrome P450 (CP450) superfamily are known as phase 1 enzymes and play a key role in the biotransformation of a large number of endogenous  and exogenous  compounds to a more hydrophilic form . HoweverBecause P450s play a vital role in drug metabolism, co-administration of a drug with another CYP substrate may alter their metabolism, thus causing a drug–drug interaction . As a deUnderstanding the properties of adverse drug reactions (ADRs) gives us a clear indication for quantification of the side effects of a prospective drug and a good knowledge of the pathogenic pathways involved during the interaction . NowadayIt has been reported by Badyal and Dadhich (2001) that drug–drug interactions could be the fourth or the sixth leading cause of deaths in United States. Drug–drug interactions can be the result of inhibition and induction of P450 enzyme activity . PotentiRelevant studies have suggested that potential drug–drug interactions (pDDIs) are more likely to occur in persons suffering from cardiovascular disease compared to other major diseases . The expAspirin  is a nonsteroidal, anti-inflammatory drug used for the treatment of fever, pain, and inflammation . It is hHowever, there is a lack of information concerning the effect of salicylic acid on other CYPs. There is thus an urgent need for a detailed study on the inhibitory effect of salicylic acid on other P450s enzyme activities. Our previous in vitro inhibition study showed that salicylic acid acts as a non-competitive inhibitor for CYP2C11 enzyme activity . This meThe aim of this study is to investigate the inhibitory effect of salicylic acid on the metabolism of the CYP2E1 isoform in male rat liver microsomes. In this systematic study, the in vitro inhibitory effect of salicylic acid on CYP2E1 enzyme activity was evaluated to determine the potency of salicylic acid in affecting CYP mediated phase 1 metabolism in male rat microsomes, employing chlorzoxazone as a probe substrate for the CYP2E1 enzyme in the presence of different concentrations of salicylic acid.UV-VIS spectrophotometry analysis for CYP2E1 assay was carried out by dissolving chlorzoxazone (200 µM), phenacetin (50 µM), salicylic acid (100 µM), and 6-hydroxychlorzoxazone (50 µM) powder in pure acetonitrile (wavelength cut-off is 210 nm) .The following figure demonstrates the measurements of maximum wavelength of each compound in the CYP2E1 assay.According to the overlain spectra , it is p2O, 15% methanol, and 25% acetonitrile)  (see Method development for CYP2E1 assay was assessed using HPLC low-pressure isocratic elution programming (60% H °C) see .2O, 25% acetonitrile, and 15% methanol). It results in a good separation of the salicylic acid peak from the CYP2E1 metabolite (6-hydroxychlorzoxazone) peak at T = 25 °C using C18 , at a 0.7 mL/min flow rate and wavelength of λ = 282 nm. In order to determine the retention time of each compound in the CYP2E1 assay, each compound was run separately using phenacetin as an internal standard on the HPLC instrument using isocratic elution programming. Specificity was achieved by choosing the right mobile phase composition  according to the International Conference on Harmonization (ICH) guidelines. The % RSD (relative standard deviation at each different concentration (% RSD < 5%)) met the ICH guidelines.Different solution concentrations of chlorzoxazone  and its metabolite 6-hydroxychlorzoxazone  were injected into the HPLC instrument for the determination of the standard calibration curves. Calibration curves were assessed by plotting the mean area peak of standards and phenacetin  (50 µM) versus the concentration of standard (chlorzoxazone and 6-hydroxychlorzoxazone). The outcomes are listed in The results, presented in n = 3). Mean activity was calculated from the following calibration curve linear equation: y = 0.0544 x + 0.0626 (r2 = 0.9997). The outcomes, summarized in Intra-assay variation of chlorzoxazone was determined by measuring three concentration levels  three times in a single batch (n = 3). Mean activity was calculated from the following calibration curve linear equation: y = 0.0164 x − 0.0021 (r2 = 0.9994). The outcomes, summarized in Determination of intra-assay variation of the metabolite was assessed by injecting three levels of 6-hydroxy chlorzoxazone concentrations  into a HPLC instrument three times in a single batch (2 = 0.9997) for day 1, y = 0.0522 x + 0.074 (r2 = 0.9994) for day 2, and y = 0.024 x − 0.0268 (r2 = 0.9994) for day 3. The results, summarized in Inter-assay variation was determined by measuring chlorzoxazone standards of three concentrations levels  for three consecutive days, in separate batches. Mean activity was calculated from the following calibration curve linear equations on days 1, 2, and 3: y = 0.0544 x + 0.0626 (rn = 3). Mean activity was calculated from the following calibration curve linear equations on days 1, 2, and 3: y = 0.0164 x − 0.0021 (r2 = 0.9994) for day 1, y = 0.0248 x − 0.0048 (r2 = 0.9999) for day 2, and y = 0.0277 x − 0.0141 (r2= 0.9996) for day 3. The outcomes are shown in Assessment of inter-assay variation of 6-hydroxychlorzoxazone was carried out by injecting three concentration levels of 6-hydroxychlorzoxazone  for three consecutive days in separate batches (n = 3) for each batch. The calibration curve of chlorzoxazone was run at t = 0, t = 24, t = 48, and t = 72 h. The stability test results are summarized in the The stability of chlorzoxazone was investigated for three different concentrations  stored for 72 h at room temperature in natural light conditions. Phenacetin  was added to each batch. Each sample was analysed in triplicate (2 = 0.9998), day 2: y = 0.0356 x − 0.0103 (r2 = 0.9998), day 3: y = 0.0374 x − 0.132 (r2 = 0.9994), and day 4: y = 0.0358 x + 0.0298 (r2 = 0.9993), where the r2 met ICH guidelines. Percentage recovery values for chlorzoxazone at concentrations 25, 100, and 200 µM were found to be within acceptable criteria (80–120%) according to ICH guidelines. Thus, the results show high and acceptable accuracy (80–120%) for chlorzoxazone concentrations of 25, 100, and 200 µM, because the recovery was high (within acceptable range with ICH guidelines) compared with the standard known concentration. The results indicate that chlorzoxazone solution was stable for 72 h at ambient temperature, in accordance with the study done by Shaikh et al., (2008) [The outcomes in teria 80–0% accordn = 3) for each batch. The calibration curve of 6-hydroxychlorzoxazone was run at t = 0, t = 24, t = 48, and t = 72 h. The stability test results are summarized in the Stability of 6-hydroxychlorzoxazone was investigated for three different concentrations  stored for 72 h at room temperature in natural light conditions. Phenacetin  was added to each batch. Each sample was analysed in triplicate (2 = 0.9999), day 2: y = 0.0259 x + 0.0141 (r2 = 0.9995), day 3: y = 0.0255 x − 0.0305 (r2 = 0.9991), and day 4: y = 0.0251 x – 0.0145 (r2 = 0.9993), where the r2 met within ICH guidelines. Percentage recovery values for 6-hydroxychlorzoxazone at concentrations 10, 40, and 80 µM were found to be within acceptable criteria (80–120%) according to ICH guidelines. Thus, the results shows high and acceptable accuracy (80–120%) for 6-hydroxychlorzoxazone concentrations of 10, 40, and 80 µM, because the recovery was high (within acceptable range with ICH guidelines) compared with the standard known concentration. The results indicate that 6-hydroxychlorzoxazone solution was stable for 72 h at room temperature, in accordance with the study done by Schelstraete et al. (2018) [The outcomes in . (2018) .Different concentrations of chlorzoxazone  were incubated in the presence of 0, 10, 20, 40, and 60 µM of salicylic acid using 0.5 mg/mL of rat liver microsome with 1.0 mM nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), 5.0 mM glucose-6-phosphate (G6P), 1.7 units/mL glucose-6-phosphate dehydrogenase (G6PDH), 1.0 mM ethylenediamine tetraacetic acid (EDTA), and 3.0 mM of magnesium chloride. The reaction was terminated at different sets of time. The incubation time was 40 min. The inhibitory effect of salicylic acid on CYP2E1 enzyme activity is shown in Studying CYP enzyme inhibition is considered as an important route in evaluating drug–drug interactions in the pharmaceutical field and in the drug development process. Several marketed drugs have been withdrawn from the market in the past few decades because CYP enzyme inhibition causes harmful drug–drug interactions . To our The CYP2E1 isoform appears to be the most vulnerable when salicylic acid’s modulatory effect on each individual isoform is considered. Based on its characteristics, the CYP2E1 isoform has a broad substrate