Thus, the finding that LigaSure but not rubber band is safe in sealing off the inflamed cecal stump in rat model of acute appendicitis suggests the possibility of applying LigaSure for appendectomy via single port procedure or natural orifice transluminal endoscopic surgery (NOTES).Safety of either LigaSure or rubber band in closing inflamed appendiceal stump in acute appendicitis has been less investigated. In this study, cecal ligation followed by resecting inflamed cecum was performed to mimic appendectomy in a rat model of acute appendicitis. Rats were sacrificed immediately (Group A) and 7 days (Group B) after cecal resection, respectively. The cecal stumps were closed by silk ligature (S), 5 mm LigaSure (L), or rubber band (R). Seven days after cecal resection, the LigaSure (BL) and silk subgroups (BS) had significantly less intra-abdominal adhesion and better laparotomy wound healing than rubber band subgroup (BR). The initial bursting pressure at cecal stump was comparable among the three methods; along with tissue healing process, both BL and BS provided a higher bursting pressure than BR 7 days after appendectomy. BL subgroup had more abundant hydroxyproline deposition than BS and BR subgroup. Furthermore, serum TNF- Although single port laparoscopic appendectomy or natural orifice transluminal endoscopic surgery (NOTES) has been applied for the treatment of acute appendicitis , 2, techLigaSure had been examined for its safety in sealing off mesoappendix  or appenTherefore, the objective of this study is to investigate the safety and effectiveness of using either LigaSure or rubber band ligature in closing appendiceal stump and mesoappendix for the treatment of acute appendicitis, whose results will then justify further investigations in applying these tools for appendectomy via single port procedure or NOTES.Male Sprague-Dawley rats (weights ranged around 320 g) were purchased from the National Animal Center. All of rats were maintained in an animal facility at China Medical University. All rats underwent cecal ligation to mimic appendicitis in rat model on postoperative day 0 and received cecal resection to remove the obstructed cecal stump 24 hours after cecal ligation on postoperative day 1. Rats were randomly allocated into Group A and Group B. In Group A, we sacrificed the rats immediately after cecal resection. In Group B, instead of sacrificing the rats immediately, we kept the rats alive for 7 days till postoperative day 8. The scheme of grouping was shown in Intraperitoneal ketamine hydrochloride (50 mg/kg body weight) combining with ether inhalation was used for anesthesia. All operations were performed under aseptic condition. After shaving for abdominal skin and sterilization for abdominal wall, a midline longitudinal laparotomy was performed. Cecum, which was measured around 1 cm in diameter and 2 cm in length, was identified. To create and mimic acute appendicitis in the rat model, ligation at the point proximal to the tip of cecum about 5 mm with 3-o silk was performed. By occluding the lumen of cecum, all rats suffered from cecal obstruction and inflammation that mimic acute appendicitis in clinical situation . A singlIn the “S” subgroup, the cecal stump and mesocecum were ligated together with 3-0 silk at both ends and transection line was put just in the middle of the two ties. In the “L” subgroup, 5 mm LigaSure precise  device was used to seal off and transect the cecal stump and mesocecum. The cecal stump was transected till the formation of two adjacent white fusion lines at the proximal margin of cecal stump to seal off cecal lumen. Subsequently, the transection line was put distal to the two white lines and the distal lumen was also closed by coagulation line created by LigaSure. In the “R” subgroup, with the assistance of band ligator, two rubber bands  were applied to the proximal end of the cecal stump and mesocecum, and then partial cecal resection was performed distally. Before application of rubber band, they were all sterilized by chlorhexidine as well as 75% alcohol and cleaned again with saline irrigation. The remnant distal length of cecal stump in all groups was around 1 mm in prevention of sloughing of rubber band or silk ligature. The cecal stumps were not buried into the remaining cecum, mimicking similar situation in laparoscopic appendectomy.Laparotomy wound healing status and changes of body weight in the perioperative period were recorded serially. In addition, at the latest laparotomy, signs of bowel leakage, integrity of stump, presence of local abscess as well as peritonitis, and intra-abdominal adhesion condition were examined. The wound healing process was graded as follows: 1 = good healing without wound disruption, 2 = slightly poor healing with little gap at skin wound edge, and 3 = poor healing with obvious gap at wound edge and exposed subcutaneous layer. The grading of intra-abdominal adhesion was defined in accordance with a scale proposed by van der Ham et al. . The scaIn Group A after sacrificing the rats, a 2 cm ileocolic bowel segment containing cecum in the middle portion was taken down. The method of measurement of bursting pressure was modified from the protocol proposed by Elemen et al. . BrieflyAfter measuring the bursting pressure, the excised bowel segment was divided into two equally sized specimen containing part of the stump at each other. One of them was sent for histological examination after being stained by haematoxylin and eosin (H&E). The remnant bowel segment was stored in −80°C freezer and ready for the measurement of tissue hydroxyproline level. The wound healing condition at cecal stump was evaluated according to the modified Ehrlich/Hunt scale [μg/g in wet tissue.The measurement of hydroxyproline level was conducted according to the protocol that has been published previously . Brieflyα) by ELISA (enzyme-linked immunosorbent assay) with rat TNF-α ELISA kit (eBioscience). The procedure was conducted according to the instructions of the manufacturer.To determine systemic inflammatory response in rats, we examined the serum concentration of tumor necrosis factor . The statistically significant level was set at 0.05 by a two-tailed test.P = 0.008) (P = 0.018)  . On post= 0.018) . In otheP = 0.024) (P = 0.024)  . As for = 0.024) .In Group A, the bursting pressure of cecal stump was comparable between each subgroup . In GrouP = 0.032), but bursting pressure between BS and AS subgroups  as well as that between BR and AR subgroups  was comparable. In other words, after tissue healing, LigaSure could provide stronger strength to seal cecal stump when compared to the initially mechanical sealing force.In addition, bursting pressure of BL subgroup was higher than that of AL subgroup   .α, was serially analyzed in Group B. The TNF-α levels in BR subgroup showed a significant increasing trend along with time and reached to the highest level on postoperative day 8 . Additionally, the serum TNF-α of BR subgroup on postoperative day 8 was significantly higher than that of either BS or BL subgroup   .P = 0.041), whereas the differences in fibroblast activity, collagen deposition, and neovascularization scores within Group B were not significant. In addition, there was no significant difference in the four scores within Group A . The kappa score was 0.7.The microscopic views at cecal stump sections stained with haematoxylin and eosin (H&E) in BS, BL, and BR subgroups were shown in Single port laparoscopic appendectomy for the treatment of acute appendicitis has been developed but not popular, because most of them need specially designed curve instrument , 11. LigWound healing involves a series of reactions, including initial inflammation after injury or infection, followed by recruitment of inflammatory cells, such as neutrophil and macrophage, fibroblast, and collagen deposition . The amoα measured before and 1 day after cecal ligation did not differ significantly which implied early appendicitis could mainly be a locally inflammatory illness, and the finding was also consistent with that proposed in previous human study [α in BR subgroup, and we believed that rubber band related foreign body reaction might cause persistently increased TNF-α in BR subgroup. More inflammatory cells infiltrating in cecal stump observed at BR subgroup might also support this hypothesis. In addition, rubber band ligation related tissue ischemia could also contribute to release of HMGB1, a DAMP (damage associated molecular pattern), and subsequently cause prolonged TNF-α production [α, might also be responsible for delayed resumption of oral intake and recovery of body weight in the rats at BR subgroup [Ligature in cecum of a rat has been accepted as a rat model of acute appendicitis . Additioan study . Furtheroduction . The persubgroup .α and quantification of local collagen deposition around appendiceal stump among various treatment modalities, we conducted this study to further address this issue. According to clinical observation, overwhelming release of proinflammatory cytokines could cause profound septic shock in patients with appendicitis. Therefore, our study provided important and clinically relevant information regarding the systemic influences and safety of LigaSure and rubber band ligature in closing appendiceal stump in rat model of acute appendicitis.Elemen et al. had conducted an excellent experimental study to investigate safety of LigaSure in sealing off appendiceal stump following appendectomy for appendicitis . BecauseBecause of lack of well-designed and safe sealing tools in bowel walls, single port appendectomy or NOTES in abdominal surgery was not prevailing yet in current surgical field. After proving the safety of LigaSure in transecting and sealing off mesoappendix  and cecaIn this study, we identified that LigaSure may be a safe and convenient tool in transecting mesoappendix and appendiceal stump in rat appendicitis model. Rubber band ligature should not be suitable to close appendiceal stump due to profound systemic and local inflammation as well as associated impaired wound healing process at the appendiceal stump.


