GSB) pinto bean flours were used to make gluten‐free cookies, and their sensory characteristics evaluated to determine how their consumer acceptability scores compared. Taste panelists (31) graded cookies made from raw pinto beans with an overall value of 6 on a 9‐point hedonic scale (p < .05). This rating was not significantly different from cookies formulated with germinated and GSB flours. Therefore, gluten‐free cookies can be made using raw pinto bean flours at a 40% inclusion level, with similar sensory characteristics as those prepared with flours treated by cooking and germination. Instrumental measurement of cookie hardness and color showed no significant difference in hardness, but significant differences in color. The germinated bean flour produced cookies with a significantly lower L* value and significantly higher a*, b*, Chroma and hue values compared to the other treatments. There was no significant difference in the cookie spread ratio. Proximate composition, water absorption index (WAI), water solubility index (WSI) and gelatinization properties of the flour treatments were characterized.Beany and grassy flavors in raw edible bean flours reduce consumer acceptability of bean‐based baked products. In order to improve consumer acceptability, beans may be further processed by cooking and germination. However, these operations drive up the cost of end‐products. Therefore, it is necessary to develop formulations, using raw edible bean flours that have acceptable sensory attributes. In this study, cooked, germinated, and germinated/steam‐blanched ( These benefits include high protein, high fiber, low fat, significant amounts of vitamins and minerals, and low glycemic index .In this study raw, cooked, germinated, and germinated/steam‐blanched (GSB) pinto bean flours were used to make gluten‐free cookies, and their sensory properties investigated. Several researchers have studied the effect of adding pulse ingredients to gluten‐free cookies. Pulse ingredients added to gluten‐free cookies so far include germinated/extruded black bean cotyledon flour, extruded navy bean flour, extruded pinto bean flour, commercially available yellow pea flour, dehulled green lentil flour, raw navy bean flour, raw pinto bean flour, precooked and dehulled soybean flour, commercially available soybean flour, raw lupine flour, extruded black bean flour, dehulled pigeon pea flour, and chickpea flour  were purchased from Ruler Foods . Oats, rice, tapioca, and quinoa flours, distributed by Bob's Red Mill , were purchased from Kroger . All other ingredients, i.e., sugar, butter, egg, xanthan gum, salt, and vanilla were purchased from Walmart Stores Inc. .2.1® polypropylene tub (35.6 cm × 20.3 cm × 12.4 cm). After soaking, the beans were drained to remove excess water and then boiled in fresh excess distilled water for 20 minutes in a stainless steel pot heated on a gas kitchen range. Cooking time started when the water temperature reached of 99°C. The cooked beans were then dried in a convection oven  at 85°C for 14 hours. After drying, the beans were milled, collected in Ziploc® bags, and stored as previously described. Germinated bean flours were prepared by rinsing 1 kg of pinto beans under running tap water and then steeping them for 20 hours at room temperature in a Sterilite® polypropylene tub (35.6 cm × 20.3 cm × 12.4 cm). The beans were aerated during steeping, using an Aqua Culture® aquarium pump  . After steeping, beans were rinsed with distilled water, drained and then placed in a closed cupboard to germinate for 48 hours. The beans were sprayed with a layer of fresh distilled water every 12 hours to ensure that they did not dry out during germination. The beans were then dried, milled, and stored as previously described. GSB bean flours were prepared according to the previously described germination procedure and then steam‐blanched in a steam pot for 25 minutes on a gas kitchen range. Blanching time started when the center temperature of the bean batch reached 93°C. The beans were finally dried and milled as previously described, and held at room temperature until flour analysis and cookie mix preparation. Four cookie flour mixes were made by homogenously combining 40% pinto bean flour , 30% oats, 15% rice, 7.5% tapioca, and 7.5% quinoa in 3.8 L PETE plastic jars.To prepare raw flour, 1 kg of pinto bean was milled, using a Retsch centrifugal mill  running at 10,000 rpm and fitted with a 0.5 mm sieve. The flour was collected in Ziploc® bags and stored at room temperature until ready for flour mix preparation. Cooked