specificity, an ability to metabolise major marketed drugs, and is responsible for the catalysis of many carcinogens and poisons [Only one enzyme exists in the CYP2E isoform and this is known as the CYP2E1 isozyme . CYP2E1 In vitro evidence has also revealed that garlic and watercress serve as potent inhibitors of the CYP2E1 enzyme in the human body . In addii = 83.56 ± 2.730 µM and a 50% inhibitory concentration (IC50) value exceeding 100 µM (IC5 0= 167.12 ± 5.460 µM). In addition, salicylic acid at its concentration of 25 µM can considered as a saturated concentration, since its Michaelis constant Km value represents the highest value compared to other salicylic acid concentrations. According to m and the maximum rate of the reaction (Vmax) hanged between each concentration of salicylic acid, and thus salicylic acid acts as a mixed inhibition for CYP2E1 enzyme activity. Thus, in this study it is considered that the in vitro inhibitory effect of salicylic acid on CYP2E1 enzyme activity will be beneficial for a future in vivo study for healthcare screening of the effective use of salicylic acid in clinics and the safe administration of salicylic acid with other drugs, consistent with in vivo drug–drug interactions pharmacokinetic parameters. Some further investigational in vitro and in vivo studies in human CYP450s are needed due to the varied CYP2E isoform expression in species.Recent studies have suggested that the sensitivity of the CYP2E1 enzyme activity can be decreased by certain drugs and food stuffs for example green and black powder tea, ellagic acid, chrysin, N-acetyl cysteine, and dandelion . Our pre+), magnesium chloride (MgCL2), and chlorzoxazone were purchased from Merck . Methanol, acetonitrile, and water were of HPLC grade; salicylic acid, potassium phosphate monobasic, potassium phosphate dibasic, phenacetin with purity greater than 98%, and phosphoric acid (85% w/w) were purchased from Merck .6-Hydroxychlorzoxazone was purchased from Carbosynth limited . Glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH), ethylenediamine tetraacetic acid (EDTA), nicotinamide adenine dinucleotide phosphate  in this study were purchased from Merck  and stored at –80 °C. The manufacturer had previously characterized the microsomes for CYP2E1 activity, CYP450 content, and protein concentrations.18 SUPLCO column (25 cm × 4.6 mm) was employed from Merck  to separate target analytes using a low-pressure isocratic elution system. A UV-VIS Spectrometry instrument with a 1-cm-length quartz cuvette was purchased from VWR International Ltd.  and UV spectra were processed using a Bio 100 Cary software from Agilent Technologies LDA . A 570 pH meter, purchased from JENWAY Limited  was used.A Shimadzu LC-2010A HT high performance liquid chromatography system equipped with a low-pressure pump quaternary gradient (series 200 LC pump), a series 200 Peltier LC column oven for chromatographing the analysed solutions, a series 200 autosampler , a degasser, and a model series 200 UV detector were used. The data were processed using the Shimadzu HPLC 2 Data Lab Solutions  software processing system. A 5-µm C2O, (B): 15% of methanol, and (C): 25% of acetonitrile. The flow rate was set to 0.7 mL/min, and the oven temperature was set at 25 °C. Wavelength detection was set to 282 nm. An injection volume of 10 µL was used. The mobile phase consisted of methanol: water: acetonitrile (15:60:25 % v/v). This provided a good separation and resolution of CYP2E1 components.Validations were conducted using HPLC system LC-2010A HT Module . Chromatographic experiments were evaluated in a low-pressure isocratic system. The separations of the four target components  were injected into a SUPELCO C18 column . The mobile phase for the chromatographic separation of the four compounds was made up as follows: (A): 60% H+) into the mixture [v/v. Tubes were incubated for 40 min in an Eppendorf thermomixer  at 150 rpm (37 °C). Termination of the reaction was carried out by adding ice-cold acetonitrile containing 50 µM of phenacetin as an internal standard at each time interval . Tubes were centrifuged in a microcentrifuge  for 12 min to precipitate protein. Then the supernatant was collected and dissolved in a mobile phase  made up to 1000 µL volume. A 10-µL injection volume of the dissolved supernatant was used for HPLC analysis. CYP2E1 activity was assessed using chlorzoxazone (substrate) via the formation of 6-hydroxychlorzoxazone (the metabolite for the CYP2E1 enzyme). Oxidative metabolism of chlorzoxazone was evaluated using a NADPH-regenerating system consisting of: 1.0 mM nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), 5 mM glucose-6-phosphate (G6P), 1.7 units/mL glucose-6-phosphate dehydrogenase (G6PDH), 1.0 mM ethylenediamine tetraacetic acid (EDTA), and 3.0 mM magnesium chloride. A 500-µL incubation volume containing NADPH-regenerating system and a final concentration of 0.067 M potassium phosphate buffer at pH = 7.4 was incubated in triplicate. The incubation mixture consisted of 0.5 mg/mL of pooled liver microsomes with a serial range of chlorzoxazone concentrations  and a serial range of salicylic acid concentrations . Pre-incubation of all components was done for 10 min in a water bath at T = 37 °C before the addition of nicotinamide adenine dinucleotide phosphate , salicylic acid (100 µM), chlorzoxazone (200 µM), and 6-hydroxychlorzoxazone (50 µM) standard solutions in the UV region of 200–350 nm.2O as mobile phase (B), and HPLC grade acetonitrile as mobile phase (C) .Various mobile phases for CYP2E1 assay were tested. The most suitable mobile phase was: HPLC grade methanol (low UV cut-off of 205 nm) as mobile phase (A), H2O, 25% acetonitrile, and 15% methanol). Serial dilutions were performed, yielding final concentrations of 40, 25, and 10 µM. Chlorzoxazone (3.39 mg) (C = 400 µM) was weighed accurately and dissolved in a mobile phase . A serial dilution of chlorzoxazone stock solution was made, yielding to final concentrations of 300, 200, 150, 100, 50, and 25 µM. Phenacetin powder (0.9 mg) was weighed and dissolved in pure HPLC-grade acetonitrile. The metabolite for the CYP2E1 enzyme (6-hydroxychlorzoxazone) was prepared as a stock solution of C = 100 µM, followed by a range of serial dilutions .Salicylic acid (SA) (1.38 mg) (C = 200 µM) was measured accurately and dissolved in a 50-mL volumetric flask by a mobile phase  as an internal standard. Validation parameters  were calculated using Microsoft Excel 2010 software. All results are presented as mean ± S.D.Vm, Km, Clint (hepatic intrinsic clearance), Ki) were determined by nonlinear regression analysis from secondary Lineweaver–Burk and Michaelis–Menten plots of the enzyme activity-metabolite concentration data. The type of CYP2E1 inhibition was assumed to be mixed inhibition based on the shape of Lineweaver–Burk plots, the standard error, the Akaike information criterion (AIC), and Schwarz criterion (SC) values, which were fitted by a simple non-linear regression model. A non-linear regression model was chosen as a better fit because of its low AIC value. The Akaike weights (∆AIC) are the relative difference between the best model and each other model in the set. The ∆AIC provided substantial evidence for the model (∆AIC < 2). The 50% inhibitory concentration (IC50) value was calculated by using the following mixed inhibition equation:0 is uninhibited velocity, S is slope factor, and I is the inhibitor concentration.Formation of the 6-hydroxychlorzoxazone metabolite of the tested CYP2E1 substrate (chlorzoxazone) was measured for CYP inhibition analysis. Microsoft Excel 2010 software was used for determination of area peak ratios of both metabolite and internal standard. Pharmacokinetic parameters  for chlorzoxazone and 6-hydroxychlorzoxazone were in line with ICH guidelines. The in vitro data provided allow us to understand the drug interactions with salicylic acid. Nevertheless, our findings indicated that salicylic acid potentially acts as a mixed inhibitor (competitive and non-competitive inhibitor) for CYP2E1 enzyme activity in rat liver microsomes. This finding provides useful data for the efficacy and the safe use of salicylic acid in clinical practice. However, the extent to which our obtained data can be related to human CYP isoforms is unknown. The majority of clinically important drugs such as S-warfarin, steroids, and retinoic acid are metabolised by both rat CYP2C11 and human CYP2C9 that display a high identity of amino acid sequences. Thus, it is primarily important to investigate whether human CYP isoforms are induced by salicylic acid, and further verification of the obtained in vitro data should be done with in vivo experiments.