Mortality of full-term calves at calving is an increasing problem in dairy industries internationally. Multiple herd management factors contribute to such losses. This case-control study identified factors which differed between herds with high and low calf mortality. These included breeding, dietary, health and calving factors. It was concluded that calving, not pre-calving, management appears to be the most important area of concern in herds with high perinatal mortality. This indicates that farmers and their veterinarians need to focus on calving management when investigating such problems and when attempting to reduce losses in herds with high rates of bovine perinatal mortality.Bovine perinatal mortality is an increasing problem in dairy industries internationally. The objective of this study was to determine the risk factors associated with high and low herd-level calf mortality. Thirty herds with a history of either high (case) or low (control) calf mortality were recruited. A herd-level questionnaire was used to gather information on management practices likely to impact bovine perinatal mortality. The questionnaire was divided into four subsections dealing with pre-calving  and calving factors. Most of the significant differences between case and control herds were found in calving management. For example, in case herds, pregnant cattle were less likely to be moved to the calving unit two or more days and more likely to be moved less than 12 hours pre-calving, they were also less likely to calve in group-calving facilities and their calves were more likely to receive intranasal or hypothermal resuscitation. These management procedures may cause social isolation and periparturient psychogenic uterine atony leading to dystocia, more weak calves requiring resuscitation and high perinatal calf mortality. The key finding is that calving, not pre-calving, management appears to be the most important area of concern in herds with high perinatal mortality. Perinatal mortality may be defined as death of the fetus or calf before, during or within 48 h after calving at full-term >260 days) . Perinat60 days .Concerns have been raised both about high  and incrHowever, the reasons why some herds have more calf losses than others has received little attention in the literature; few studies have been conducted comparing herds with high and low loss rates, specifically in perinates. An investigation of risk factors for young calf mortality (1–90 days) between 60 Swedish herds with high and low loss rates found that inadequate calf serum alpha-tocopherol and beta-carotene concentrations, number of faecal pathogens and cases of diarrhoea were significantly more likely in high loss herds . A DanisIn American beef herds it has been reported that calving in confinement and high dystocia rates were significantly associated with high, compared to, low calf morbidity (birth to weaning) herds . In an eThese limited observations indicate a paucity of information on why perinatal bovine mortality differs between high and low loss herds. Hence, the objective of this study was to investigate the management factors associated with a low risk (LR) and a high risk (HR) of perinatal bovine mortality on commercial dairy farms using an observational study design. Perinatal bovine mortality was defined as death of a fullterm (≥260 day gestation) calf before, during or within 48h after calving . An unmaP ≤ 0.05. All data editing and statistical analyses were carried out using appropriate procedures ).Data on risk factors likely to impact perinatal mortality were collected from a pre-designed farmer questionnaire . A writtThe significant odds ratios and 95% confidence limits for the associations between the management factor variables and the response variables (High or Low herd BPM status) in the logistic regression analysis are shown in In HR herds (compared to LR herds) there was more likely to be use of Je or JexHF sires on cows than HF or AA sires . In HR herds (compared to LR herds) there was more likely to be heifers in the target BCS range than obese heifers and more likely to be thin than obese heifers . P < 0.10), (More farmers with HR herds tended to vaccinate heifers and cows against leptospirosis (< 0.10), .In HR herds (compared to LR herds) there was more likely to be a combination of calving units than a group calving unit . In HR hP < 0.05), irrespective of whether they were in HR or LR herds. No other management changes over time were evident from the questionnaire responses.Heifers and cows were more likely to be vaccinated against salmonellosis and IBR in 2012 than in 2010 (www.icbf.com) and internationally [The overall results are discussed first followed by the comparison between the case and control herds. As expected almost all herds had Holstein-Friesian heifers but a surprisingly high proportion of herds also had other dairy or crossbreed heifers, mainly Jerseys. This reflects a recent change within some herds in the Irish dairy industry  in the majority (90%) of herds. As with the heifers, all herds of cows were fed grass silage with only a relatively small proportion offered ration or straw. In all herds cows were supplemented with trace elements pre-calving, most commonly by mineral powder. Previous studies at this research centre have shown the benefits of this practice  and it hNeospora caninum, a cause of perinatal calf mortality [The most commonly used vaccines  in heifers reflect the perceived importance of these pathogens in causing abortions and weak calves in Irish dairy herds. However, while salmonella spp. are one of the most commonly diagnosed causes of abortion in Irish cattle herds , leptosportality ,31. A smTraditionally tie-up stalls in the 1960s and individual calving pens from the 1970s to the 1990s were the norm on Irish dairy farms, in the latter case due to their dual use as isolation pens for suspected cases of brucellosis. However, in this study only half of all herds had individual calving pens with a large proportion using group pens, outdoor pads or combinations of both. This apparent move away from individual pens may reflect the large herd sizes in this study  , the eraA surprisingly small proportion of farmers (33%) moved their pregnant animals to the calving unit at least 24 h pre-calving. This may have been due to limited calving accommodation following gradual herd expansion over years. This early move allows animals to adapt to their new environment, social group and diet. On a large proportion (43%) of farms pregnant animals were moved to the calving unit between 6 h and 24 h pre-calving reflecting the detection of impending signs of calving such as relaxation of the sacrosciatic ligaments and/or colostrum dripping from the teats. A smaller proportion (20%) of farmers moved pregnant animals to the calving unit on the point of calving which may reflect limited observation of animals pre-calving or limited calving facilities. This ‘just in time’ calving movement protocol has become popular in North America and has recently been shown to have no detrimental effects on calving performance and is preferable to moving animals during stage one of calving .The majority (73%) of farmers observed pregnant animals once they were in the calving unit for signs of impending calving every 4 h to 6 h. This is similar to the findings of a recent Canadian survey in dairy herds . Given tResuscitation techniques were used on the majority (86%) of farms, a figure comparable with that on US dairies (81%) . The mosMost of the management factors investigated here did not differ between case and control herds. This finding is congruent with the results of recent surveys on perinatal calf mortality in German  and in IBoth heifers and cows in HR herds tended to be more likely to be vaccinated against leptospirosis, reflecting the importance attributed to this pathogen in causing abortions and bovine perinatal mortality  and probPregnant animals were moved to the calving accommodation earlier on LR compared to HR farms. This may facilitate adaption to the new environment, diet and social hierarchy, particularly in heifers, where such stresses can negatively impact calving performance . The latCalves were more likely to be resuscitated using intranasal or hypothermal stimulation than no resuscitation in the HR herds. This may be due to ongoing calf losses in the HR herds. Both of the techniques listed, prodding a straw up the calf’s nostrils and pouring cold water in the ear or over the calf’s head, are commonly used revival practices on Irish farms  known tohttp://www.animalhealthireland.ie/index.php). This probably influenced farmers to commence vaccination as no increase in clinical IBR was reported in these herds.No apparent changes in the genotypes present on these farms over time were detectable which is not surprising given the short time frame of the study. No changes were apparent either in the feeding management of the heifers or cows pre-calving over this period. This may reflect the generally optimal feeding management adopted by these farmers and the previous changes they had made to prevent problems at calving. For both heifers and cows there was an increase in the number of herds vaccinated against salmonellosis and IBR over the three years. In the former case the incidence of clinical disease tends to have annual cycles  a peak oNo changes in calving supervision, calving assistance, newborn calf care or milk fever control, were apparent over the three years. As these farmers appeared to generally implement best practice and these tended to be practices of habit and routine, changes were possibly unlikely to occur in this relatively short time frame.This study demonstrates that some pre-calving and calving management factors differ between herds with high and low rates of bovine perinatal mortality. However, the majority of such factors were similar between these two herd categories. The findings here suggest that calving management factors are more important than pre-calving factors. For example, farmers who moved their cows to the calving area earlier pre-calving had lower rates of bovine perinatal mortality. The finding that calving management factors are of greater importance is a key conclusion and indicates where farmers and their veterinary practitioners need to focus both when investigating such problems and when attempting to reduce losses in herds with high rates of bovine perinatal mortality.