bean flour was prepared by rinsing 1 kg of pinto beans under running tap water followed by soaking in excess distilled water for 12 hours at room temperature in a Sterilite2.2® kitchen mixer  operating at speed 1. Egg and vanilla were gradually added while mixing for another 4 minutes at speed 1 to ensure that all the cream was combined with the egg. Salt and xanthan gum were added to the cookie flour mix and the mix then manually folded in, using a spatula to achieve a soft homogenous dough. The dough was transferred to a 3.8 L Ziploc® bag and refrigerated for 1 hour to harden. After hardening, the dough was sheeted, using a rolling pin on a counter top lined with parchment paper. Dowel rods (0.5 cm × 22 cm) were used to ensure that the cookie dough sheet maintained consistent thickness. Cookies were then cut from the sheet, using a 36 mm diameter nonserrated circular cookie cutter, and transferred to a 40.6 cm × 30.5 cm × 2.54 nonstick metal baking tin . The cookies were then baked at 190.6°C for 7 minutes after which the cookies were removed from the oven and allowed to cool for 2 minutes on the baking tray. Immediately afterwards, the cookies were transferred to a kitchen countertop lined with grease paper where they were allowed to finish cooling. The prepared cookie dough was large enough to bake four batches of cookies with each tray holding 20 cookies (yield = 80 cookies). However, only one tray was placed in the oven at a time to ensure that the tray was placed in the same position in each run, and that the oven temperature variation was minimized.Two separate batches of cookies, representing each pinto bean flour treatment were prepared in order to replicate cookie dough formulation and baking procedure. Baking protocol proceeded by combining and creaming sugar and butter Table  for 3 mi2.3® combo mantle, Terre Haute, IN). Hexane was evaporated with a rotary evaporator  at 40°C under vacuum. Flasks were then reweighed to determine increase in weight. Weight difference was used to calculate percentage of total lipids extracted. Total protein was determined in duplicate, using a nitrogen combustion analyzer  according to AACC International Approved Method 46‐30.01 based on approved AACCI Method 76‐21.01 AACCI, .2.4The spread ratio was determined by measuring the width and thickness of 10 randomly selected cookies from each baking run (n = 20), and then dividing the width by the thickness. Each of the randomly selected cookies from the spread ratio test was also measured for texture using a CT3 texture analyzer  equipped with a three‐point bend assembly. The instrument was set at a speed of 1 mm/sec; 4 mm target value and 10 g trigger load. The color of the flours was determined, using a CR‐410 Konica Minolta Chroma meter  and reported as L*, a*, b* values. The hue angle was calculated by finding the ratio of a* to b* and then determining the inverse tangent. The chroma was calculated using the following equation:2.5Sensory evaluation of cookies was based on a hedonic test, using 31 sensory panelists obtained after Institutional Review Board (IRB) human subject research approval. Panelists included students, faculty, and staff over 18 years of age who responded to email invitation to attend the sensory evaluation exercise. There was no criteria in the invitation that they were consumers of gluten‐free products. The evaluation was done in a classroom. Tables were spaced to reduce opportunities for interference by, or communication with other panelists. Nevertheless, they were advised not to communicate during the tasting. Each panelist was provided with four cookies representing each treatment. The cookies were assigned a random three‐digit number and presented in random order to the panelists. They were not otherwise provided with the identity of the cookies, including not telling them that they were gluten‐free. Along with the samples, they were provided with a bottle of drinking water and asked to drink between sampling each cookie, and allow their palate to rest for at least 30 seconds before moving to the next sample. They panelists rated the appearance, flavor, texture and overall acceptability of the cookies on a 9‐point scale .2.6p ≤ .05.Cookie baking was done on two separate days and the spread ratio, hardness, and color values reported as means of the two baking runs. Analysis of these attributes was based on a completely randomized design. Sensory data was based on a completely randomized block design with panelists representing 31 blocks. SAS software  was used to conduct analysis of variance (ANOVA) and comparison of means done, using Tukey's multiple comparison test. Differences were considered significant at 33.1Chemical and functional properties of flour treatments are presented in Table Raw and germinated flours had similar pasting characteristics Table  while coIt should be noted here that while many of the chemical and functional attributes shown in Tables 3.2Spread ratio Table  was in tColor measurements showed that cookies made with germinated bean flours had a significantly lower L* value compared to the other flours Table . It also3.3In general, taste panelists attributed an acceptability score of 6.0 (liked slightly) for cookies made with each bean flour treatment, with no significant differences between them Figure . StudiesHowever, an important question to address is whether or not the inclusion of raw untreated pulse flours in bakery flour mixes at such a high level (40%) will have any large negative effect on the safety of their end products. Raw pulse flours are known to have several antinutrients, including flatulence‐causing α‐galactosides, trypsin inhibitors, chymotrypsin inhibitors, α‐amylase inhibitors, phytates, phytohemagglutinins, tannins, saponins, and oxalates. These antinutrients can be significantly reduced by application of techniques such as soaking, dry heat, fermentation, roasting, boiling, autoclaving, extrusion cooking, microwaving, and germination (Adamidou, Nengas, & Grigorakis, 4Sensory evaluation suggest that consumers will find cookies that are made with flours containing 40% raw pinto beans to be acceptable at the same level as cookies made with pretreated pinto beans. Therefore, food manufacturers can reduce the cost of inputs, using raw pinto beans and similar pulses in bakery formulations such as cookies. Further studies are needed to investigate antinutrients in cookies and other bakery products made with raw bean flours. However, current evidence suggests little or no significant risk.The authors declare that they have no conflict of interest.


Patients with moderate-to-severe atopic eczema (AE) often require photo- or systemic immunomodulatory therapies to induce disease remission and maintain long-term control. The current evidence to guide clinical management is small, despite the frequent and often off-label use of these treatments. Registries of patients on photo- and systemic immunomodulatory therapies could fill this gap, and the collection of a core set concerning these therapies in AE will allow direct comparisons across registries as well as data sharing and pooling.Using an eDelphi approach, the international TREatment of ATopic eczema (TREAT) Registry Taskforce aims to seek consensus between key stakeholders internationally on a core set of domains and domain items for AE patient registries with a research focus that collect data of children and adults on photo- and systemic immunomodulatory therapies.Participants from six stakeholder groups will be invited: doctors, nurses, non-clinical researchers, patients, as well as industry and regulatory body representatives. The eDelphi will comprise three sequential online rounds, requesting participants to rate the importance of each proposed domain and domain items. Participants will be able to add domains and domain items to the proposed list in round 1. A final consensus meeting will be held with representatives of each stakeholder group.Identifying a uniform core set of domains and domain items to be captured by AE patient registries will increase the utility of individual registries, and provide greater insight into the effectiveness, safety and cost-effectiveness of photo- and systemic immunomodulatory therapies to guide clinical management across dermatology centres and country borders.Not applicable. This eDelphi study was registered in the Core Outcome Measures for Effectiveness Trials (COMET) database.The online version of this article (doi:10.1186/s13063-016-1765-7) contains supplementary material, which is available to authorized users. Atopic eczema (AE) (synonymously ‘atopic dermatitis’) is among the most common chronic inflammatory disorders, with a lifetime prevalence of 15–30% in children and 2–10% in adults . AlternaThe current evidence to guide clinical management for moderate-to-severe AE originates from a small body of randomised controlled trials (RCTs)  and obseNevertheless, as shown in a recent survey among over 700 dermatologists and paediatricians from eight European countries, these immunomodulatory treatments are frequently prescribed as off-label medicines in children . It is lThe establishment of national AE patient registries with a research focus that use the same methodology would not only partially fill the gaps in the current evidence, but could also operate as a research platform to inform the design of future RCTs and basic research and serve as an example for other initiatives. Such a platform will allow the collection of prospective daily practice data on effectiveness, safety, cost-effectiveness, subgroup characteristics, quality of care and personalised medicine. Our research group, the TREatment of ATopic eczema (TREAT) Registry Taskforce, is currently developing an interoperable patient registry platform based on best-practice guidelines for this purpose .a priori agreed core set are likely to yield disparate datasets, hampering direct comparisons of results and data pooling [Experience has shown that disease registries that were set up without an  pooling . To over pooling  to reducIn alignment with the PARENT guidance, the objective of this eDelphi study is to reach international consensus between different stakeholders on a core set of domains and domain items (what to measure), for existing and future atopic eczema patient registries with a research focus, that collect data of children and adults on photo- and systemic immunomodulatory therapies. This protocol outlines the methodology.www.comet-initiative.org/studies/details/825?result=true. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist can be found in Additional File This study will be conducted following the recommended checklist proposed by Sinha et al. , of itemTo investigate domains and domain items of importance, an online Delphi exercise (further referred to as ‘eDelphi’) in combination with a consensus meeting will be conducted Fig. .Fig. 1ThThe eDelphi approach comprises sequential questionnaires answered anonymously by key stakeholder groups. The answers are anonymised to avoid participants being influenced by the opinions of other group members, and thus minimise bias. After each questionnaire round, a summary of the responses is fed back to the group. Individual participants may then decide to keep their original answers or to change their opinion in the next round. Gradually, a consensus evolves as in general the range of answers decreases and the group converges towards a consensus opinion over the course of several rounds .After the eDelphi exercise, a consensus meeting will be organised with representatives from each stakeholder group to resolve potentially remaining disagreements and to agree on a core set of domains and domain items.Health care professionals – doctors who care for patients with moderate-to-severe AE, including doctors who are involved in AE clinical researchHealth care professionals – nurses who care for patients with moderate-to-severe AENon-clinical researchers with a specialist and active research interest in AE, e.g. methodologists, epidemiologists, health economistsPatients (adult patients and carers of children or adults with AE)Industry representatives from pharmaceutical companies involved in the development of systemic immunomodulatory drugs for AERegulatory body representatives from the EMA, US Food and Drug Administration (FDA) and national regulatory bodies such as the UK National Institute for Health and Care Excellence (NICE)Representatives from six key stakeholder groups  will be Currently, there are no official recommendations for the number of participants to include in an eDelphi study . BecauseTo identify health care professionals and non-clinical researchers to participate in the eDelphi, we will approach relevant international societies registered with the International League of Dermatological Societies (ILDS) and other relevant special interest groups  Table . SocietiMembers of the TREAT Research Group will participate in the eDelphi exercise as well as in the final consensus meeting.The eDelphi questionnaire has been developed using a comprehensive list of domains  and domain items  identified by the TREAT Research Group through panel discussions and by literature review. For the ‘physician- and patient-reported’ domain and domain items a direct reference was made to guidance from the Harmonising Outcome Measures for Eczema (HOME) initiative (homeforeczema.org). After obtaining an initial list of domains and items, the members of the TREAT Research Group were again asked to review and to add any additional domains and items that they thought should be included. This led to a proposed list of core domains and domain items for the eDelphi