The renin-angiotensin system (RAS) is important in the onset and course of cardiovascular, kidney, and metabolic disorders. Previous reports showed that the RAS blockade protects organs and suppress the development of type 2 diabetes mellitus. A novel component of the RAS, namely, chromosome 9 open reading frame 3 (C9orf3), was recently identified, however, its effects are unclear. We evaluated whether the genetic variant of C9orf3 is associated with morbidity of hypertension among subjects with type 2 diabetes. We enrolled 382 subjects with type 2 diabetes, 222 of whom were diagnosed with hypertension. Human leukocyte genomic DNA was isolated and a genetic variant was analyzed for a C/T variant of C9orf3 (rs4385527) via PCR analysis. The relationship between the genotype and hypertension morbidity among subjects with diabetes was examined. The proportion of the respective C9orf3 genetic variants were as follows 247 CC, 119 CT, and 16 TT. The risk of hypertension was determined to be 1.58, with a 95% confidence interval of 1.11–2.27. Moreover, the p value was 0.012 for allelic comparison and for Armitage’s trend test, with the C allele identified as the risk factor. Consequently, hypertension was markedly associated with type 2 diabetes in subjects with the C9orf3 variant, exhibiting a nearly 1.6-fold increased risk. The C variant of a new component of the RAS, C9orf3 (rs4385527) might have a considerable impact on the pathogenesis of hypertension in diabetes. The RAS also functions in blood pressure regulation and electrolyte metabolism4. Moreover, a specific genotype of the RAS has been shown to contribute to tissue expression, plasma renin activity (PRA) in combination with environmental factors5 and angiotensin receptor blockers6. Previous studies have reported that RAS inhibitors exhibit organ-protective effects9 while suppressing the onset of type 2 diabetes mellitus12. In 2014, we reported that the aldosterone synthase (CYP11B2) C-344T variant was markedly associated with type 2 diabetes morbidity13. Furthermore, multivariate logistic-regression analysis indicated that age, gender, and CYP11B2 genotype were independently associated with type 2 diabetes; and the risk associated with development of type 2 diabetes when individuals had TT haplotype compared to CC/CT was determined to be 1.40 .Excessive activation of the renin-angiotensin system (RAS) plays a pivotal role in the onset and course of cardiovascular15. This aminopeptidase was also called aminopeptidase O, an intracellular member of the M1 metalloprotease family of enzymes. This protein catalyzes the hydrolysis of amino acids and N-terminal residues of peptides or protein substrates. It is expressed in the heart, testis, placenta, liver, and pancreas. C9orf3 was suggested to affect the concentrations of angiotensin-related molecules in tissues. Thus, C9orf3 may be involved in the development of specific clinical conditions. Specifically, a marked relationship between the C9orf3 (rs4385527) genotype and polycystic ovarian syndrome (PCOS) has been reported16, which is a condition that affects hyperandrogenism and is associated with metabolic disorders. PCOS also raises the risk of type 2 diabetes and cardiovascular diseases18, with higher rates of morbidity associated with hypertension in women with PCOS compared to their healthy counterparts19. In this study, the GG haplotype, or CC haplotype, was markedly higher in the PCOS patients than in controls, revealing that the CC haplotype is a risk factor for PCOS18.Recently, a novel component of the RAS, namely, chromosome 9 open reading frame 3 (C9orf3), which is an aminopeptidase cleaving angiotensin III-generating angiotensin IV, was identified in addition to alanyl aminopeptidaseHowever, the role of C9orf3 in hypertension has not yet been examined. We, therefore, sought to determine whether specific genetic variant of C9orf3 was associated with the morbidity of hypertension among subjects with type 2 diabetes.20. Subjects aged less than 20 years and those with type 1 diabetes mellitus, gestational diabetes mellitus, secondary diabetes mellitus, severe organ failure, or acute phase disorders were excluded. At the time of blood sampling, no subjects had been treated with anti-hypertensive or anti-dyslipidemic agents in the previous one week. Subjects with diabetes continued to receive their usual care. Following the Japanese guideline, hypertension is defined as systolic blood pressure (SBP) of ≥ 140 mmHg or diastolic blood pressure (DBP) of ≥ 90 mmHg, measuring at an examination room21.Without any specific inclusion criteria we enrolled 382 subjects who presented at our hospitals with type 2 diabetes between November 2000 and November 2013. The study was approved by the ethics committee of Fukui University (n. 13.1/14.2) and written informed consent was obtained prior to enrollment of all participants. The study has been registered with UMIN-CTR (#UMIN000001580). The study was conducted in accordance with the International Ethical Guidelines and Declaration of Helsinki. Diabetes was diagnosed according to the criteria established by the World Health Organization. Patients with fasting plasma glucose ≥ 126 mg/dL, 2-h post-load plasma glucose after a 75 g oral glucose tolerance test ≥ 200 mg/dL, glycosylated hemoglobin A1c ≥ 6.5%; or a random blood glucose ≥ 200 mg/dL in the presence of signs and symptoms are considered to diabetes13. A genetic variant was analyzed using genomic DNA for a C/T variant of C9orf3 (rs4385527) with the StepOnePlus real-time PCR system and the TaqMan method . The probe mix (primer) of rs4385527 was TaqMan SNP Genotyping Assay (Assay ID: C___7961143_20). The global minor allele frequency of C9orf3 (rs4385527) was 0.23 . The Japanese minor allele frequency of C9orf3 (rs4385527) was 0.21 . The real-time PCR was analyzed by using StepOnePlus software version 2.2.2. The PCR program consisted of Pre-PCR Read (Holding Stage 1) at 50 °C for 2 min, Holding Stage 2 at 95 °C for 10 min, 45 cycles of Cycling Stage , and post-PCR Read (Holding Stage 3) at 60 °C for 30 s.Genotyping was carried out with genomic DNA isolated from human leukocytes using a commercial kit , as previously reported13. Briefly, the sample size of the study was calculated by setting the difference to be detected between gene groups as at least 10% for hypertension prevalence. By using χ2 analysis with a 5% significance level and 80% power, we determined that the study required approximately 400 subjects in total. All statistical analyses were conducted using SPSS Version 22.0 software . The allele frequencies for each genotype were tested by contingency table analysis. A p < 0.05 was regarded as statistically significant. Data were presented as numbers/percentages, means ± SD, or medians (interquartile ranges), as appropriate. The differences between 2 paired continuous variables were analyzed by Student’s t-test or Wilcoxon signed rank test as appropriate. Dichotomous characteristics were compared by χ2 analysis for tests including Hardy–Weinberg equilibrium. Odds ratios for hypertension and 95% CIs were calculated using Armitage’s trend test.This statistical analysis was carried out as previously reported2, and urinary albumin excretion rate was 23.0 (9.0–75.4) mg/g CRE. Mean systolic blood pressure and diastolic blood pressure were 143.8 ± 21.1 mmHg and 82.6 ± 12.4 mmHg, respectively.Overall, 382 subjects were included in this cohort, of which 222 (58.1%) were hypertension cases. Moreover, 227 males (59.4%) and 155 females (40.6%) were enrolled. The mean age of the participants was 61.9 ± 12.0 years, mean Body Mass Index (BMI) was 24.2 ± 4.0, fasting plasma glucose was 155.6 ± 65.6 mg/dL, glycosylated hemoglobin A1c was 7.6 ± 1.5%, estimated glomerular filtration rate was 79.0 ± 26.3 mL/min/1.73 mWe compared the baseline characteristics of subjects who were non-hypertensive to those that were hypertensive Table . BetweenWithin all of the type 2 diabetic patients, the proportion of the C9orf3 haplotype variants were as follows CC (247), CT (119), and TT (16). Moreover, in hypertensive patients, CC accounted for 154 cases, CT for 62 cases and TT for 6 cases. In non-hypertensive controls, CC accounted for 93 cases, CT for 57 cases, and TT for 10 cases. The distributions were similar to the expected values according to Hardy Weinberg equilibrium. The risk of hypertension was determined to be 1.58, with the 95% confidence interval at 1.11–2.27. The p-value was 0.01 for allelic comparison as well as for the Armitage’s trend test, with the C allele found to be a significant risk factor. Consequently, a significant association with hypertension among subjects with type 2 diabetes was observed for the C9orf3 variant with a 1.6-fold increased risk , while the diastolic blood pressure levels in subjects with each genotype were, CC, 83.8 mmHg; CT, 80.8 mmHg; and TT, 78.4 mmHg . For all blood pressure values, the CC allele tended to be associated with high blood pressure compared to the CT and TT alleles , CT (100.6 mmHg), and TT (98.4 mmHg) with a p value of 0.009 when comparing the blood pressure of patients with CC to those with CT. Systolic blood pressure levels in subjects with each genotype were as follows: CC, 146.0 mmHg; CT, 140.2 mmHg, and TT, 138.4 mmHg  compared with CT/TT alleles group (r = 0.371) as well as all subjects (r = 0.374), as we expected  and written informed consent was obtained prior to enrollment of all participants.Not applicable.


Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 Å resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a Expanding our knowledge of SARS-CoV-2 target proteins remains important both for understanding SARS-CoV-2 virology and developing alternative treatments to mitigate against potential resistance that evolves, or new viruses that emerge, in the future19.Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is an ongoing global pandemic. Vaccine and therapeutic development are predominantly focused on the essential virus-encoded Spike, main protease, and RNA-dependent RNA polymerase proteins. High-resolution structures of these targets, some in complex with drug candidates or neutralizing antibodies, have yielded mechanistic insight into their function and have provided a platform for successful structure-guided vaccine and drug design21, the envelope protein (E) and open reading frame (ORF) 3a and 8a2. Viroporins have been shown to modify host membrane permeability to promote viral assembly and release22 and SARS-CoV-1 3a has been reported to form an emodin-sensitive K+-permeable cation channel24. 3a is highly conserved within the Betacoronavirus subgenus Sarbecovirus which includes SARS-CoV-1 and other related bat coronaviruses aviruses 25. BiopsStill, the precise roles of 3a in disease pathogenesis are unknown, precluded in part by the lack of a mechanistic understanding of 3a. Here, we utilize cryo-electron microscopy (cryo-EM), electrophysiology, and fluorescent ion flux assays to elucidate 3a protein structure and function.Spodoptera frugiperda (Sf9) cells with a cleavable C-terminal GFP tag. Purification of 3a in detergent resulted in two species separable by gel filtration  permitted de novo modeling of 195 of the 275 amino acids per protomer chain. The N-terminus (amino acids 1–39), C-terminus (amino acids 239–275), and a short cytoplasmic loop (amino acids 175–180) are either not observed or weakly resolved in the density map due to conformational differences between particles or because they are disordered –E. The 228. Viewed from the membrane plane, 3a is approximately 70 Å tall with a 40 Å high transmembrane region and a cytosolic domain (CD) extending 30 Å out of the membrane  trace the circumference of an ellipse with TM1–3 from one protomer followed by TM1–3 of the second protomer in a clockwise order  and highly complementary interface with residues V168, V225, F230, and I232 forming a continuous hydrophobic core . While the exact positions of side chains cannot be determined at this resolution, residues W131, R134, K136, H150, T151, N152, C153, and D155 are poised to mediate tetramerization through a network of hydrophobic, polar, and electrostatic interactions : Ca2+ (1.88±0.08) > K+ (1.0±0.01) > Na+ (0.59±0.04) > NMDG+ (0.29±0.01)  . Alkalin29±0.01) . Using F2+ influx measurements (50=90 ± 10 μM) with flickery block at negative potentials, similar to that observed with other large pore channels , a 786 Da polycationic dye, blocks 3a activity in current recordings –H and Caurements –I. RuR dchannels . In addicordings . RuR, spe emodin ,E, two rtructure –9.+ (37. Robust YO-PRO-1 uptake was observed in a subset of 3a liposomes that was significantly higher than background fluorescence observed in empty liposomes (50=175 ± 98 μM). These data demonstrate that 3a can form an ion channel that is capable of passing large cations. RuR (786 Da) block and YO-PRO-1 (629 Da) and NMDG+ (406 Da) permeation sets limits on 3a pore size that are similar those observed for TRP and P2X channels.Given the ability of 3a channels to conduct NMDG+ –D, we neiposomes –D. To coiposomes . Using tiposomes . Uptake iposomes , C–E. Siiposomes –F, we ob2+ and NMDG+ permeability, but did not alter Na+ or K+ permeability. These effects were specific to these mutations as seven additional mutations, including a common circulating variant Q57H38, had no effect on relative ion permeability.We speculate that an open conformation of 3a involves substantial rearrangement of the hydrophobic seal to create a central pore through the membrane or more subtle rearrangement of the partially hydrophilic membrane facing sides of TM2 and TM3 to create membrane facing conduction pathways. To probe the structural determinants for 3a ion conduction, we set out to identify 3a mutations that alter channel activity. To this end, we purified nine mutated or truncated 3a constructs, reconstituted each into proteoliposomes, and compared them to wild-type 3a in electrophysiological recordings , S16. Co2+ and NMDG+ relative permeability in proteoliposome recordings, albeit to a lesser extent than Q57E and S58L/Q116L . Distant homology to the CD was identified in the membrane protein ORF5 found in Betacoronavirus subgenus Merbecovirus species including MERS-CoV. However, we identified high confidence structural homologs in all remaining Betacoronavirus and Alphacoronavirus subgenera . Perhaps the presence of 3a in coronaviruses with bats as their principal reservoir reflects a unique aspect of bat coronavirus biology.While 3a is well conserved in the subgenus , relatedCoV-NL63 ), severa2+ permeable cation channel when reconstituted in liposomes. 3a structures show a novel fold with a polar cytoplasmic cavity open to the cytosol and membrane below a hydrophobic seal, consistent with a closed or inhibited state. The basis for 3a channel gating and conduction remains to be determined. SARS-CoV-2 3a channels display permeability to large cations including NMDG+ and YO-PRO-1 and sensitivity to RuR and Gd3+, reminiscent of other Ca2+-permeable channels including TRPV1, TRPA1, and P2X730. Channel activity may be important for promoting viral maturation in cells through inhibition of autophagy and disruption of lysosomes8. The calcium permeability of 3a is of particular interest in the context of infection. SARS-CoV-2 3a has been recently shown to trigger programmed cell death in vitro26 and calcium-influx through 3a may be the switch that activates calcium-dependent caspases and apoptosis. Previous studies showed SARS-CoV-1 3a channel expression in infected lung pneumocytes, a key cell type also infected by SARS-CoV-2, and calcium signaling in type II pneumocytes plays an important role in maintaining airway homeostasis43. Thus, the expression of a calcium permeable channel, like SARS-CoV-2 3a, could impact lung homeostasis and COVID-19 pathogenesis. Our data suggest that 3a may represent a new target for treating COVID-19 and other coronavirus diseases and points the way to future experiments that will elucidate the role of 3a in the viral life cycle and disease pathology.Overall, we demonstrate that SARS-CoV-2 3a forms a non-selective CaSpodoptera frugiperda (Sf9 cells) and synthesized . The sequence was then cloned into a custom vector based on the pACEBAC1 backbone  with an added C-terminal PreScission protease (PPX) cleavage site, linker sequence, superfolder GFP (sfGFP) and 7xHis tag, generating a construct for expression of 3a-SNS-LEVLFQGP-SRGGSGAAAGSGSGS-sfGFP-GSS-7xHis. Mutants and truncation were also introduced into this construct using PCR. MultiBac cells were used to generate a Bacmid according to manufacturer’s instructions. Sf9 cells were cultured in ESF 921 medium  and P1 virus was generated from cells transfected with Escort IV reagent  according to manufacturer’s instructions. P2 virus was then generated by infecting cells at 2 million cells/mL with P1 virus at a MOI ~0.1, with infection monitored by fluorescence and harvested at 72 hours. P3 virus was generated in a similar manner to expand the viral stock. The P2 or P3 viral stock was then used to infect Sf9 cells at 4 million cells/mL at a MOI ~2–5. At 72 hours, infected cells containing expressed 3a-sfGFP protein were harvested by centrifugation at 2500 × g for 10 minutes and frozen at −80°C.The coding sequence for the 3a protein from SARS-Cov-2 was codon optimized for For preparation of the 3a dimer and mutant constructs, infected Sf9 cells from 1 L of culture (~15–20 mL of cell pellet) were thawed in 100 mL of Lysis Buffer containing 50 mM Tris, 150 mM KCl, 1mM EDTA pH 8. Protease inhibitors , Pepstatin A (1 μg/mL), Soy Trypsin Inhibitor (10 μg/mL), Benzamidine (1 mM), Aprotinin (1 μg/mL), Leupeptin (1μg/mL), AEBSF (1mM), and PMSF (1mM)) were added to the lysis buffer immediately before use. Benzonase (4 μl) was added after the cell pellet thawed. Cells were then lysed by sonication and centrifuged at 150,000 × g for 45 minutes. The supernatant was discarded and residual nucleic acid was removed from the top of the membrane pellet using DPBS. Membrane pellets were scooped into a dounce homogenizer containing Extraction Buffer , pH 8). A 10% stock solution of DDM was dissolved and clarified by bath sonication in 200 mM Tris pH 8 prior to addition to buffer to the indicated final concentration. Membrane pellets were then homogenized in Extraction Buffer and this mixture  was gently stirred at 4°C for 1 hour. The extraction mixture was centrifuged at 33,000 × g for 45 minutes and the supernatant, containing solubilized membrane protein, was bound to 4 mL of Sepharose resin coupled to anti-GFP nanobody for 1 hour at 4°C. The resin was then collected in a column and washed with 10 mL of Buffer 1 , 40 mL of Buffer 2 , and 10 mL of Buffer 1. The resin was then resuspended in 6 mL of Buffer 1 with 0.5 mg of PPX protease and rocked gently in the capped column for 2 hours. Cleaved 3a protein was then eluted with an additional 8 mL of Wash Buffer, spin concentrated to ~500 μl with Amicon Ultra spin concentrator 10 kDa cutoff (Millipore), and then loaded onto a Superdex 200 increase column  on an NGC system  equilibrated in Buffer 1. Peak fractions containing 3a channel were then collected and spin concentrated prior to incorporation into proteoliposomes or nanodiscs. For the tetramer, the preparation was carried out in a similar manner, except with overnight protease cleavage and collection of a peak of larger hydrodynamic radius see .44. 3a dimer was first purified into Buffer 1. Protein was then exchanged into lipid with the addition of Biobeads SM2  and an overnight incubation at a protein:lipid ratio of 1:10  in DR Buffer . For the YO-PRO-1 assay, 3a was incorporated at a ratio of 1:50. TRAAK control proteoliposomes were prepared at 1:50 as described previously44. Control liposomes were prepared from the same lipid mix and protocol with protein replaced with Buffer 1.For proteoliposome patching experiments, we incorporated protein into lipid and generated proteoliposome blisters for patch recordings using dehydration and rehydration as described previously with the following modifications2 for permeability experiments. Borosilicate glass pipettes were pulled and polished to a resistance of 2–5 MΩ when filled with pipette solution. For cation permeability experiments, liquid junction potentials were calculated and data were corrected offline. For current-voltage plots, the following voltage protocol was applied: Vhold= 0 mV mV; Vtest= −100 to +100 mV, Δ20 mV, ttest = 1 second. Currents from each patch correspond to mean values during the step to the indicated voltage.All electrophysiology recordings were made from 3a-reconstituted Soy PC proteoliposomes. Patches formed in an inside-out configuration and were quickly (within 5–10 seconds) transferred to a solution exchange chamber. Recordings were made at room temperature using Clampex 10.7 data acquisition software with an Axopatch 200B Patch Clamp amplifier and Digidata 1550B digitizer (Molecular Devices) at a bandwidth of 1 kHz and digitized at 500 kHz. A pressure clamp (ALA Scientific) was used to form seals. Potassium pipette and bath solution was 5 mM HEPES pH 7.2, 150 mM KCl. Sodium bath solution was 5 mM HEPES pH 7.2, 150 mM NaCl. NaCl in the bath solution was substituted for 150 mM NMDG-Cl or 75 mM CaClX+/PK+ = exp(ΔVrevF/RT). For divalent cations, permeability ratios were calculated as: PX2+/PK+ = αK+[K+]exp(ΔVrevF/RT)(1 + exp(ΔVrevF/RT))/ 4αX2+[X2+] where Vrev is the reversal potential, F is Faraday’s constant, R is the universal gas constant, and