The broad tolerance of domain-rearranging mutations by a yeast signaling network suggests that signaling complexes have loose spatial constraints, making manipulation and perhaps evolution easier. in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as  Cells use complex protein interaction networks to sense and process external signals. Proteins involved in signaling are often composed of multiple functional units called domains. Because domains are modular, mutations that rearrange domains among proteins have the potential to result in the creation of novel proteins with altered functions. At an evolutionary timescale, domain rearrangements contribute to the functional diversification of signaling networks; at the shorter timescale of the life of an individual, domain rearrangements can impair cellular functions and lead to disease. Here, we investigated how domain-rearranging mutations alter the function of signaling networks, in particular when these mutations disrupt pre-existing proteins. We used as a model system the yeast mating signaling pathway, which shares many properties with more complex pathways active in human cells. Our results demonstrate that signaling networks are often robust to domain rearrangements that disrupt pre-existing genes. In addition, our experiments suggest a possible mechanism to explain this robustness: rather than being a rigid multi-protein machine, the yeast mating signaling complex is a dynamic ensemble with loose spatial constraints. Because of this, the changes in protein interaction partners caused by domain-rearrangement mutations can be accommodated without disrupting network function. Cell signaling networks possess a remarkably modular organization. This modularity has attracted the attention of synthetic biologists, for it offers a plausible approach to engineer novel and useful cellular behaviors. At the center of this modular organization are protein domains, which are recurring structural units that often perform modular and thus portable functions Experimental While in nature domain rearrangements can occur by a variety of mechanisms To answer this question, we used a synthetic biology approach to systematically determine the robustness of the mitogen activated protein kinase (MAPK)-mediated yeast mating pathway   reporter controlled by a mating-responsive heromone . As a cop≤0.013) . Remarkap≤0.013) . In addip≤0.013) . Taken tWhile the mating pathway seems capable of tolerating domain rearrangement-mediated replacements, it is possible that at least some of these replacements could have detrimental effects on other cellular processes, and thus their evolutionary relevance would be questionable. To investigate this possibility, we measured the growth rate (as a proxy for fitness) of the most active domain rearrangement variants (nine variants in four different deletion strains). As shown in While it is likely that some domain combinations would be unable to fold and/or function properly, there is no simple correlation between a domain rearrangement variant expression level and its ability to mediate mating pathway response . More liTo explore this hypothesis, we first compared mating pathway activation mediated by the domain-rearranged kinase variants with those of kinase variants lacking N-t localization domains. As shown in We then investigated the mechanisms that enable the signaling complex to tolerate changes in recruitment interactions. Domain-domain interactions depend on specific binding interfaces, thus they are unlikely to tolerate drastic changes in interaction partners. Thus, if alternative recruitment has to maintain specific domain-domain interactions, two hypotheses are possible : (i) reaWe hypothesized that if signaling complexes possess loosely defined spatial constraints, the network should tolerate more complex domain rearrangement events, such as those in which domains from pairs of proteins are reciprocally rearranged . To testSome of the changes in network topology resulting from domain rearrangement events in our experiments mimic changes in network topology that have occurred during evolution. For instance, in yeast, the adaptor Ste50 mediates the interaction between the MAP3K (Ste11) and the small GTPase (Cdc42) . In contFinally, among the seven kinase-containing domain combinations that in our experiments resulted in active pathways , three hOur results indicate that the yeast mating pathway is remarkably robust to domain rearrangement-mediated replacements, tolerating multiple changes in recruitment interactions. In particular, we observed that the N-t domains or motifs of the three multi-domain kinases in the mating pathway , which normally localize the respective kinase domains to the mating signaling complex, can be replaced by alternative interaction domains . In contrast, kinase domains cannot be replaced, suggesting that the specificity of kinase-substrate interactions is key for proper signaling function. Thus, while the inter-molecular connectivity of the domains is important, the intra-molecular connectivity is not as important, suggesting that proper network function depends more on the formation of a signaling complex composed of key domains, rather than key proteins. Interestingly, even though intra-molecular interactions between different domains within a protein may play regulatory roles Because domain-domain interactions are highly specific, proteins with rearranged domain compositions may have altered localization within the signaling complex. How can then domain rearrangements be tolerated? Initially, we hypothesized that the presence of long, disordered inter-domain linkers may enable each domain within a rearranged protein to localize to the correct site within the complex. However, we found that the IDRs present within inter-domain linkers are dispensable for pathway function and, more importantly, for the robustness of the network to domain rearrangements. Taken together, these observations suggest that the function of the mating signaling complex is not constrained by a defined geometry. Thus, we propose that, rather than a precisely assembled multi-molecular machine, the yeast mating signaling complex is an ensemble of dynamically interacting molecules with loose spatial constraints While in the short term mutational robustness buffers the impact that genotypic changes could have on phenotype, in the long term, mutational robustness may facilitate evolution As affordable genome and transcriptome sequencing are rapidly expanding the list of domain-rearrangement mutations involved in disease MATa, bar1::NatR, far1Δ, mfa2::pFUS1-GFP, his3, trp1, leu2, ura3. Seven strains were created in which the following genes from the mating pathway were deleted individually: Ste4, Ste5, Ste7, Ste11, Ste18, Ste20, and Ste50, in all cases using Trp as a selectable marker. Deletion strains were validated by genomic PCR and flow cytometry . Double deletion strains  were also made by homologous recombination, using Leu as the second selectable marker.Deletion strains were derived from a W303 strain with the following genotype: CycI promoter, and an AdhI transcription terminator.The domain-rearrangement libraries were designed and constructed using a previously described combinatorial cloning strategy n≥4) and found to be in good agreement.Each strain carrying an individual deletion  described above was transformed with appropriate plasmids encoding each domain-rearrangement variant to be tested. Equal amounts of “A-type” cells transformed with each variant (or controls) were mixed with WT “α-type” cells and deposited on the surface of a polycarbonate filter placed on a YPD plate and incubated for 3 hours at 30°C. Cells were then detached from the filters by vortexing and aliquots were plated on minimum synthetic media, or synthetic media lacking lysine. Mating efficiency was calculated as the number of colonies on minimum synthetic media divided by number of colonies on synthetic media lacking lysine n≥6) and found to be in good agreement.Mating assays were performed with minor modifications to a previously described method Site-direct mutagenesis was done by Quick Change, following the manufacturer's protocol . Mutations were verified by DNA sequencing.Proteins with domain compositions similar to those found in our experiments were identified in the Domain Club Database 0 * Nν0 is a constant that depends on persistence length, N is the number of residues in the polymer, ν is a scaling factor, and F is a correction factor that accounts for the net charge and Pro content of the IDR.Hydrodynamic Radii for IDRs was calculated using the power law relation Rh = F * ρThe IDRs of Ste50, Ste20, and Ste11 were identified in the Pfam database. Specifically, we deleted the disordered regions between amino acids 156 and 230 in Ste50, 408 and 578 in Ste20, and between 258 and 354 in Ste11.Figure S1Function of each individual domain in the analyzed proteins.(TIF)Click here for additional data file.Figure S2Schematic representation of each subset of library variants transformed into the corresponding deletion strains Click here for additional data file.Figure S3Basal levels of mating pathway activity (as determined by GFP fluorescence measured by flow cytometry). GFP expression levels determined before addition of pheromone indicate that, in almost all cases, domain rearrangement-mediated gene replacements do not result in substantial constitutive activation of the mating pathway. Furthermore, even in those cases where basal pathway activation is higher than WT, addition of pheromone further increases GFP expression (compared data here with data on (TIF)Click here for additional data file.Figure S4Statistical analysis of the GFP reporter measurements of mating pathway activation upon pheromone induction. In all cases, the GFP values for pairs of variants were compared and the significance of the observed differences were assessed by performing two tailed t-tests. In (C) comparisons were made between the WT strain and strains carrying each domain rearrangement variant. If p>0.05 then we concluded that the GFP values measured for the corresponding variant are not significantly different from that measured for the WT. In (D) comparisons were made between each Δ strain and the corresponding strain carrying each domain rearrangement variant. If p<0.05 then we concluded that the GFP values measured for the corresponding variant are significantly different from that measured for the Δ strain. Data shown in (TIF)Click here for additional data file.Figure S5Domain rearrangement variants can mediate pheromone-induced polarized growth (“shmooing”). Deletion strains carrying individual domain rearrangement variants were incubated for 1 h in the presence of 1 µM alpha-factor and representative images were taken using an automated inverted Leica TCS SP8 confocal microscopy, using a 63× objective. As controls, we included WT cells (able to shmoo), as well as strains carrying inactive domain rearrangement variants that, as expected, fail to induce polarized growth (two images at the bottom).(TIF)Click here for additional data file.Figure S6Quantitative mating assays. Mating assays: each “a-type” individual deletion strain was transformed with appropriate plasmids encoding each domain-shuffling variant to be tested. Equal numbers of “A-type” cells transformed with each variant (or controls) were mixed with WT “α-type” cells and deposited on the surface of a polycarbonate filter placed on a YPD plate, and incubated for 3 hours at 30°C. Cells were then washed from the filters and plated on minimum synthetic media or on synthetic media lacking lysine. Plates were incubated at 30°C for 48 hours and colonies on each plate were counted. Mating efficiency was calculated as the number of colonies on minimum synthetic media/number of colonies on synthetic media lacking lysine. Results were normalized according the WT strain. Averages from duplicates and standard errors were calculated. The experiments were repeated at least twice. Data shown in (TIF)Click here for additional data file.Figure S7Pathway activation, as determined by GFP fluorescence measured by flow cytometry 2 h after addition of 1 µM pheromone correlates, in most cases, with mating efficiency as determined in quantitative mating assays. Data shown in (TIF)Click here for additional data file.Figure S8Determination of the growth rates for active domain rearrangement variants, as compared to the growth rates of the WT and the corresponding deletion strains. Each strain was grown in liquid culture (in triplicates) and ODs were measured at 600 nm every hour for 8 hours. Data was fitted using the exponential equation: OD = ODo eλt, where ODo is the initial OD value, λ is the growth rate, and t is time. (A) Cultures were grown in rich media under isosmotic conditions. (B) Cultures were grown in rich media under high osmolarity stress (0.4 M KCl). Data shown in (TIF)Click here for additional data file.Figure S9Statistical analysis comparing the GFP values for the variants analyzed in , before or after addition of pheromone. In all cases, two tailed t-tests were performed. Significant differences (p<0.05) are marked with asterisks. Data shown in (TIF)Click here for additional data file.Figure S10Ability of isolated N-t domains to mediate mating pathway activation. The corresponding deletion strains were transformed with N-t localization domains alone and the fluorescence of the mating pathway reporter pFus1-GFP was measure before or 2 h after addition of pheromone. As shown in the figure, none of the localization domains can restore pathway activity in the deletion strains. Data shown in (TIF)Click here for additional data file.Figure S11The recruitment of the GFP-tagged variants to the mating projections seen in , depend on the presence of localization domains in the domain rearrangement variants. When GFP is not fused to localization domains, it remains uniformly distributed in the cytoplasm, failing to localize to the mating shmoos.(TIF)Click here for additional data file.Figure S12Alternative mechanisms of kinase recruitment to the signaling complex, for the domain shuffling variants shown to localize to the mating projections (shmoos) by fluorescence microscopy. Note that our results suggest that the complex stoichiometry is flexible and can accommodate a diverse number of components.(TIF)Click here for additional data file.Figure S13There is no simple correlation between a variant's expression levels and its ability to mediate mating pathway response. Domain rearrangement variants were tagged at their N-t with GFP and transformed in the corresponding deletion strain . GFP fluorescence was measured by flow cytometry. Data shown in (TIF)Click here for additional data file.Figure S14IDRs are found in Ste5, Ste7, Ste11, Ste20, and Ste50. While in Ste7 and Ste5 IDRs are located at the protein termini, in Ste11, Ste20, and Ste50, IDRs are found within inter-domain linkers and, thus, they can separate folded domains by long and flexible distances. (A) Schematic representation of the IDR-containing mating pathway proteins Ste5, Ste7, Ste11, Ste20, and Ste50. IDRs are represented as light blue segments. (B) Estimation of the volume and maximum extended length of Ste11, Ste20, and Ste50 IDRs. (C) Schematic representation of the relative volume occupied by either IDRs or folded domains in Ste11, Ste20, and Ste50.(TIF)Click here for additional data file.Figure S15Pathway activation (as determined by GFP fluorescence) for IDR-deleted variants, before the addition of pheromone, as compared with the corresponding full length variants. In the single Δ strains, deletion of Ste20's or Ste50's IDRs does not affect basal levels of pathway activation. In contrast, deletion of Ste11's IDR causes small increases in basal pathway activation, suggesting it may alter the regulation of Ste11's kinase activity. Furthermore, in the double Δ strains, simultaneous deletion of Ste11 and Ste20 IDRs or of Ste11 and Ste50 IDRs leads to large increases in basal levels of pathway activation, suggesting that the simultaneous deletions have a marked effect in the regulation of pathway function. Statistically significant differences are marked with asterisks. Data shown in (TIF)Click here for additional data file.Figure S16Schematic representation of proteins with alternative domain combinations that lead to functional pathways in our experiments, and are also found in natural proteins.(TIF)Click here for additional data file.Data S1Data shown in –, and Figures S3, S4, S9, and S15.(XLSX)Click here for additional data file.Data S2Data shown in Figures S6–S8, S10, and S13.(XLSX)Click here for additional data file.Data S3GenBank accession numbers for sequences used to build the domain rearrangement library.(XLSX)Click here for additional data file.Text S1Supplemental material and methods.(DOCX)Click here for additional data file.