questionnaire Table .Table 3PSome domains and domain items identified were found obvious by the TREAT Research Group through panel discussions, e.g. age and gender, and will, therefore, not be included in the eDelphi but listed separately Table . This liIn addition, patients with AE have critically reviewed the protocol, questionnaire and invitation email concerning content and language to allow patient representatives to take part in the eDelphi in a meaningful way.The eDelphi questionnaire will be distributed using an online e-management survey system, DelphiManager, maintained by the COMET Initiative. This system has previously been successfully used in several studies to facilitate the determination of core outcome sets . The surThe study will run over three online rounds Fig. . At the Participants will be asked to complete each round of the eDelphi exercise and reminder emails will be sent to increase the response rate. To reduce the risk of attrition bias, the importance of completing all eDelphi rounds will be highlighted to all participants at the outset of each round. Participants who save an incomplete questionnaire will be contacted to encourage completion. Furthermore, we will provide clear timelines for the completion of each round and send out personal reminder emails.Round-1 content includes (1) demographic data of the participants , age group of AE patients they predominantly care for , how much experience they have in the care of AE patients , membership of an international dermatology society or AE interest group), (2) the obvious list of domains and their items to be reviewed and potentially commented on, (3) a list of domains with their items to be scored and (4) an option to add any additional domains or domain items.The total number of registered participants for round 1 will be recorded as the number of participants who have actually completed the rating of the domains and domain items.All domains and domain items will be carried through to the second round. Additional domains and items listed by participants will be reviewed by the TREAT Research Group and included in round 2.In this second round, each participant will be asked to rate the domains and domain items again, together with any additional domains and domain items from round 1. They will be presented with the number of participants who scored and the distribution of scores (%) for each domain and domain item for their particular stakeholder group with a reminder of their own round 1 score. They will be asked to consider responses from the other members of their stakeholder group before rescoring each domain and domain item.Reasons for changes to scores will be documented by asking participants at the end of round 2 to give a general view of why they changed scores.Those who have not participated in, or completed, the first round will not be invited to round 2. The total number of invited participants for round 2 will be recorded as the number of participants who have actually completed the rating of the domains and domain items. All domains and domain items will be carried forward to round 3.An overview of the domains and domain items that have reached consensus will be presented at the beginning of the round-3 survey.In this third round, all participants will receive identical feedback, containing the distribution of scores (%) for each domain and each domain item for all stakeholder groups, along with a reminder of their round 2 score. They will then be asked to rescore.All participants included in round 2 will be invited for round 3. As with the previous rounds, the total number of invited participants for round 3 will be recorded as the number of participants who have actually completed the rating of the domains and items. Results of each stakeholder group will be determined. This will lead to the classification of each domain or domain item as ‘consensus in’, ‘consensus out’ or ‘no consensus’ for each stakeholder group separately, which will be used in the final consensus meeting.All eDelphi participants will be asked in round 3 of the survey if they are willing to attend the consensus meeting. The meeting will be held face-to-face at a location agreed by the TREAT Research Group and/or by telephone conference within 4 months after the close of round 3. The consensus meeting will be attended by representatives of all eDelphi stakeholder groups. All those who completed the three rounds are eligible. Representation of health care professionals and patients will be of particular importance.One non-voting neutral participant