T is absolute temperature (where RT/F = 25.2 mV at room temperature), and α is the ion activity coefficient (assumed to be 0.75 for K+ and 0.25 for Ca2+).Permeability ratios were calculated according to Goldman-Hodgkin-Katz relationship. For monovalent cations, permeability ratios were calculated as P45 25 mM HEPES pH 7.4, 150 mM KCl, and 262 mM Sucrose with a final concentration of 25 uM Fluo-5N . Next, the liposome mixtures in Eppendorf tubes were placed in a foam flotation in an ice bath and sonicated with a Branson Digital Sonifier 450 for 30 seconds total . Excess (unincorporated) Fluo-5N was then removed using microspin G-50 columns (Cytiva). The sample was then diluted into solution containing 50mM HEPES, 300mM KCl, and 2mM EGTA, pH=7.4 and plated onto poly-D-lysine  coated 96-well plates, and centrifuged  at room temperature. Ca2+ influx was measured upon addition of a 50 mM HEPES, 285 mM KCl, 10 mM CaCl2, pH=7.4 solution. Images were acquired every 3 seconds for a total of 150 seconds. Fluorescence intensity of each liposome was normalized to its average fluorescence prior to Ca2+ addition.Fluo-5N was incorporated into 3a-reconstituted proteoliposomes and control liposomes by first thawing frozen liposomes at a ratio of ~1:20 (v:v) into modified sucrose formation bufferLiposomes were diluted in saline solution containing 300mM KCl, 50mM HEPES, pH=7.4 and plated onto poly-D-lysine  coated 96-well plates, centrifuged  and incubated in 10μM YO-PRO-1 iodide (Invitrogen) for 10 min at room temperature. Liposomes were rinsed with saline solution to remove free YO-PRO-1. Images were acquired using an ImageXpress Micro XLS microscope with a solid-state light source and 20X air objective. Images were analyzed using MetaXpress 6 software (Molecular Devices). Empty liposomes displayed low-level fluorescence in the 488/540nm range. Thus, liposomes were defined as YO-PRO-1+ if their fluorescence intensity was above the average intensity of unlabeled, empty liposomes. To calculate the total number of liposomes, we quantified the total number of liposomes using auto segmentation on MetaXpress 6 from brightfield images. Stock solutions of Ruthenium red (Tocris) were prepared in water, diluted in bath solution and applied to liposomes or cells 10min before the start of the experiment. All statistical tests were performed using Prism (GraphPad). Values are reported as the mean ± SD or mean ± SEM as indicated. Mann Whitney or a one-way or Welch’s ANOVA followed by the Dunnett’s, Sidak’s, or Tukey’s post hoc tests (where appropriate) were used for statistical comparisons.46 without cleavage of the His-tag. Tetrameric 3a in nanodiscs was prepared similarly, except with a ratio of 1:2:200 .Freshly purified 3a dimer in Buffer 1 was reconstituted into MSP1E3D1 nanodiscs with a mixture of lipids  at a final molar ratio of 1:4:400 . First, 20 mM solubilized lipid in Nanodisc Formation Buffer  was mixed with additional DDM detergent and 3a protein. This solution was mixed at 4°C for 30 minutes before addition of purified MSP1E3D1. This addition brought the final concentrations to approximately 15 μM 3a, 60 μM MSP1E3D1, 6 mM lipid mix, and 10 mM DDM in Nanodisc Formation Buffer. The solution with MSP1E3D1 was mixed at 4°C for 10 minutes before addition of 200 mg of Biobeads SM2. Biobeads  were weighed after liquid was removed by pipetting (damp weight). This mix was incubated at 4°C for 30 minutes before addition of another 200 mg of Biobeads . This final mixture was then gently tumbled at 4°C overnight (~ 12 hours). Supernatant was cleared of beads by letting large beads settle and carefully removing liquid with a pipette. Sample was spun for 10 minutes at 21,000 × g before loading onto a Superdex 200 increase column in 20 mM HEPES, 150 mM KCl, pH 7.4. Peak fractions corresponding to 3a protein in MSP1E3D1 were collected, 10 kDa cutoff spin concentrated and used for grid preparation. MSP1E3D1 was prepared as describedDimeric 3a in MSP1E3D1 was prepared at final concentration of 1.1 mg/mL. For the sample with emodin , a stock solution of 50 mM emodin in DMSO added to protein sample for final concentrations of 1.1 mg/mL 3a and 100 μM emodin and 1% DMSO. Concentrated sample was cleared by a 10 minute 21,000 × g spin at 4°C prior to grid making. For freezing grids, a 3 μl drop of protein was applied to freshly glow discharged Holey Carbon, 300 mesh R 1.2/1.3 gold grids . A FEI Vitrobot Mark IV (ThermoFisher Scientific) was used with 4°C, 100% humidity, 1 blot force, a wait time of ~5 seconds, and a 3 second blot time, before plunge freezing in liquid ethane. Grids were then clipped and used for data collection. Tetrameric 3a in MSP1E3D1 was frozen at 0.7 mg/mL with the same grid preparation. Unimaged grids from the same session were then shipped to the Netherlands for data collection on the 300 kV microscope with Falcon 4/CFEG/Selectris energy filter.− Å2 s−1 and 10.135 e− Å2 s− and total dose was 50.02 e− Å2 and 53.72 e− Å2 in the first set (1–2007) and second set (2008–6309) of movies respectively. The two different doses are the result of needing to restart the electron gun during collection. For the 3a with added emodin dataset, the electron dose was 8.991 e− Å2 s−1 and total dose was 47.21 e− Å2. For the 3a tetramer, the electron dose was 8.841 e− Å2 s−1 and total dose was 49.95 e− Å2. Nine movies were collected around a central hole position with image shift and defocus was varied from −0.6 to −2.0 μm through SerialEM47.For data collected on the Talos Arctica, grids were clipped and transferred to the microscope operated at 200 kV. Fifty frame movies were recorded on a Gatan K3 Summit direct electron detector in super-resolution counting mode with pixel size of 0.5685 Å. For the apo 3a dataset, the electron dose was 9.528 e48 of Falcon 4 detector at a magnification of 165 kX corresponding to a pixel size of 0.727 Å. Each movie stack was recorded with an exposure time of 6s with a total dose of 50e/A2 on sample and a defocus range between 0.5 to 1.2 μm.For data collected for the high resolution 3a dimer structure, clipped grids from the same batch used on Talos Artica were sent to Thermo Fisher Scientific RnD division in Eindhoven, The Netherlands. Grids were loaded onto the Krios G4 microscope equipped Cold Field Emission gun (CFEG) operated at 300 kV. Data were collected on a Falcon 4 detector that was mounted behind a Selectris X energy filter. The slit width of the energy filter was set to 10eV. 5599 movie stacks containing 1429 raw frames were collected with EER (electron event representation) modeSee Table 1 for detailed data collection statistics.51. CTFFIND-4.1 was used to estimate the contrast transfer function (CTF) parameters52. Micrographs were then manually sorted to eliminate subjectively bad micrographs, such as empty or contaminated holes, resulting in 3,611 good micrographs. Additionally, micrographs with a CTF maximum resolution lower than 4 Å were discarded, resulting in 2,595 remaining micrographs. Template-free auto-picking of particles was performed with RELION3.1’s Laplacian-of-Gaussian (LoG) filter yielding an initial set of particles. This initial set of particles were iteratively classified to generate templates, which were subsequently used to template-based auto-pick 1,750,730 particles.For the apo 3a dimer, motion-correction and dose-weighting were performed on all 6,309 movies using RELION 3.1’s implementation of MotionCor2, and 2x “binned” to 1.137 Å per pixel53, resulting in 820,543 particles. These particles were subsequently 3D-classified in cryoSPARC v2 with iterative ab-initio and heterogeneous refinement jobs. The resulting maps were visually evaluated with regard to the transmembrane domain density. A set of 86,479 particles were identified, polished in RELION3.1 and refined in cryoSPARC v2 with subsequent homogeneous and non-uniform refinement54 jobs , and the dynamic mask was tightened with the near (2–5 Å) and far (3–9 Å) parameters), yielding a map with overall resolution of 3.6 Å. UCSF pyem tools were used to convert data from cryoSPARC to RELION format55.Template picked particles were iteratively 2D-classified in RELION3.1 and then in cryoSPARC v256. Training, picking, and extraction were performed independently on each subset of the micrographs. 4,134,279 total particles were extracted in RELION3.1 with a box size of 256 pixels and “binned” 4x to 4.548 Å/pixel. These particles were then iteratively 2D-classified in RELION3.1 resulting in 2,674,606 particles which were extracted at 2.274 Å/pixel. 2D-classification was continued in both RELION3.1 and cryoSPARC v2 resulting in 1,429,763 particles. Further classification was performed in cryoSPARC v2 with subsequent ab-initio  and heterogeneous refinement  jobs. The two best classes were selected and the particles pooled resulting in 743,800 particles which were extracted in RELION3.1 at 1.137 Å/pixel.From this set of 86,479 particles, 2D-classification was performed in RELION3.1 to identify a set of particles with subjectively equal view distribution. From the resulting set, 1,000 particles were randomly sampled and their coordinates used for training in the Topaz particle-picking pipelineIterative 3D-classification was performed with subsequent ab-initio and heterogeneous refinement jobs as described above. Following each round, 2D classification jobs were used to “rescue” good particles from the worst classes before the next round. After 3 rounds, a final 2D-classificaiton job was used to identify 112,502 particles, which were subsequently pooled with the previous 86,479 RELION3.1 template-picked particles, resulting in 185,871 particles after duplicates (within 100 Å) were removed with RELION3.1.These particles were then refined with subsequent homogeneous and non-uniform refinement jobs resulting in a map with overall resolution of 3.4 Å. This map was post-processed in RELION3.1 using a mask with a soft edge , the output of which was used for Bayesian particle polishing in RELION3.1 (training and polishing were each performed independently on each subset of the micrographs). The resulting “shiny” particles were then refined in cryoSPARC v2 with subsequent homogenous refinement  and non-uniform refinement  to yield a map with 2.9 Å overall resolution.For 3a dimer with added 100 μM emodin, initial processing was similar to the dimer without added drug see . As withFor the 3a tetramer, the initial 7,092 micrographs were first cleaned using manual inspection and removal of images with < 4 Å CtfMaxResolution to obtain a set of 4,324 micrographs. Reference particles for Topaz particle picking were generated by first template picking in RELION3.1, followed by 2D classification in both RELION3.1 and cryoSPARC v2, and subsequent ab-initio in cryoSPARC v2. Particles from