Apolipoprotein (apo) E was initially described as a lipid transport protein and major ligand for low density lipoprotein (LDL) receptors with a role in cholesterol metabolism and cardiovascular disease. It has since emerged as a major risk factor (causative gene) for Alzheimer’s disease and other neurodegenerative disorders. Detailed understanding of the structural features of the three isoforms , which differ by only a single amino acid interchange, has elucidated their unique functions. ApoE2 and apoE4 increase the risk for heart disease: apoE2 increases atherogenic lipoprotein levels (it binds poorly to LDL receptors), and apoE4 increases LDL levels . ApoE4 also increases the risk for neurodegenerative diseases, decreases their age of onset, or alters their progression. ApoE4 likely causes neurodegeneration secondary to its abnormal structure, caused by an interaction between its carboxyl- and amino-terminal domains, called domain interaction. When neurons are stressed or injured, they synthesize apoE to redistribute cholesterol for neuronal repair or remodeling. However, because of its altered structure, neuronal apoE4 undergoes neuron-specific proteolysis, generating neurotoxic fragments (12–29 kDa) that escape the secretory pathway and cause mitochondrial dysfunction and cytoskeletal alterations, including tau phosphorylation. ApoE4-associated pathology can be prevented by small-molecule structure correctors that block domain interaction by converting apoE4 to a molecule that resembles apoE3 both structurally and functionally. Structure correctors are a potential therapeutic approach to reduce apoE4 pathology in both cardiovascular and neurological disorders. Apolipo1, HDLc) , 9. Orig1, HDLc) , the pro1, HDLc) .ApoE has three common alleles encoded by the apoE gene on chromosome 19 . These aPlasma apoE is synthesized primarily by liver hepatocytes, which account for ~75 % of the body’s apoE production. In normolipidemic subjects, the plasma concentration of apoE is approximately 4–8 mg/dl. The second most common organ synthesizing apoE is the brain, where it is produced primarily by astrocytes, but also by oligodendrocytes, microglia, and neurons, especially injured or stressed neurons [see refs. ApoE, a 34-kDa protein of 299 amino acids with a single glycosylation site at threonine-194 , has twoApoE3 and apoE4 bind to the LDL receptor with similar affinity . The key amino acids for receptor binding were identified by site-directed mutagenesis and by the existence of naturally occurring receptor-defective human mutations in type III hyperlipoproteinemic patients . ApoE plApoE4 has an arginine at residue 112 (Arg-112). This produces a property of apoE4 referred to as domain interaction, in which Arg-112 causes the side chain of Arg-61 to extend away from the amino-terminal domain , 24, enaBecause it binds defectively to LDL receptors, apoE2 homozygosity can precipitate type III hyperlipoproteinemia , 21, 22.ApoE4 increases plasma LDL levels and the risk for atherosclerosis , 12, 25.How does the amino acid difference at residue 112 in the amino-terminal domain—arginine in apoE4 and cysteine in apoE3 and apoE2—alter the lipoprotein preference of apoE4 when lipid binding is mediated by residues 240–260 in the carboxyl-terminal domain? The reason is the structural effect of domain interaction. When domain interaction is disrupted with small molecules that prevent the Arg-61–Glu-255 ionic interaction or by site-directed mutagenesis (Arg-61 to threonine), apoE4’s binding preference shifts from VLDL to smaller, phospholipid-rich HDL , 24, 31.ApoE4 is the major genetic risk factor (or causative gene) for Alzheimer’s disease (AD) and other neurological disorders, including poor clinical outcomes after traumatic brain injury, or stroke, frontotemporal dementia, Down syndrome, certain patients with Parkinson’s disease, and Lewy body disease [for review, see refs. Multiple factors acting through various pathways cause cognitive decline and neurodegeneration. Several mechanisms have been proposed, but the so-called amyloid hypothesis has received the most attention [for review, see refs. There is no consensus concerning the mechanisms by which apoE4 affects the amyloid pathway –41. SeveLipidation of apoE-containing lipoproteins in the brain has been postulated to modulate amyloid deposition and Aβ clearance by delivering Aβ to microglia and astrocytes for degradation or to the blood brain barrier for transport out of the brain , 51, 52.Regardless of the reason, apoE4 carriers have increased numbers of amyloid plaques in their brains , 39, 64.Studies from the Gladstone Institutes showed that apoE4 acts directly and independently of Aβ on a parallel path leading to neuropathology , 36–38. ApoE4 is highly susceptible to neuron-specific proteolysis, which generates 12–29-kDa neurotoxic fragments , 76, 77 These fragments escape the secretory pathway and enter the cytosol, where they stimulate tau phosphorylation and interact with mitochondria, causing mitochondrial dysfunction and neurodegeneration –38 Fig.. FragmenEscherichia coli dihydrofolate reductase. The FRET assay measures the ability of an active structure corrector to prevent the FRET emission signal that occurs in apoE4 when the amino- and carboxyl-terminal domains are in close proximity [Small-molecule structure correctors that block the ionic interaction of Arg-61 and Glu-255 in apoE4 Fig.  have beeroximity . Testingroximity . StructuOverexpression of apoE4 in transgenic mice or expression of apoE4 in targeted-replacement mice causes central nervous system pathology, including increased tau phosphorylation, loss of interneurons, and impaired learning and memory , 73–75.


Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.N-terminal domains of BVU_4064 and BF1687 proteins from Crystal structures of both BVU_4064 and BF1687 proteins, solved at the JCSG center, show strikingly similar three-dimensional structures. The main difference between the two is that the two domains in the BVU_4064 protein are connected by a short linker, as opposed to a longer insertion made of 4 helices placed linearly along with a strand that is added to the C-terminal domain in the BF1687 protein. The N-terminal domain in both proteins, corresponding to the PF12985 (DUF3869) domain is a β–sandwich with pre-albumin-like fold, found in many proteins belonging to the Transthyretin clan of Pfam. The structures of C-terminal domains of both proteins, corresponding to the PF12986 (DUF3870) domain in BVU_4064 protein and an unclassified domain in the BF1687 protein, show significant structural similarity to bacterial pore-forming toxins. A helix in this domain is in an analogous position to a loop connecting the second and third strands in the toxin structures, where this loop is implicated to play a role in the toxin insertion into the host cell membrane. The same helix also points to the groove between the N- and C-terminal domains that are loosely held together by hydrophobic and hydrogen bond interactions. The presence of several conserved residues in this region together with these structural determinants could make it a functionally important region in these proteins.Structural analysis of BVU_4064 and BF1687 points to possible roles in mediating multiple interactions on the cell-surface/extracellular matrix. In particular the N-terminal domain could be involved in adhesive interactions, the C-terminal domain and the inter-domain groove in lipid or carbohydrate interactions.The online version of this article (doi:10.1186/s12859-014-0434-7) contains supplementary material, which is available to authorized users. B. fragilis and B. vulgatus from the genus Bacteroides are among the most prevalent organisms of the human gut microbiome and constitute one of the largest bacterial contributions to the human fecal biomass [B. fragilis: [B. vulgatus: [http://www.jcsg.org) has solved at high-resolution several structures of proteins belonging to Bacteroides protein families that are over-represented in human gut microbiome. We report here a detailed analysis of two novel protein structures from B. vulgatus and B. fragilis. These structures map to Pfam families PF12985 (DUF3869) and PF12986 (DUF3870) that were previously considered “domains of unknown function”. These are the first members of those families to be experimentally characterized.Humans harbor complex bacterial communities in various body habitats such as skin, gut and oral cavities -5 that c biomass . Both or biomass . However biomass ,11. Bothragilis: , B. vulgulgatus: ), contaiBacteroides proteins BVU_4064  and BF1687  have been determined to 1.85 Å and 2.2 Å resolution, using MAD and SAD phasing methods respectively as described in the The crystal structures of the N-terminally truncated Despite a relatively low overall sequence identity of 24%  of the MetaHIT human gut microbiome analysis of 124 human subjects [Bacteroides lipoproteins to carry an N-terminal β-propeller domain that may form an adhesion module [http://scop.mrc-lmb.cam.ac.uk/scop/; [Bacteroides β-sandwich domains have a key role in the cytoadherence functions of these bacteria.From our comparative genomics study , subjects , the ave14, subjen module . Similark/scop/; ,23). HenBacillus cereus pilin structure (PDB code: 3kpt) a long loop with two short strands forming a β-hairpin is present between the strands 6 and 7. Yet another variation to this fold is seen in the repetitive B regions (PDB code: 1d2p) of the Staphylococcus aureus collagen binding protein , where the strand 6 is part of the β–sheet with strands 4, 1, and 7 of the pre-albumin fold  has been implicated in membrane insertion  and the Å RMSD; ) and aer Å RMSD; ). Figuresertion ,32). By . By Laet2 vs. 2362 Å2 respectively). The domain interface is fairly hydrophobic and is held loosely together by a few hydrophobic interactions and a number of weak to moderate hydrogen bonds (data not shown). While the residues from the C-terminal domain in the 3kog structure that are buried at the interface are mostly not conserved in the family (PF12986), about 40% of the buried residues of the N-terminal domain at this interface in both the 3kog and 3g3l structures are conserved in 70% or more of the sequences of the Pfam family they belong to (PF12985). This points to a key role for the interactions of the N-terminal domain for maintaining characteristic bilobal structure of these proteins. The computed molecular surface of 3kog (data not shown) reveals the presence of a deep groove lined by the hydrophobic residues  data were collected to 1.90 Å resolution at wavelengths corresponding to high-energy remote, inflection, and peak of the Selenium edge at beam line BL11-1 at SSRL. For the structure with PDB code 3g3l, single anomalous diffraction (SAD) data were collected to 2.20 Å resolution at 0.97966 Å wavelength corresponding to the peak of the Selenium edge at beam line BL9-2 at SSRL. Both sets of data were collected using BLU-ICE , integraThe QC server reports the stereochemical quality of the model using AutoDepInputTool , MolProbhttp://www.ncbi.nlm.nih.gov/Ftp/).The gene neighborhoods were extracted using an in-house Perl script that scans the NCBI genome file for a query gi and determines the adjacent genes using the query PF12985) as the anchor. Thus extracted neighbors (Additional file 985 as thAll the molecular structure visualizations presented in this report have been made using PyMOL .