will act as a facilitator and ensure that the voices of all representative groups are heard, and that the process is not dominated by individual participants.The nominal group technique will be used , 20. TheFor the domains and domain items that have not reached consensus, voting will take place, ideally anonymised by using electronic handsets and TurningPoint© software to analyse the results in real time.The definition of consensus for the eDelphi exercise is based on that proposed by the Management of Otitis Media with Effusion in Children with Cleft Palate (MOMENT) study group , but ameThe definition of consensus for the consensus meeting is applied as used by the Outcome Measures in Rheumatology Collaboration (OMERACT) group and the HOME initiative –24. ConsA core set is defined as a list of variables that are essential to collect for any patient/subject .The results of the third and final eDelphi round will be analysed separately for each stakeholder group using the abovementioned definition of ‘consensus in’ and ‘consensus out’. The consensus meeting results will be evaluated for the whole group using the ‘one-sided consensus rule’.Confidentiality of the survey data will be ensured by the use of unique numerical identifiers, anonymously allocated to participants, to ensure unrecognizability of individual responses. Data will be password-protected and accessible only to the TREAT Research Group, whose members will under no circumstances breach confidentiality.Consent to participate will be assumed if individuals agree to participate and complete the online questionnaires. Ethical guidance has been obtained from the Medical Ethics Review Committee of the Academic Medical Center in Amsterdam (reference number W15_249 # 15.0294). The committee confirmed that the Medical Research Involving Human Subjects Act (WMO) does not apply to this study and that no official approval is required.By the end of this Delphi study we hope to have reached consensus on the core set of domains and domain items to capture in AE patient registries with a research focus, that collect data of children and adults on photo- and systemic immunomodulatory therapies. This internationally agreed core set has the potential to unify data collection within existing and future AE patient registries to allow direct comparisons and data sharing and pooling. Analysis of these registry data will facilitate insight into the effectiveness, safety and cost-effectiveness of photo- and systemic immunomodulatory therapies across dermatology centres and country boundaries.Applying the Delphi method has several advantages. First, it allows us to include a large group of international experts and patients who are geographically dispersed to participate. In this way, broad consensus can be obtained. Second, the anonymity of the survey may avoid the dominance of certain persons in contrast to face-to-face meetings. However, this could diminish the positive effects of interaction to be found in face-to-face meetings, which helps to identify reasons for disagreement . TherefoDespite the obvious advantages of the eDelphi process, there are several sources of possible biases. Specifically, there is a risk of disproportionate representation among individual groups, including a dominance of clinicians and researchers, and relative underrepresentation of other groups such as patients and families . By contThe eDelphi study is currently ongoing. After the data of the eDelphi are analysed, a consensus meeting will be planned for defining the final core set of domains and domain items.


DK of 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment . However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS ( The large, multidomain glycoprotein Von Willebrand factor (VWF)1–VWF, expressed in endothelial cells, multimerizes in the Golgi apparatus and is stored in Weibel-Palade bodies as heterogeneous high molecular weight multimers (as large as 20–40-mers) before being released into the plasma –9. Once –11–The multimeric size and hemostatic potential of VWF is regulated through a multistep mechanism of proteolysis of its A2 domain by the metalloprotease ADAMTS13 , 16. Thi19–24–The importance of ADAMTS13 conformation in regulating VWF proteolysis has been recently investigated. The proteolytic activity of ADAMTS13 against VWF73 is enhanced in the truncated variant MDTCS , 32, sugTo further explore the molecular basis of conformational activation of ADAMTS13, we have prepared and studied a panel of ADAMTS13 domain deletion variants. We have employed binding and