various views were then selected from iterative RELION3.1 2D classification to create a set of 6,843 particles. Using these coordinates for training, Topaz particle picking was then performed to generate a set of 1,282,913 initial particles. These particles were then cleaned using 2D classification in RELION3.1 and cryoSPARC v2, followed by rounds of cryoSPARC v2 ab-initio and RELION3.1 3D classification. A major hurdle for tetramer processing was obtaining a reconstruction where most particles were properly oriented in the same direction  that achieved a non-uniform reconstruction at 2.17 Å. Finally, rounds of CtfRefine in cryoSPARC v2 and a final non-uniform refinement (with higher-order CTF terms enabled) achieved a map with an estimated resolution of 2.1 Å. Depending on inputs into sharpening and local resolution jobs , the map57 analysis performed in cryoSPARC .We note that the merging of two Topaz picked particle sets likely allowed us to break a particle-limited barrier to achieve the final reconstruction based on ResLogPARC See . Finally59. Real space refinement of the models was carried out using Phenix.real_space_refine60. Molprobity61 was used to evaluate the stereochemistry and geometry of the structure for subsequent rounds of manual adjustment in Coot and refinement in Phenix. For final sharpening and visualization of the high-resolution map we used Phenix Resolve Density Modification62. Docking of the apo dimeric 3a into the tetrameric 3a cryo-EM map was performed in Phenix using a map in which large empty regions of the nanodisc were erased in Chimera63. Similar results were found using maps with only the CDs present. Cavity measurements were made with HOLE64 implemented in Coot. Comparisons to the structure database was performed with DALI28. Structure prediction was performed with Phyre265. Figures were prepared using PyMOL, Chimera, ChimeraX66, Fiji, Prism, GNU Image Manipulation Program, and Adobe Photoshop and Illustrator software.Apo dimeric 3a cryo-EM maps were sharpened using cryoSPARC and were of sufficient quality for de novo model building in CootSf9 cells (~4 million) from the third day of infection were pelleted, frozen, and then thawed into extraction buffer . Extraction was performed at 4°C for 1 hour and lysate was then pelleted at 21,000 × g at 4°C for 1 hour to clear supernatant. Supernatant was then run on a Superose 6 Increase column with fluorescence detection for GFP into 20 mM HEPES pH 7.4, 150 mM KCl, .025% DDM.The constructs for full length 3a and 3aΔN were cloned into a vector with a CMV-promoter and C-terminal EGFP. Constructs (2 μg) were transfected into HEK293 cells on glass coverslips using Fugene HD  per manufacturer’s instructions. Two days after transfection cells were washed with DPBS and then fixed in 4% Formaldehyde in DPBS for 10 minutes. Cells were then washed with DPBS before mounting the coverslip with Prolong Glass Antifade with NucBlue (ThermoFisher Scientific) per manufacturer’s instructions. Fluorescent images were collected using a Zeiss LSM 880 NLO AxioExaminer confocal microscope at either 20X (NA 1.0) or 63X oil immersion objective (NA 1.4). The samples were excited with 488nm argon laser and image analysis was performed using ImageJ.1


Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR4O and RecR4O2 complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR4O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination. Recombination mediator proteins (RMPs) are essential factors in genome maintenance as they facilitate initiation of homologous recombination. Examples of RMPs include the UvsY protein of phage T4 , RecFOR Escherichia coli  (E. coli SSB (SSB-Ct), termed the acidic tip  ,28 that idic tip . This hiidic tip ,52. One idic tip ,51,53–62idic tip , replicaidic tip  and repaidic tip  via the idic tip ,82, up tidic tip . The lasidic tip  22)..EschericEscherichia coli RecR does not bind SSB or DNA . Spectrophotometric grade glycerol was from Alfa Aesar . Buffer BTP is 20 mM Bis-Tris Propane (pH 8.0 at 25°C), 50 mM NaCl, 25% (v/v) glycerol, 1 mM TCEP. Any variations in pH, temperature, NaCl or MgClEscherichia coli RecO protein was overexpressed from plasmid pMCSG7 in E. coli strain BL21(DE3)pLysS  and purified using Ni-NTA affinity chromatography and a HiTrap Heparin HP affinity column  after His-tag cleavage with TEV protease as described (E. coli strain BL21(DE3) transformed with PRK793 and pRIL  and purified as described (Escherichia coli RecR protein was overexpressed from plasmid pMCSG7 in E. coli strain BL21 Rosetta 2(DE3)pLysS (kindly provided by Dr Sergey Korolev) and purified using Ni-NTA affinity chromatography, followed by cleavage of the His-tag with TEV protease as described . The P15 peptide concentration was determined in buffer BTP from its absorption spectrum using an extinction coefficient of ϵ258 = 390 M−1 cm−1 due to its two Phe residues.A peptide containing the 15 C-terminal amino acids (PSNEPPMDFDDDIPF) of Sedimentation velocity and equilibrium experiments were performed with an Optima XL-A analytical ultracentrifuge using An50Ti or An60Ti rotors  at 42 000 rpm for sedimentation velocity and at the indicated rotor speeds between 18,000 rpm and 30 000 rpm for sedimentation equilibrium experiments at 25°C as described . Absorbain = number of moles of species ‘i’, iM = molecular weight of species ‘i’, and i’.The densities and viscosities of the buffers at 25°C were calculated using SEDNTERP . The parmentclass2pt{minim4O and RecR4O2 complexes in buffer BTP are 0.716 and 0.720 ml/g, respectively.The calculated values of c(s) distributions (c(s) distribution function defines the populations of species with different sedimentation rates and represents a variant of the distribution of Lamm equation solutions (c(s) distributions over the entire sedimentation coefficient range used for fitting the data in Sedfit (Sample (380 μl) and buffer (394 μl) were loaded into each sector of an Epon charcoal-filled two-sector centerpiece. Absorbance data were collected by scanning the sample cells at intervals of 0.003 cm. Data were analyzed using Sedfit to obtain ibutions . The c(solutions . Weight n Sedfit .Sedimentation velocity experiments were performed at multiple RecO (ranging from 1.5 to 8 μM) and RecR concentrations (ranging from 2 to 20 μM monomer) as indicated in the text and figure legends. In titrations of RecO by RecR, 1.5 μM RecO was titrated by increasing concentrations of RecR (1.5–9 μM) at [RecO]:[RecR] molar ratio of 1:1, 1:2, 1:3, 1:4 and 1:6. In forward titrations of RecR by RecO, 2 μM of RecR was titrated by 4 and 6 μM of RecO at [RecO]:[RecR] molar ratio of 2:1 and 3:1.Lobs is the RecR tetramerization equilibrium constant. K2, K3 and K4 are the step-wise association equilibrium constants, corrected for statistical factors, for RecO binding to RecR2, R4 and R4O, respectively. Simulations were performed for RecO (1.5 μM) and RecR (1.5–9 μM) at [RecO]:[RecR] molar ratio of 1:1, 1:2, 1:3, 1:4 and 1:6 as in experiments in Figure K3 and K4 values. The value of Lobs was fixed as this was obtained from analysis of an independent set of sedimentation equilibrium experiments with RecR alone . K2 was fixed at a low value (10 M−1) as the RecR2O species is not observed in any experiments (see Results). The reverse rate constants for all reactions were set to 0.01 s−1; lower values did not affect the results. The extinction coefficients at 230 nm were determined as 1.55 × 105 M−1 cm−1 for RecO and 5.3 × 104 M−1 cm−1 for RecR from known protein concentrations and extinction coefficients at 280 nm. The meniscus and the bottom of the cell were set at 6.14 cm and 7.2 cm, respectively. These values are similar to what we observe in typical sedimentation velocity experiments. Standard deviation of noise of 0.005 was added. The simulated data were then analyzed using Sedfit to obtain c(s) distributions  peaks observed by sedimentation velocity in Figure Lobs, from NLLS analysis as described below.Sedimentation equilibrium experiments with RecR were analyzed in two ways. In the first analysis, we determined that the two Ar, were analyzed using NLLS fitting to Equation  and multiple rotor speeds (ranging from 20 000 to 30 000 rpm) as indicated in the text and figure legends. The protein sample (110 μl) and buffer (120 μl) were loaded into each sector of an Epon charcoal-filled six-channel centerpiece. Absorbance data were collected by scanning the sample cells at intervals of 0.003 cm in the step mode with 5 averages per step. Samples were sedimented to equilibrium at the indicated rotor speeds, starting with the lowest speed. Equilibrium was deemed to have been reached when successive scans made several hours apart showed no differences. The resulting absorbance profiles, Equation  to estimLobs = [R4]/[R2]2, the RecR sedimentation equilibrium data were fit to a ‘Dimer–Tetramer’ equilibrium model \docu\docuLobs2]/[Rtot] and [R4]/[Rtot], respectively, and Rtot is the RecR concentration in dimer units.Species fractions of RecR were simulated using the tetramerization equilibrium constants obtained from sedimentation equilibrium experiments using Scientist  using Equations  and 5),,5),(4)\dLobs) as a function of pH and were fit to a model of m protonation sites on a tetramer and n protonation sites on a RecR dimer as described by Equation m and Pdi = (1 + kdi[H+])n for m and n identical and independent protonation sites, and the binding polynomial for m cooperative protonation sites on R4 is Ptet,coop = 1 + (ktet[H+])m. The ktet and kdi are the equilibrium constants for protonation of the RecR tetramer and dimer, respectively. The binding polynomials Ptet,coop and Pdi were used to fit the data in Figure Ptet and Pdi were used to fit the data in The tetramerization equilibrium constants obtained were plotted in Figure Equation ,(6)\docuSedimentation equilibrium experiments were performed at two different molar ratios of RecO (1.5 μM) to RecR (1.5 and 6 μM in monomers) and multiple rotor speeds (ranging from 18 000 to 29 000 rpm) as indicated in the text and figure legends.Buffer BTP used in our experiments contains 25% (v/v) glycerol, which increases the solubility of the proteins and thus concerns can be raised about whether a glycerol gradient that can form may affect the conclusions drawn from the sedimentation experiments. Although a glycerol gradient will form in these experiments, its effect does not influence our conclusions as discussed in Supplementary Materials.monomer) in buffer BTP, pH 8.0 at 25°C. The heats of dilution were obtained by blank titrations in which P15 was titrated into a solution containing only RecR (8 μMmonomer), and corrections for heats of dilution were applied.Isothermal titration calorimetry (ITC) experiments were performed using a VP-ITC titration