By establishing anti-PLAG4-neutralizing monoclonal antibodies, we confirmed its role in CLEC-2 binding, platelet aggregation, and tumor emboli formation. Our results suggest the requirement of simultaneous inhibition of PLAG3/4 for complete suppression of podoplanin-mediated tumor growth and metastasis.Podoplanin/Aggrus is a sialoglycoprotein expressed in various cancers. We previously identified podoplanin as a key factor in tumor-induced platelet aggregation. Podoplanin-mediated platelet aggregation enhances tumor growth and metastasis by secreting growth factors and by forming tumor emboli in the microvasculature. Thus, precise analysis of the mechanisms of podoplanin-mediated platelet aggregation is critical for developing anti-tumor therapies. Here we report the discovery of a novel platelet aggregation-inducing domain, PLAG4 (81-EDLPT-85). PLAG4 has high homology to the previously reported PLAG3 and contributes to the binding of its platelet receptor CLEC-2. Mutant analyses indicated that PLAG4 exhibits a predominant platelet-aggregating function relative to PLAG3 and that conserved Glu Tumor cell growth and survival are affected by a wide variety of tumor microenvironments. During hematogenous metastasis processes, circulating tumor cells rarely survive due to exclusion by the immune system. Platelets are known as the critical component that affects the survival of circulating tumor cells, leading to metastasis formation . In factWe have previously identified a type-I transmembrane sialoglycoprotein, podoplanin, also known as Aggrus, as a platelet aggregation-inducing factor in highly metastatic tumor cells . PodoplaPLatelet AGgregation-stimulating (PLAG) domain (PLAG1–3) [O-glycanase treatment reduces podoplanin's platelet aggregation ability [O-glycan for podoplanin-induced platelet aggregation [52 in PLAG3 has been detected in human podoplanin [Podoplanin contains three tandemly repeated EDXXVTPG motifs in the extracellular domain. Analysis of the epitope of a neutralizing anti-mouse podoplanin monoclonal antibody (mAb) 8F11 helped to identify the domains critical for exhibiting platelet-aggregating ability. Therefore, we designated the motif-containing domain  as the PLAG1–3) . These dPLAG1–3) , 12. O-g ability , and sturegation , 15. In doplanin .45 in PLAG2 and Asp48 and Asp49 in PLAG3, the perimeter structure around PLAG2 and PLAG3 could be the CLEC-2-binding site [O-glycosylated PLAG2–PLAG3 peptide in complex with CLEC-2 extracellular domain revealed that the acidic side chains of Glu47, Asp48, and the sialic acid attached to Thr52 on PLAG3 were recognized by basic residues on CLEC-2 [The C-type lectin-like receptor 2 (CLEC-2), originally identified as a platelet receptor for the snake venom toxin rhodocytin , has beeing site , 23. A rn CLEC-2 .in vivo. These results suggest that the newly identified PLAG4 domain plays a critical role in the binding to CLEC-2, hence being a promising target motif for suppressing podoplanin-expressing tumor cell growth and metastasis.Here we report the discovery of an additional PLAG domain (81–85 aa in human podoplanin), which is highly conserved among mammals. The region includes the highly conserved EDXXT motif, which is closely related to the PLAG domain consensus sequence EDXXVTPG. Deletion of the region or a point mutation in it drastically attenuated its binding to CLEC-2 and podoplanin-induced platelet aggregation. Thus, we designated the region as the fourth PLAG domain, PLAG4. Importantly, deletions or point mutations in both PLAG3 and PLAG4 domains almost completely suppressed the CLEC-2-binding ability and platelet-aggregating activity of human podoplanin, whereas the deletion of or point mutation in either one of the two domains could not completely suppress the binding ability and platelet-aggregating ability. To further investigate the requirement of the PLAG4 domain for podoplanin's platelet-aggregating ability, we tried to establish PLAG4-recognizing mAbs. The generated PLAG4-recognizing mAbs exhibited CLEC-2-binding inhibitory activity and platelet aggregation-neutralizing ability. Moreover, the injection of the mAbs suppressed podoplanin-mediated hematogenous metastasis podoplanin cDNA-containing plasmid. We generated a PLAG1-deletion mutant by deleting the 29–34 aa-coding region and a PLAG3-deletion mutant by deleting the 47–52 aa-coding region  reduced the CLEC-2 binding, and compared with the D48A, the substitution of the Asp82 residue in PLAG4 to Ala (D82A) partially but rather significantly reduced the binding to CLEC-2  also lowered the platelet-aggregating ability when compared with WT podoplanin  of both mAbs failed to reach values measurable by Biacore X100 due to the low level of dissociation, and so the precise equilibrium dissociation constant (KD) values could not be determined. However, the KD values of both mAbs were less than 0.3 nM.To further estimate the role of the PLAG4 domain in podoplanin-induced platelet aggregation and in pulmonary metastasis promoted by platelet aggregation, we attempted to establish a mouse mAb recognizing the PLAG4 domain. We purified a 12- or 40-times tandemly repeated human podoplanin PLAG4 peptide  as an antigen from a bacteria lysate and immunized the mice with the peptide. After fusing splenocytes with P3U1 myeloma cells, we screened hybridomas that secreted mAbs recognizing the immunogen, using enzyme-linked immunosorbent assay (ELISA)-based competition analysis. Upon further selection by recognition capability for podoplanin-expressing CHO/PDPN cells, but not for CHO/mock cells, we finally obtained two anti-PLAG4 mAbs, designated PG4D1 and PG4D2. The epitopes of PG4D1 and PG4D2 were identified by examining their reactivity to recombinant human ΔN24-podoplanin expressed in 3 Figure . Each amt Figure . We conft Figure . The subt Figure . The dis10 proteins. The addition of each mAb to the reactants suppressed podoplanin-CLEC-2 interaction in a concentration-dependent manner r Figure . At highr Figure . To exclr Figure . PG4D1 ar Figure . To furtr Figure . The rear Figure . These rin vivo metastasis assay to verify the role of the PLAG4 domain. Podoplanin-induced pulmonary metastasis was significantly blocked by a single administration of PG4D1 or PG4D2 mAb on the day before tumor inoculation, like MS-1 mAb  attenuated the activity and that podoplanin expressed in glycosylation-deficient CHO mutants, Lec-2 or Lec-8, exhibited no platelet-aggregating activity [O-glycosylation at the Thr52 residue [O-glycans attached to Thr52 in addition to Glu47 and Asp48 residues in the human PLAG3 domain are associated with the binding to Arg residues in CLEC-2 [O-glycosylated amino acid . In the case of our newly identified PLAG4 domain (81-EDLPT85SE-87), Thr85 and Ser86 residues appeared to be O-glycosylated since the NetOGlyc 4.0 Server [O-glycosylation at the Thr85 and Ser86 residues in the human PLAG4 domain than at the Thr52 residue in the human PLAG3 domain. Moreover, Zimmer et al. previously suggested glycosylation at the Thr92 residue within the canine PLAG4 domain (88-EDGPT92QE-94) [O-glycosylated Thr85 and/or Ser86 in addition to negatively charged Glu81 and Asp82 residues in the human PLAG4 domain would be associated with the binding to CLEC-2 and the platelet-aggregating activity of podoplanin. Based on the low homology of the Ser86 residue among mammalian podoplanin, O-glycans attached to the Ser86 residue in the human PLAG4 domain may not be required for CLEC-2 binding but may contribute to robust PLAG4–CLEC-2 interaction. At least, we indicated the contribution of Thr85 residue to CLEC-2 binding using its Ala-substituted mutant  . Therefod mutant . The nums Figure . Moreoves Figure , similar81, Asp82, and Thr85 residues in the PLAG4 domain, with the equivalent positions of Glu47, Asp48, and Thr52 residues in the PLAG3 domain, as essential for CLEC-2 binding . The single administration of the anti-PLAG4 mAbs into mice could almost completely suppress hematogenous metastasis of CHO/PDPN cells. Since a PG4D1 mAb of murine IgG1 subclass exhibiting low ADCC/CDC activity and a PG4D2 mAb of murine IgG2a subclass having strong ADCC/CDC activity could suppress pulmonary metastasis, ADCC/CDC activity would not be associated with the metastasis-inhibitory function of the mAbs, similarly to the anti-PLAG3 mAb MS-1 [in vivo [Our established anti-PLAG4 neutralizing mAbs, PG4D1 and PG4D2, exhibited a high affinity to human