functional assays to determine the role of the ADAMTS13 C-terminal domains in its activation by VWF D4-CK. We propose that CUB1 binding to the spacer domain forms the autoinhibitory basis of its quiescent conformation. Moreover, several distinct interactions between VWF D4-CK and ADAMTS13 have been identified, with the TSP8 domain of ADAMTS13 proving essential for VWF D4-CK-induced activation.We first conducted a preliminary co-immunoprecipitation investigation of the binding between the isolated CUB1 and CUB2 domain fragments and the truncated ADAMTS13 variant, MDTCS. Both CUB1 and CUB2 were shown to interact with WT MDTCS in solution with an affinity that was sufficient to allow specific co-immunoprecipitation of the CUB fragments when MDTCS was pulled down. As expected, binding of neither CUB fragment to the GoF MDTCS variant was observed.A and B), DK values of 135 ± 10.1 nm and 86.9 ± 9.0 nm, respectively (G). As expected, a higher affinity interaction was observed between WT MDTCS and the CUB1-2 fragment (C), with a DK value of 55.6 ± 10.9 nm, similar to that reported previously (max of binding between WT MDTCS and CUB1-2 compared with the isolated CUB1 and CUB2 domain fragments (G). In support of co-immunoprecipitation (co-IP) experiments, no binding between CUB1 or CUB1-2 and GoF MDTCS (D and F) and minimal binding between high concentrations of CUB2 and GoF MDTCS were observed (E).These findings were corroborated by surface plasmon resonance analysis in which MDTCS variants (WT or GoF) were immobilized on a sensor chip . Both CUectively G. As expfragment C, with aeviously . There wragments G. In supoF MDTCS , D and Fobserved E.A and B). The CUB1-2 domain fragment significantly inhibited the activity of WT MDTCS, but not the GoF variant, in this assay (C). Importantly, the isolated CUB1 fragment significantly inhibited WT MDTCS (p < 0.05) to a similar extent as CUB1-2, but the isolated CUB2 fragment was without effect, even when added up to a concentration of 10 nm (data not shown). This inhibitory influence of CUB1 was reflected in the activities of C-terminal-truncated ADAMTS13 variants. Truncation of WT ADAMTS13 after CUB1 (WTΔCUB2) did not significantly alter proteolytic activity in FRETS-VWF73 assays. However, removal of both CUB domains (WTΔCUB1-2) increased activity at a level similar to that of GoF ADAMTS13 (D). Truncation of GoF ADAMTS13 (GoF ΔCUB2 and GoFΔCUB1-2) did not alter its activity.As previously reported , WT and is assay C. ImportADAMTS13 D. TruncaA and B), with DK values of 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively (D). However, the capacity of binding between CUB2 and VWF D4-CK was markedly lower than that of CUB1, with Bmax values of 60.1 ± 25.3 RU and 188 ± 24.5 RU, respectively (D). Furthermore, there was no increase in affinity between VWF D4-CK and the CUB1-2 domain fragment (C) compared with the isolated CUB1 fragment .SPR analysis has also revealed that both CUB1 and CUB2 (immobilized on a sensor chip) are able to bind the VWF D4-CK domain fragment , A and Bectively D. Howeveectively D. Furthefragment C compareA) with a DK of 114 ± 5.2 nm (D), similar to that published previously (B and C) with the DK values (132 ± 3.9 nm and 131 ± 3.8 nm) but not the Bmax values (111 ± 0.6 RU and 106 ± 0.5 RU) differing significantly (p < 0.05) from that of WT ADAMTS13 (D).WT ADAMTS13, immobilized on the sensor chip, bound to the VWF D4-CK domain fragment A with a ± 5.2 nm D, similaeviously . Surpriseviously , B and CADAMTS13 D.E). The DK(APP) of binding between WT ADAMTS13 and VWF D4-CK, derived from these data (146 ± 15.5 nm), was somewhat higher than that determined by SPR. However, the same relative decrease in affinity for VWF D4-CK exhibited by the truncated variants WTΔCUB2 and WTΔCUB1-2 (DK(APP) values of 167.0 ± 15.9 nm and 183.5 ± 21.8 nm, respectively) was observed.These results, suggesting that binding of ADAMTS13 to VWF D4-CK occurred in the absence of the CUB domains, were corroborated using an equilibrium plate binding assay E. The KDE), indicating TSP8 as a major VWF D4-CK binding partner. Indeed, the in-frame deletion of TSP8 (WTΔTSP8) also largely reduced binding (DK(APP) of ∼1 μm).Using the same equilibrium plate binding assay, minimal binding was observed between VWF D4-CK and either WT MDTCS or WTΔTSP8-CUB2 E, indicaD), removal of the CUB2 domain did not induce hyperactivity compared with WT ADAMTS13 (A and B). Moreover, WTΔCUB2 activity was significantly enhanced (p < 0.05) in a dose-dependent manner by the addition of VWF D4-CK, similar to the enhancement