microcalorimeter  . All pron), observed association equilibrium constant (Kobs) and binding enthalpy (ΔHobs), were obtained by fitting the titration curves to a model of P15 (X) binding to n identical and independent sites on the RecO (M) using Equation (ith injection and X) was obtained by solving Equation \docui’th injection and x is the free P15 concentration. Nonlinear least-squares fitting of the data was performed using the MicroCal Data Analysis software. The conversion of integral heats (In Equations  and 9),,9), \docescribed .s0 = 0.87 ± 0.04 S in buffer BTP at 25°C. From this, we calculate Previous studies using sucrose gradient sedimentation  and X-raHydroPRO  and a crHydroPRO ,107.Sedimentation equilibrium experiments with RecO (4 μM) in buffer BTP (pH 8.0) at 25°C were performed at three rotor speeds to determine an absolute molecular weight . Each eqE. coli RecR protein using sedimentation velocity in buffer BTP (pH 8.0) at 25°C at eight concentrations between 2 – 20 μM (monomer). At each protein concentration, c(s) analysis indicates the presence of two major peaks at ∼1.2 and ∼1.7 S  in buffer BTP (pH 8.0) at 25°C at three rotor speeds Figure Biv, and Sedimentation velocity c(s) profiles at eight RecR concentrations at pH 7.0 and 7.5 similarly show two major peaks at ∼1.2 and ∼1.7 S Figure Ai and iiLobs = [R4]/[R2]2, at each pH by constraining the MW of the dimer and tetramer to their known values while fitting for Lobs using Equation (Lobs = (2.16 ± 0.05) × 105 M−1. The values of Lobs obtained at pH 6.4, 7.0, 7.5, 8.0, 8.5 and 9.0 are given in Table Lobs) versus pH in Figure Lobs obtained from sedimentation equilibrium experiments in Figure Lobs < 500 M−1 at pH 9.We next performed sedimentation equilibrium experiments with RecR in buffer BTP at 25°C at concentrations of 4, 8 and 12 μM (monomer) at six pH values between 6.4 and 9 Figure . We analEquation . The gloLobs are nearly constant at low pH < 7.5, but then decrease steeply at pH > 7.5. The slope of the curve in Figure Lobs/Lobs/Lobs on pH can be described by a model  × 109 M−1. Other models were also considered  × 104 M−1 (Lobs = (3.49 ± 0.48) × 104 M−1 (Lobs = (1.49 ± 0.17) × 105 M−1 , 25°C by titrating RecO (1.5 μM) with increasing concentrations of RecR in a series of sedimentation velocity experiments. At low concentrations of 1.5 μM and 3 μM RecR (monomer)  peak at ∼0.8 S is observed, reflecting unbound RecO, along with a broader c(s) peak centered at ∼2.5 S, consistent with a RecOR complex species since it has a higher sedimentation coefficient than unbound RecO (0.8 S), RecR dimer (1.2 S) or RecR tetramer (1.7 S). At RecR concentrations ≥4.5 μM  peak at ∼2.5 S increases in amplitude and a new c(s) peak at ∼1.2 S appears, corresponding to free RecR dimer. Importantly, no c(s) peak corresponding to free RecR tetramer is observed even at the highest RecR concentration of 9 μM  peak corresponding to unbound RecO (0.8 S) is observed at RecR concentrations ≥4.5 μM, indicating all RecO is bound to RecR. Together with the observation of an unbound RecR2 peak (1.2 S), saturation of RecO indicates that there exists a mixture of RecOR complexes with different stoichiometries. In fact, the RecOR peak at 9 μM RecR  occurs, due to a redistribution of RecO among the RecR molecules. This result is further discussed below along with results from sedimentation equilibrium experiments.A previous study using sucrose gradient sedimentation  concludemonomer = 1:1 , rather than a RecR4O complex (115.2 kDa).Sedimentation equilibrium experiments were performed at two RecO to RecR molar ratios to determine the stoichiometries of the RecOR complex species. At [RecO]:[RecR]1 Figure Ai, the d1 Figure Ai, red. 4O2 complex (142.6 kDa)  Figure Aii.4O2 and RecR4O species and tried fitting the sedimentation equilibrium data to a three species model while fixing the molecular weight of one species to that of RecR2 and floating the molecular weights of the other species. However, we could not resolve the MW of the two species using this model. We conclude from these results that the RecR4O2 species is the primary RecOR species formed at a molar ratio of 1:4. However, recall that we noted that the RecOR c(s) peak undergoes a slight shift to lower s values at higher RecR concentrations   .Lobs is the RecR dimer–tetramer equilibrium constant determined independently from sedimentation equilibrium experiments of RecR alone. K2 is the association equilibrium constant of RecO binding to RecR dimer to form RecR2O. K2 was fixed at 10 M−1 since RecR2O was not observed in our experiments as discussed in the next section. K3 and K4 are the association equilibrium constants for RecO binding to RecR4 and RecR4O, respectively. The simulated data were then analyzed to obtain c(s) distribution profiles (c(s) distribution profiles from the SedAnal analysis are shown in Figure K3 and K4 determined from the SedAnal simulations that best describe the experimental data are K3 = 5 × 105 M−1, K4 = 4 × 108 M−1 , hence RecO binds with positive cooperativity to the RecR tetramer. The statistical factors associated with these equilibria are noted in Scheme c(s) distribution profiles in Figure To obtain a more quantitative interpretation of the RecO–RecR sedimentation velocity profiles in Figure profiles . The bes1 Figure . These K2O complex is 71.3 kDa, and we would expect the RecR2O species to be observed in a sedimentation velocity experiment at ∼1.4 S, as estimated from its molecular weight. Although this value is closer to the sedimentation coefficient of RecR dimer at 1.2 S and would be difficult to resolve, a significant peak at 1.4 S is not observed . F. F2O comd Figure Ai and ii2O complex can form, sedimentation velocity experiments were performed with increasing excess concentrations of RecO added to a constant concentration of RecR (2 μM (monomer))  Figure . When Re) Figure . HoweverE. coli SSB C-terminal acidic tip to RecO has any effect on RecO-RecR interactions. Our previous studies showed that a 15-residue peptide containing the C-terminal tip of SSB (PSNEPPMDFDDDIPF), referred to as P15, forms a 1:1 complex with RecO with equilibrium constant K = (1.2 ± 0.3) × 107 M−1 in buffer BTP (pH 8.0) at 25°C (We next examined whether the binding of the  at 25°C . Based o4O2 complex (146.0 kDa) bound with two P15 molecules ) and multiple excess RecO concentrations in the presence of P15. In contrast to the results in the absence of P15 Figure , a RecORs Figure  indicatic(s) peak for the RecOR complexes shows a notable shift from 2.5 to 2.2 S with increasing [RecR] suggesting formation of a smaller RecOR complex. At the higher RecR/RecO molar ratio of 4:1  and increasing concentrations of RecR in the presence of a 6-fold excess of P15 over RecO. The species distributions Figure  show fre1 Figure , blue, ad Figure , blue. T4O versus RecR4O2, we performed sedimentation equilibrium experiments in the presence of P15 at different [RecO]:[RecR] molar ratios  distribution for the RecOR complex from ∼2.5 to ∼2.2 S with increasing [RecR]  profiles are shown in Figure K3 = 2.5 × 108 M−1 and K4 = 1 × 107 M−1, which differ considerably from the values estimated in the absence of P15. Comparison of the values of K3 and K4 estimated in the presence and absence of P15 shows a dramatic effect of P15 on both equilibrium constants. In the presence of P15, the value of K3 increases whereas the value of K4 decreases compared to their values in the absence of P15. This results in a much lower ratio of K4/K3 = 4 × 10−2 in the presence of P15 indicating negative cooperativity. Hence, in the presence of P15, the first RecO molecule now binds stronger and the second RecO binds weaker to RecR4. Recall that in the absence of P15 RecO binding to the RecR tetramer displays positive cooperativity.To obtain a more quantitative interpretation of these sedimentation velocity experiments we used SedAnal  to simulKO-P15 = (1.2 ± 0.3) × 107 M−1 with ΔH = –5.2 ± 0.1 kcal/mol. In the presence of RecR, the measured ΔH for the P15–RecO interaction is less than 1 kcal/mol, near the limit of detection of the instrument, and therefore we are unable to obtain accurate binding parameters. Qualitatively, however, both the binding affinity and enthalpy are clearly reduced significantly in the presence of RecR  at 25°C containing a 1:4 molar ratio of RecO to RecR (monomer), with RecO at 2 μM, the same concentration used in a previous study of P15 binding to RecO . The repEscherichia coli RecO is an essential recombination mediator protein in the RecF DNA repair pathway, which also involves RecF and RecR along with RecQ helicase, RecJ exonuclease, and SSB  that differ from those used in our study concluded that concluded that E. coli RecO is monomeric and E. coli RecR is dimeric (2) for a range of RecR concentrations (2–20 μM) in the presence of 25% glycerol to enhance protein solubility. The formation of tetramers is accompanied by an uptake of protons in the pH range from 8.0 to 9.0. We observe that inclusion of 10 mM MgCl2 promotes RecR dimerization, which may explain why RecR tetramers were not observed in the previous study  under solution conditions  that are composed of two domains, an N-terminal 112 amino acid DNA binding domain, which forms an OB-fold, and a 65 amino acid intrinsically disordered C-terminal domain, the last 9 amino acids of which, the Ct acidic tip, form the major site of interaction with an array of genome maintenance proteins (SIPs), including RecO. The 56 amino acid intrinsically disordered linker (IDL) is essential for cooperative binding of SSB to ssDNA (4O is observed to be the primary species. Hence binding of the SSB-Ct to RecO shifts the RecR4O–RecR4O2 equilibrium to favor RecR4O.to ssDNA ,117,118.to ssDNA . The genE. coli RecO shows the SSB-Ct tip bound in a hydrophobic pocket in the central alpha helical region (Figure E. coli RecQ helicase and RadD, have also recently been demonstrated (E. coli RadD is stimulated by SSB-Ct peptide both in the presence and absence of DNA (A crystal structure of n Figure , separatn Figure  indicatinstrated ,80,119. nstrated  and the 4O complex, with a RecR tetramer encircling the DNA (Figure 2 dimer. After formation of a RecR4O2, one of the RecO molecules would likely dissociate due to negative cooperativity that stabilizes a RecR4O complex. The absence of the second RecO may allow the RecR4 tetramer to recruit RecA to be loaded onto the ssDNA gap. The ensuing formation of a RecA filament on the ssDNA results in SSB displacement (Figure 15 (Based on the results reported here, we suggest a model for the RecA loading pathway facilitated by RecOR as depicted in Figure A Figure  and C. At Figure . Previout Figure . Althougigure 15 , the bingkaa1291_Supplemental_FileClick here for additional data file.