podoplanin with very low mAb MS-1 . ConsidemAb MS-1 , the tramAb MS-1 . Of cour[in vivo . However[in vivo . These rIn this report, we succeeded in identifying the novel PLAG4 domain that is associated with the binding to CLEC-2 and platelet aggregation. Based on the PLAG4 domain sequence, we would propose ED(X)XXT as the revised PLAG domain consensus sequence. Our finding is expected to shed light on the clinical development of podoplanin-targeting cancer therapy.CHO cells were purchased from the American Type Culture Collection  and cultured in PRMI 1640 medium  containing 10% FBS . Stable podoplanin-expressing clones were established as described previously [podoplanin cDNA (pcDNA3-podoplanin) was established as described previously [podoplanin deletion mutated cDNAs  or podoplanin point-mutated cDNAs  were generated from pcDNA3-podoplanin as a template using the QuikChange site-directed mutagenesis kit . pGEX-6P-3 vectors  containing signal peptide-deleted podoplanin cDNA (PDPN-ΔN24) or its point-mutated cDNAs were constructed as described previously [The pcDNA3 vector containing WT human eviously . pcDNA3 eviously .nu/nu, and Jcl:ICR mice were purchased from Charles River . All animal procedures were performed according to the protocols approved by the Japanese Foundation for Cancer Research Animal Care and Use Committee.Female BALB/c, BALB/c-http://www.ncbi.nlm.nih.gov/refseq/). For the alignment of podoplanin sequences, sequences including multiple gaps were excluded, and typical experimental animal species and representative species of each family were selected. The selected 42 sequences are presented as All podoplanin protein sequences were obtained from NCBI's reference sequence database , and the cell pellets were boiled in SDS sample buffer for immunoblots. The membranes were incubated with antibodies to podoplanin , α-tubulin , and GST .Cells were lysed in TENSV buffer  and electrophoresed followed by the transfer to a PDVF membrane . The GST-tagged human PDPN-ΔN24 and its point mutants were produced in BL21 (DE3) 10-tagged human CLEC-2 protein , following incubation with 1/500 dilutions of Alexa Flour 488-conjugated anti-penta-His antibody . Flow cytometric analyses were performed using Cytomics FC500 flow cytometry system . In some experiments, MS-1 and PG4D2 mAbs were used as primary antibodies, following the conjugation of DyLight594 using the DyLight antibody labeling kit .Cells were harvested and treated with 1 μg/ml of anti-podoplanin antibodies or control mouse IgG , following incubation with 4 μg/mL of Alexa Flour 488-conjugated anti-mouse IgG (H + L) . For CLEC-2-binding analysis, cells were incubated with 0.4 μg/mL of (His)g for 8 min twice. Platelets were collected from the pellets of PRP by centrifugation at 500 xg for 10 min, and the supernatant was stocked as platelet-poor plasma (PPP). Washed platelets were prepared by washing the pellets with the modified Tyrode's buffer  followed by centrifugation under the same conditions and resuspended in the modified Tyrode's buffer at a concentration of 1 × 108/mL. Prior to the assay, 10% PPP and 400 μM CaCl2 were supplied to the washed platelets. Next, the washed platelets were incubated with cell suspension (1 × 108 cells/mL). For the antibody inhibition assay, cells were incubated with 10 μg/mL of mAb or control IgG for 30 min on ice before incubation with platelets. The platelet aggregation rate was examined using a platelet aggregometer .Murine whole blood was drawn by cardiac puncture from Jcl:ICR mice terminally anesthetized with sevoflurane and taken with heparin solution. Platelet-rich plasma (PRP) was obtained from the supernatant of whole blood by centrifugation at 120 xpodoplanin cDNA region encoding amino acids 76–89 (226–267 bp) was cloned and tandemly connected 12 and 40 times. These cDNA fragments were inserted into a pGEX-6P-3 vector . Next, the GST-tagged human podoplanin peptide (76–89 aa) produced in BL21 (DE3) E. coli was purified using glutathione sepharose. Six-week-old female BALB/c mice were injected with the GST-tagged peptide as an immunogen in conjugation with Titer MAX Gold adjuvant . Further, intraperitoneal immunization was performed intermittently for two months. Mice were euthanized, and splenocytes were fused with mouse myeloma P3U1 cells using PEG4000 . Hybridoma screening and antibody purification from ascites were performed as described previously [A human ds 76–89 6–267 bp KD) were decided using the bivalent binding analyte model.SPR analysis was performed using Biacore X100 . Recombinant human podoplanin-Fc protein  was immobilized on a CM5 sensor chip . Final levels of immobilization were approximately 562.2 or 562.6 response units . Five concentrations of PG4D1, PG4D2 mAbs, and their control mouse IgGs were flowed over the chip in the single cycle kinetics. Sensorgrams were fit by global analysis using the Biacore X100 evaluation software. Efforts toward determining the equilibrium dissociation constant (10-tagged human CLEC-2 protein (30 ng/well) was added to the mixture, following the addition of alpha screen beads . After incubation for 30 min at RT, binding intensity was measured as a fluorescence signal using the EnVision plate reader .Podoplanin-Fc protein (1 ng/well) was firstly incubated with PG4D1, PG4D2, and MS-1 mAbs, or control IgG1 and IgG2a in a 96-well plate. Next, (His)nu/nu mice were intravenously inoculated with antibodies (10 μg/mouse) the day before cell injection. CHO/PDPN cells were resuspended in Hanks’ Balanced Salt Solution  and intravenously injected into mice (2.5 × 105 cells/mouse). Eighteen days following CHO/PDPN injection, the mice were euthanized, and their lungs were stained with the saturated picric acid solution. Surface lung metastatic foci were counted.Five-week-old female BALB/c-U test was performed to determine the statistical significance in flow cytometry and metastasis assays. Significant probability (P) values are shown as *P < 0.05, and **P < 0.01. All statistical tests were two-sided.A Mann–Whitney


Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into “classical” and “non-classical” inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61 Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents. Hemophilia A is a blood clotting disorder caused by a lack of functional blood coagulation factor VIII (fVIII), a protein cofactor essential to the intrinsic pathway of the blood clotting cascade. Congenital hemophilia A, which varies in severity depending on the amount of functional fVIII present, is an X-linked disorder affecting 1 in 5,000 males worldwide245a3-A3-C1-C2 prior to proteolytic processing789a3-A3-C1-C2)12131516Coagulation fVIII is a 2,332-residue glycoprotein that is expressed with the domain arrangement of A1-A2-B-Characterization of the immune response to fVIII has revealed that the A2 and C2 domains harbor the majority of epitopes recognized by inhibitory antibodies against fVIII181920142223202520ABs) of both a classical (3E6) and non-classical (G99) antibody demonstrated that these inhibitors bind to opposite sides of the C2 domain with minimal overlap with the BO2C11 epitope313234AB of the classical antibody 3E6. Comparisons between the C2 domain/3E6 FAB binary structure with the C2 domain/3E6 FAB/G99 FAB ternary structure illustrate the structural conservation and dynamic behavior of C2 domain epitopes for classical antibody inhibitors in the presence and absence of non-classical antibody inhibitors.Detailed structural analyses of the fVIII C2 domain in complex with inhibitory antibodies have allowed for the characterization of both classical and non-classical epitopes2728AB, 3E6, was refined to a resolution of 2.61 Å with a final Rwork and Rfree of 19.9% and 26.7%, respectively  was built to represent a complete model with the exception of residues 132–133 from chain A, which reside in a loop at the base of the constant domain. By contrast, the FAB from complex 2 possessed poorly resolved density for the constant domains of both the heavy and light chains . Within the FAB constant domains of complex 2, residues 177 and 130–135 were absent from the heavy chain and residues 106, 149–154, 187–191 and 198 203 were absent from the light chain. Regardless of this region of poorly defined density, the fVIII C2 domain/3E6 FAB interface was well defined by unambiguous electron density for both the C2 domain as well as the CDR loops for each complex.The structure of the fVIII C2 domain in complex with the inhibitory antibody Fectively . Iteratiow angle . Validatow angle . The strAB binding interface is highly conserved. Upon superposition of the C2 domain and variable domains for each binary complex with the C2/3E6 portion of the previously determined structure of the C2 domain/3E6/G99 FAB ternary complexAB elbow, respectively  and His2315 (−0.6 pH units).Following the characterization of each binding interface for both binary complexes and comparing them to the ternary complex, it was concluded that the mechanism for cooperativity between classical and non-classical anti-fVIII C2 domain inhibitory antibodies is likely not due to significant changes in conformation directly at the binding interface. Given that cooperativity is observed for several classical/non-classical antibody pairs, we hypothesized that the cooperative behavior may be due to either electrostatic or dynamic perturbation. Following pKa calculations with PROPKA for the fVIII C2 domain in isolation as well as in complex with the 3E6 antibody, it was observed that the majority of significant pKa perturbations occurred directly at the 3E6 interface, as expected. Specifically, residues with significant pKa perturbations (>0.25 pH units) in the region of the 3E6 epitope were Glu2181, Glu2322, Lys2183, Lys2207, Lys2236, His2211, Arg2209, Arg2215 and Arg2320. By contrast, two residues at or near the non-classical G99 FAB ternary complex (pdb#: 4KI5), the C2/BO2C11 FAB complex (pdb#: 1IQD) and the isolated fVIII C2 domain (pdb#: 1D7P). While overall B factors are largely crystal-dependent20383942To assess the dynamic behavior of the non-classical anti-C2 domain epitopes in the presence and absence of the classical inhibitory antibodies, crystallographic B factors were compared for each C2/3E6 binary complex described herein (pdb#: 4XZU), the C2/3E6/G99 FAB complexes within this crystal form with the previously determined C2 domain/3E6/G99 FAB ternary complex illustrate the high level of structural conservation at the binding interface with the exception of Arg2215, which shows different conformers for each complex. Provided that significant structural changes that would explain this cooperativity were not observed directly at the C2/3E6 binding interface, we hypothesized that cooperative binding may be the result of perturbation of either surface electrostatics or dynamics. Upon inspection of B factors for the fVIII C2 domain in each complex and in isolation, we determined that several loops distal to the 3E6 epitope displayed lower B factors relative to the entire C2 domain structure for each C2/3E6 complex. The associated decrease in mobility that is concomitant with lower B factors could decrease the entropic cost of binding a second, non-classical antibody, a hallmark of the induced fit binding mechanism often observed for antigen-antibody interactions4228In this study, we have determined the X-ray crystal structure of a classical anti-fVIII C2 domain inhibitory antibody (3E6) in complex with the C2 domain from human blood coagulation factor VIII to 2.61 Å resolution. Inhibitory antibodies often arise following fVIII “replacement therapy” in hemophilia A patients, causing a significant clinical complication of uncontrolled bleeding. Previous antibody binding data indicate that classical and non-classical anti-fVIII antibodies bind cooperatively, but the molecular mechanism of this behavior has not been described. Upon comparison of the two fVIII C2 domain/3E6 F6 affinity tag with a thrombin cleavage site. This expression construct was transformed into Escherichia coli NiCo21 cells (a BL21 (DE3) derivative) and subsequently grown at 37 °C in LB media in the presence of ampicillin to an OD600 of 0.6–0.8. Protein overexpression was induced upon the addition of isopropyl β–D-thiogalactopyranoside to 0.5 mM with adjustment of the incubation temperature to 15 °C for 16–20 hours. Overexpressed cell cultures were centrifuged at 8,000 rpm for 10 minutes at 4 °C , and the resultant cell pellet was resuspended in lysis buffer (20 mM Tris-HCl (pH 7.0), 300 mM NaCl, 10 mM imidazole (pH 7.0), 0.01% (v/v) Triton X-100, and 2.5% (v/v) glycerol). Resuspended cells were lysed by the addition of 1 mM PMSF and 0.75 mg/mL chicken egg white lysozyme for 15–20 minutes at 4 °C followed by sonication on ice with a ½-inch titanium horn attached to a Branson 450 sonifier (50% duty cycle) for two cycles of 1 minute. The fVIII C2 domain-containing cell lysate was centrifuged at 16-17,000 rpm for 30–35 minutes at 4 °C , and the supernatant was subsequently filtered with 5 μm and 0.45 μm cellulose syringe filters, sequentially. Filtered lysate was applied to TALON cobalt affinity resin , which was pre-equilibrated with lysis buffer, and incubated for 1 hour at 4 °C. The lysate/resin slurry was applied to a gravity flow column and washed with 10 column volumes (CV) of lysis buffer, 20 CV of wash buffer I (20 mM Tris-HCl (pH 7.2), 300 mM NaCl, 10 mM imidazole, 2.5% (v/v) glycerol), 10 CV of wash buffer II (20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 10 mM imidazole, 2.5% (v/v) glycerol). Lastly, the hexahistidine-tagged fVIII C2 domain was eluted with 20 mM Tris-HCl (pH 7.2) 150 mM NaCl, 150 mM imidazole (pH 7.0), and 2.5% (v/v) glycerol, which was immediately dialyzed into ion exchange buffer (25 mM Tris-HCl (pH 7.2), 50 mM NaCl). The initial purified fraction of the fVIII C2 domain was further purified by ion exchange chromatography with a Macro-PrepTM strong cation exchange column (Bio-Rad), through a salt gradient from 50 to 500 mM NaCl. The eluted C2 domain was concentrated to 6–8 mg/mL and a final purification step was completed by size exclusion chromatography with a Superdex 75 column , which was equilibrated ion exchange buffer.Generation of purified fVIII C2 domain was performed as previously described28TM Protein A Plus spin column according to the manufacturer’s instructions (Thermo Scientific). The 3E6 FAB fragments were subsequently cleaved with immobilized papain (Thermo Scientific) and further isolated by an additional Protein A spin column step to remove the Fc regions of the IgG. The resultant FAB fragments were further purified by size exclusion chromatography with a Superdex 75 column  and concentrated to 10–20 mg/mL. The C2 domain/3E6 FAB fragment binary complex was formed by incubation at 37 °C for 30 minutes with a 1.5-fold molar excess of the C2 domain. The C2/3E6 binary complex was then separated with a Superdex 75 column, concentrated to 5–10 mg/mL, flash frozen in liquid nitrogen and stored at −80 °C for crystallization trials.The murine monoclonal hybridoma for the 3E6 antibody was generated and large-scale antibody productions were performed previously28AB constant domain (PDB: 4KI5), and the 3E6 FAB variable domain, which were searched for iteratively. Model building and refinement of the X-ray crystal structure were performed with COOT and PHENIX, respectively454951Initial crystallization conditions were first identified following the manual setup of 24-well sparse matrix screens by hanging drop vapor diffusion. Crystals suitable for diffraction studies were grown by a 1:1 ratio of 8 mg/mL C2/3E6 binary complex with 10 mM MES (pH 6.4-6.8) and 20% (w/v) PEG 8000. Small, disordered crystals were grown within the first 7–9 days, diffracting to 3.2 Å resolution while larger crystals that diffracted to 2.6 Å resolution grew over the period of one year. Cryoprotection of crystals was performed by the iterative transfer of crystals to a drop containing 10 mM MES (pH 6.5), 22% PEG 8000, and 10–30% dimethyl sulfoxide, and the crystals were subsequently flash-frozen in liquid nitrogen for cryogenic X-ray data collection. X-ray diffraction data were collected to 2.6 Å resolution on a Rigaku Micromax-007HF rotating anode with Confocal Varimax Optics and an RAXIS-IV++ imaging plate detector at the Fred Hutchinson Cancer Research Center . Diffraction data were collected with CrystalClear (Rigaku) and indexed, integrated and scaled with HKL2000Accession Numbers: Model coordinates and structure factor amplitudes for the X-ray crystal structure of the factor VIII/3E6 binary complex were deposited in the Protein Data Bank (acc. #: 4XZU).How to cite this article: Wuerth, M. E. et al. Structure of the Human Factor VIII C2 Domain in Complex with the 3E6 Inhibitory Antibody. Sci. Rep.5, 17216; doi: 10.1038/srep17216 (2015).