Mechanically millled Ba1.5Sr1.5Co2Fe24O41 (Ba1.5Sr1.5Z) hexaferrite particles were used to increase permeability in the interesting frequency band. The microwave properties of Ba1.5Sr1.5Z composites were predicted and measured. Hansen’s zero-order analysis of antenna bandwidth and electromagnetic field simulation showed that the hexaferrite-based flexible composite could enhance a bandwidth and achieve the miniaturization of antennas as well. The magnetic antenna substrates can be a good solution to integrate antennas into the UAVs whose dimensions are comparable to or larger than communication wavelength.Our goal is to fabricate flexible magnetic polymer composites as antenna substrates for very high frequency (VHF)/low ultra high frequency (UHF) antennas for unmanned aerial vehicles (UAVs) and medical devices. Magnetodielectric materials, which have permeability ( Considerable efforts have been devoted to the miniaturization of high frequency devices to satisfy demands for electronic packaging and integration ,2,3,4,5.In order to incorporate antennas into electronic devices with limited space, high dielectric and low loss materials have been used as antenna substrates ,5. This r′μ > 1) makes it possible to overcome such drawbacks [2O4  have the magnetic resonance frequencies below 100 MHz [3Z (Ba3Co2Fe24O41) Z-type hexaferrite ceramic with r′μ = r′ε  = 16 was successfully demonstrated for substrates of an antenna operating at 277 MHz [1.5Sr1.5Co2Fe24O41 (Ba1.5Sr1.5Z) bulk ceramic material was developed by substituting Ba2+ with Sr2+, and it was observed to have a higher magnetic resonance frequency and lower magnetic loss than Ba3Co2Fe24O41 (Ba3Z) near 400 MHz frequency range [The use of magnetodielectric (MD) materials or magnetic materials with relative real permeability higher than one μr′ >  makes itrawbacks ,18. Whil 100 MHz ,20, ther 100 MHz . The str 100 MHz , as well 100 MHz . Ba3Z  SrCO3 (99.7%), Co3O4 (99.9%) and Fe2O3 (99.5%). The weighed mixture of the precursors was homogenized by zirconia balls in ethanol for 24 h, and calcined in an alumina crucible at 1000 °C in air for 4 h. Then the calcined powder samples were crushed by hand grinding and palletized with a pressure of 7.5 MPa. The pallets were annealed in air at 1250 °C for 16 h. Then, the sintered ceramics were mechanically ground into powders by a high pressure grinding mill. After sieving with an ASTM no. 140 sieve (opening: 106 μm), the Ba1.5Sr1.5Z powder of a length less than the opening size was used in a polymer matrix. The polyurethane  system used as matrix consists of three parts; urethane monomers consisting of 80 wt % diisocyanate and 20 wt % diol, a catalyst containing aliphatic amine, parachlorobenzotrifluoride and methyl propylketone and the accelerator containing 1 wt % organotitanate and 99 wt % acetone (polyurethane STD-102).The powder and bulk ceramic of Ba1.5Sr1.5Z/PU composite starts with mixing the Ba1.5Sr1.5Z powder with the urethane monomer. Then, the mixture was sonicated for 2 h and stabilized in cold water during ultrasonication for an additional one hour. The catalyst and the accelerator were introduced into the aforementioned mixture and the suspended solution was ultrasonically stirred for 5 min and then poured into a mold for curing and solvent evaporation. Post curing was made at 100 °C for 2 h.The fabrication of the Ba1.5Sr1.5Z powders were characterized using X-ray diffraction  with a Cu Kα radiation source (k = 0.154056 nm). The measurement of electromagnetic properties was carried out with an Agilent E4991A  impedance analyzer, combined with an Agilent 16453A dielectric material test fixture and an Agilent 16454A magnetic material test fixture in the frequency range, 0.01–1.0 GHz. Specimens for permittivity measurement were cylinders with a diameter of 17.5 mm, and those for permeability were hollow cylinders with an inner diameter of 10 mm and an outer diameter of 17.5 mm. The thicknesses of all the samples ranged from 0.5 to 2.0 mm. Antenna performance, radiation pattern and return loss of an antenna with composite substrate were simulated by using FDTD (finite difference time domain) approach.Micrographs of particles and composites were observed by a scanning electron microscope . The Ba1.5Sr1.5Z particles have irregular shapes including flake-like and needle-like particles. Grain sizes of the particles are observed in micro-size level due to the high temperature sintering at 1250 °C [1.5Sr1.5Z (21.4 Vol %)/PU. The particles were randomly and evenly dispersed in the matrix. As shown in  1250 °C . Figure 1.5Sr1.5Z/PU. Both properties increased, scaling with the volume fraction of Ba1.5Sr1.5Z in the composites. The real permittivity decreased a little with increasing frequency, while the resonant frequencies existed from 300 to 500 MHz. The real permeability was kept in a constant level as the frequency increased near 400 MHz. Magnetic resonance frequency was not observed in the interesting frequency range up to 1 GHz, because the resonance frequency of the hexagonal ferrite is more than 2–3 GHz [ 2–3 GHz ,27. In a 2–3 GHz , which iεδ) of the MD composites was observed to be relatively high over 0.05. By adopting polymers with low loss, such as polydimethylsiloxane (PDMS) [μδ) increased as the filler fraction increased, especially above 400 MHz. Therefore, it can be concluded that Ba1.5Sr1.5Z/PU in fV < 21.4% can be a good candidate for antenna substrate in a few hundred MHz range.Dielectric loss (tan e (PDMS) , or polye (PDMS)  as a hos1.5Sr1.5Z were also measured for input data of Equations (1)–(3). Its real permittivity and permeability were measured to be 17.4 and 14.5 at 402 MHz, respectively. The permeability was a little bit lower than 17.0 of Ba1.5Sr1.5Z with the same composition [The material properties of the bulk Baposition . Howeverposition . The difposition .P means the permittivity  or permeability (rμ), fV is the particle volume fraction and the subscripts, c, f, and m, denote composite, filler, and matrix. υ is assigned 2 for BG and 3 for QCA-CP.The microwave properties of the composite can be calculated by following Bruggeman equation (BG) and quasi-crystalline approximation with coherent potential (QCA-CP) ,31, usinThe material properties are also reported to be bound with Wiener’s upper and lower bounds . The uppThe experimental data of the real permittivity of the composites were found in good agreement with Wiener’s upper bound, as shown in 3Z particle/PVDF (polyvinylidene fluoride) composites [The measured real permeability of the composites was in good agreement with the data calculated by BG, as shown in mposites .r′μ·r′ε)1/2, that is, (r′μ·r′ε)MD = (r′ε)PD.BW is the bandwidth, t is the thickness of an antenna substrate and λ0 is the wavelength in free space at resonance frequency. r′μ and r′ε are real permeability and permittivity, respectively.For the comparison between them, the impedance, the miniaturization factor and the bandwidth of the polymer composite substrates were calculated. Zero-order antenna bandwidth proposed by Hansen  can be er′μ and r′ε can be evaluated by comparing that of a PD material, which has the same miniaturization factor ((1.5 × 6.2)1/2 = 3.1) as 12.3 Vol % MD composite. In the table, BW and εBW are bandwidths of MD and PD materials, respectively and BW/εBW is calculated to be equal to r′μ by Equation (4). In addition, the miniaturization factor increases by the scale of r′μ. Comparison of 12.3 Vol % MD composite with the corresponding PD material as antenna substrate implies that MDs can have the same miniaturization factors, while they can have higher relative impedance, (r′μ/r′ε)1/2, closer to that of free space (=1.0), and also higher bandwidths.G) and radiation efficiency (ηrad) might be slightly degenerated, but BW was greatly enhanced by around 63%, which is consistent with Hansen’s prediction of 50% BW improvement by Equation (4). The difference can be attributed to dielectric and magnetic losses in the real materials, as Equation (4) is derived with assumption of no loss.With optimization of feeding points of the antennas, parameters were calculated. The optimized results showed that gain  and smaller antenna area (9.0% reduced), compared with the dielectric-only material.The antenna performance simulation showed that the magnetodielectric BaIn addition, microwave properties of the composites are expected to be further optimized by controlling dielectric and magnetic losses of particles and polymer, because the polyurethane used in this study was with relatively high dielectric loss, compared with a general or low-loss dielectric polymer. The magnetodielectric materials with Z-type hexaferrite incorporated into a polymer can extend the design space of planar antennas.