The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. N-terminus with the attached catalytic domain across the membrane [Many bacterial toxins, such as diphtheria , botulinmembrane . This remembrane ,7 and fomembrane ,9. HowevThe crystal structure of the T domain in solution at neutral pH  consistsi.e., histidine and glutamic and aspartic acid) plays a key role in different stages along the pathway [N-terminal helices upon transmembrane insertion of TH8–TH9. However, the role of protonation of acidic residues has been studied to a lesser extent. The T domain has 23 acidic residues, most of which are located in the polar part of the sequence. In this study, we focus on three of them that are located within the hydrophobic consensus membrane insertion unit formed by the TH8–TH9 hairpin, which suggests that they play a direct role in the membrane interactions of the T domain. These residues are E362, which is located in the middle of TH9, and E349 and D352, both located in the loop connecting helices TH8 and TH9  computer simulations. We generated the double mutant E349Q/D352N (tip of the hairpin) and the single mutant E362Q (middle of TH9) to neutralize the negative charges on the hairpin. We complement our study with the application of Since the residues E349, D352 and E362 are located within helices TH8 and TH9 , which fWe first tested the ability of these mutants to interact with the membrane using a vesicle permeabilization assay previously applied to characterize membrane interactions of the T domain ,23. This2/dmol for the mutant E362Q at pH 8 and 6.5, respectively (about 14% and 19% less ellipticity than the WT protein). We studied the thermal stability by monitoring the molar ellipticity at 222 nm as a function of the temperature. The mutant E362Q shows a reduced stability at pH 8 and 6.5 . We have previously shown that the position of maximum emission of tryptophan, which can be reliably obtained by fitting the spectra to a log normal distribution [N-terminal region of the T domain [Next, we measured tryptophan fluorescence originating from W206 and W281 located within the ribution , is indiT domain . IntrinsT domain , appearsThe conformational changes induced by lowering the pH lead to partial exposure of the hydrophobic core of the T domain, which in the absence of membranes, leads to oligomerization . In the We performed extended molecular dynamics (MD) simulations (3.5 μs) of both the WT T domain and the mutant E362Q in explicit water. This allows us to better visualize the effect of introducing this mutation in the middle of TH9 on the solution folding of the protein. Overall, the results indicate that there is more flexibility in the mutant protein, with occasional bending and partial loss of secondary structure in helix TH1. The latter is consistent with the partial loss of ellipticity observed in CD measurements . ResidueC-terminal histidines [N-terminal region in the membrane (see the Discussion). The fact that further acidification results in a blue-shifted tryptophan spectrum suggests that E362Q adopts a WT-like conformation regardless of its early misfolding in the membrane.To determine how the altered transitions in solution affect the mutants’ folding transitions within the membrane, we applied several methods of fluorescence spectroscopy that allowed us to observe environment-induced spectral changes of intrinsic and extrinsic probes. First, we measured the spectral position of tryptophan fluorescence of the WT and the mutants in the presence of large unilamellar vesicles (LUV) composed of palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in a 3:1 ratio as a function of pH. The data presented in stidines , where tpK of membrane insertion and the cooperativity factor by fitting the data as described previously [pK by 0.3 points and decreases the cooperativity from about 2 to ~1. The latter suggests that residue E362 is protonated during the membrane insertion process (see the Discussion). Our results thus indicate that both mutants are able to adopt a transmembrane topology, but the mutant E362Q does it at a higher pH than that required for the WT and the double mutant E349Q/D352N.Next, we monitored the transbilayer insertion of the TH8–TH9 hairpin using the environment-sensitive NBD fluorophore specifically attached in the middle of what becomes a transmembrane region ,23. To aeviously . While tN-terminus, we assessed the ability of the mutants to perform this action using a translocation assay described previously [N-terminus of the T domain with a thrombin cleavage site. When the N-terminus of the T domain is translocated into the lumen of a thrombin pre-loaded vesicle, the protease cleaves off the His-tag, changing the electrophoretic mobility of the protein  associates with the membrane interface, where depending on the lipid composition, it can either get trapped in the nonproductive interfacial intermediate I-state or proceed along the pathway by inserting the TH8–TH9 helical hairpin across the lipid bilayer (T1-state). In this study, we used a lipid composition with a high fraction of anionic lipids, which lowers the kinetic barrier for insertion and assures that there is no accumulation of the interfacial I-state [N-terminal region still exists on the cis side of the bilayer, is subsequently converted into the T2-state , with tThe mutations E362Q and E349D/D352N caused changes in the membrane insertion pathway of the T domain, especially in the initial stages . The ANTpK of 5.8 and cooperativity factor n ~ 2. The latter can be interpreted as the requirement for two titratable residues to be protonated during the insertion. We suggest that one of these residues is E362, because its replacement leads to a shift of pK to 6.1 and a decrease of cooperativity n ~ 1 . ThuN-terminal helices and the translocation of the N-terminus by mutating out the histidines flanking TH8–TH9 sequence [N-terminal helices within the membrane observed in the histidine mutants. Unlike the case of the histidine mutants, however, the further acidification corrects the misfolding, and E362Q adopts a functional T2-state , which nevertheless preserves the WT-like activity. Our results indicate that the insertion pathway is modulated by multiple pH-dependent transitions and that the shift of certain steps toward less acidic pH, observed as the result of mutagenesis, does not lead to automatic shifts in the steps occurring downstream along the pathway and may result in misfolding of partially-inserted protein. We discover that conformational switching of different steps is under independent control and involves protonation of multiple titratable residues. Further studies will be directed at characterizing the complete network of protonation-driven molecular switches that trigger the membrane interactions of the T domain. A number of studies from the groups of Engelman, Reshetnyak and Andreev (see  and the N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole) was purchased from Invitrogen . The diphtheria toxin T domain (amino acids 202–378) was cloned into the Ndel- and EcoRl-treated pET15b vector containing an N-terminus six-His tag and a thrombin cleavage site [Materials: Palmitoyl-oleoyl-phosphatidylcholine (POPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) were obtained from Avanti Polar Lipids . 8-aminionapthalene 1,3,6-trisulfonic acid (ANTX) and p-xylene-bis-pyridinium bromide (DPX) were obtained from Molecular Probes . IANBD ester  were intoxicated by diphtheria toxin constructs for 24 h at 37 °C, after which incorporation of L-4,5-3H leucine was measured. Per the NIH guidelines, these measurements were performed with a weakened strain carrying the E148S mutation in the catalytic domain to reduce the toxic potency [Protein Synthesis Inhibition Assays: Cytotoxicity or cell death assays were carried out using a protein synthesis inhibition assay . Briefly potency .Leakage assays: LUV with entrapped ANTS and DPX were prepared as previously described ,46. BrieN-terminus translocation assay: The ability of the T domain to translocate its N-terminus across the lipid bilayer was studied with thrombin-loaded LUV [N-terminal His-tag and a thrombin cleavage site to release the His-tag from the rest of the protein upon proteolytic treatment. Upon insertion and translocation of the purified T domain into LUV pre-loaded with thrombin, the His-tag is cleaved by the protease, resulting in a protein of lower molecular weight that can be detected by following its electrophoretic mobility. LUVs containing ~0.02 units of bovine thrombin  were prepared by the conventional extrusion method [aded LUV . T domain method ,43. Non-N-to-U unfolding transition, where N and U are the native and unfolded state of the protein, respectively. In order to obtain the transition temperature (Tm) and the enthalpy changes (∆H°), raw data were fitted applying nonlinear least-squares analysis with six fitting parameters, NY, Nm, UY, Um, ΔH° and Tm, with the following equations [Y is the experimentally observed CD signal at a given temperature, NY and UY, represent the signals of the pure N and U states at 0 K, Nm and Um are the temperature-dependencies of these CD signals for the N and U states, respectively, and NX is the fraction of the native state at temperature T.CD measurement and analysis of thermal unfolding: CD measurements were performed using an upgraded Jasco-720 spectropolarimeter . Normally, 200 scans were performed from 260 nm to 190 nm in a sample containing 4 µM of protein in 50 mM phosphate buffer using a 1-mm optical path cuvette. All spectra were corrected for the background. Temperature dependencies of unfolding were monitored by measuring ellipticity at 222 nm with a 1-degree/min scan rate. The thermal unfolding was analyzed using thermodynamic equations for a reversible two-state, quations ,47:Y = Size exclusion chromatography: Previously purified monomeric fractions containing 25 μM WT or mutants were incubated in 50 mM phosphate buffer at either pH 8.0 or pH 6.5. Samples were placed onto the TSK-Gel G2000 SWXL column  equilibrated at the corresponding pH with an elution rate of 0.5 mL/min. BSA (68 kDa), and ovalbumin (43 kDa), cyan fluorescent protein (22 kDa) and RNAse A (14 kDa) were used to calibrate the column.Steady-state fluorescence spectroscopy: Fluorescence was measured using an SPEX Flurolog FL 3–22 steady-state fluorescence spectrometer  equipped with double grating excitation and emission monochromators. The measurements were made at 25 °C in 2 × 10 mm cuvettes oriented perpendicular to the excitation beam. For tryptophan fluorescence measurement, excitation emission wavelength were 280 nm, and emission spectra were recorded between 290 nm and 500 nm using excitation and emission spectral slits of 2 and 4 nm, respectively. For NBD fluorescence measurements, the excitation wavelength was 470 nm, and the emission spectra were recorded between 490 nm and 700 nm using excitation and emission spectral slits of 5 nm on both monochromators. Normally, we mixed the sample (taken from a concentrated stock) with LUV maintaining the T domain and lipid concentrations at 1 µM and 1 mM, respectively, and rapid acidification was achieved by the addition of small amounts of 2.5 M acetic buffer. All spectra were recorded after 30 min of incubation to ensure the equilibrium of the sample. Correction for the background and fitting of all spectra to calculate the position of maximum emission was done as described previously .Time-resolved fluorescence spectroscopy: All of the decays were measured with a time-resolved fluorescence spectrometer  using a standard time-correlated, single-photon counting scheme. Samples were excited at 440 nm by a sub-nanosecond pulsed diode laser  with a repetition rate of 10 MHz. Fluorescence emission was detected at 540 nm, selected by a Sciencetech model 9030 monochromator , using a PMA-182 photomultiplier (PicoQuant).T = 310 K over 20 ps with harmonic restraints in the protein atoms  and constant volume. The final snapshot was later equilibrated over 600 ps at constant pressure conditions with P = 1 atm and T = 310 K with restraints applied to the protein heavy-atoms , followed by three consecutive 100-ps equilibration stages with gradually reduced restraints applied to the protein heavy atoms . Final equilibration with no restraints was carried out over 10 ns and 31 ns for the wild-type and mutant, respectively. The thermostat and barostat were Langevin and Berendsen, respectively. Production simulations of the WT and E362Q mutant were performed using the GPU-accelerated PMEMD  and Anton supercomputer, respectively [T = 310 K (tau = 1 ps) and a pressure of 1 atm (tau = 2 ps), respectively. System coordinates were saved every 60 ps. Production MD simulations of WT and E362Q mutant were performed over 3630 ns and 3481 ns, respectively. Trajectory analysis was carried out using the program ptraj available in Amber12  [Molecular dynamics simulations: Atom coordinates of the T domain were taken from the X-ray structure of diphtheria toxin obtained in buffer at pH 7.5 (residues 206–380 from PDB ID 1F0L) ,48. The ectively ,54. Bothectively . A reverectively . Bonded ectively . The BerA, 2012) , and molA, 2012) .