observed previously for WT ADAMTS13  for conformational activation using the FRETS-VWF73 assay. As in earlier experiments D, removaADAMTS13 , A and BADAMTS13 .D) and previously .The increased activity of truncated ADAMTS13, lacking both CUB1 and CUB2, is now established D and preeviously . This isWF D4-CK , E and FIt had long been assumed that ADAMTS13 is secreted into the plasma in a constitutively active state and that its proteolytic activity against VWF is regulated solely by its dependence on the shear-induced conformational change in the VWF A2 domain. Recent work has indicated that ADAMTS13 itself undergoes a conformational change required for it to achieve full activity , 32. UsiA and B). The affinities of CUB1 and CUB2 binding to MDTCS  were lower than that of the CUB1-2 domain fragment reported here (C and G) and previously . However, minimal binding between CUB2 and GoF MDTCS was observed (E).To further delineate the autoinhibitory interaction between the spacer domain and C-terminal domains of ADAMTS13, we have used a combination of SPR and co-IP to study binding of the isolated CUB1 and CUB2 domain fragments to MDTCS. Surprisingly, considering the minimal surface area of the five spacer domain residues (Arg-568/Phe-592/Arg-660/Tyr-661/Tyr-665), both CUB1 and CUB2 exhibited independent binding to WT MDTCS , A and Bted here , C and Geviously . The 2-f variant D. Howeveobserved E.A–C). Despite the evidence that both CUB domains bind to the spacer domain exosite, only CUB1 appeared to exert an inhibitory effect, with both the CUB1 and CUB1-2 domain fragments inhibiting WT MDTCS activity to a similar extent (C). Neither CUB1 nor CUB1-2 was able to inhibit GoF MDTCS, as would be expected from the binding studies, and any residual binding that may exist between CUB 2 and GoF MDTCS was not inhibitory. The autoinhibitory potential of CUB1 was corroborated by determining the activities of C-terminal-truncated ADAMTS13 variants (D), as increased activity was only achieved in the WTΔCUB1-2 variant. The predicted relative geometry of the CUB domains and MDTCS (A) would allow for binding between the spacer domain exosites and both CUB1 and CUB2. When originally described by Jian et al. (A). It is, therefore, likely that binding between CUB1 and the Tyr-661/Tyr-665 residues (which is abolished in their M4 and M5 variants) is responsible for maintaining the closed ADAMTS13 conformation.Having established that both CUB1 and CUB2 interact with the spacer domain exosites, we next examined the autoinhibitory potential of these isolated CUB domains using FRETS-VWF73 assays. As reported previously , both WTr extent C. Neithevariants D, as incnd MDTCS A would an et al. , it appen et al. A. It is,A–D). However, the affinity of binding between CUB2 and VWF D4-CK is lower than that of CUB1. This may be the result of CUB2 being coupled to the chip in a suboptimal orientation, a suggestion supported by the reduction in its Bmax. This moderate affinity interaction between CUB1-2 and D4-CK (DK of ∼50 nm) is similar to that previously established for the interaction between CUB1-2 and WT MDTCS  has shown that removal of the CUB domains resulted in a significant reduction in affinity compared with WT ADAMTS13 (p < 0.05) but it did not completely eliminate binding to VWF D4-CK. A similar magnitude  decrease in affinity for VWF D4-CK was also observed in equilibrium plate binding assays, suggesting binding can occur in the absence of CUB1 and CUB2 (E). In this assay, DK(APP) values could not be determined for either WT MDTCS (lacking the entire C terminus) or WTΔTSP8-CUB2 (truncated after TSP7) due to their weak binding. This strongly suggested TSP8 as a major binding site for VWF D4-CK. Indeed, the in-frame domain deletion of TSP8 (WTΔTSP8) produced a strong decrease in DK(APP) (p < 0.001). The importance of both TSP8 and the CUB domains in ADAMTS13 activation by VWF D4-CK was also evident in FRETS-VWF73 assays (A–H). The variant WTΔCUB2, with activity similar to WT ADAMTS13, was enhanced by the addition of VWF D4-CK in a dose-dependent manner similar to that seen previously for WT ADAMTS13 (F). However, no enhancement of activity was observed upon the addition of VWF D4-CK, indicating that the interaction between TSP8 and VWF D4-CK is a crucial component of the activation mechanism.However, SPR analysis of the C-terminal-truncated ADAMTS13 variants WTΔCUB2 and WTΔCUB1-2 , A–D hasand CUB2 E. In thi3 assays , A–H. ThADAMTS13 , confirmADAMTS13 F. HoweveB) both CUB1 and CUB2 interacted with the spacer domain exosite, with the autoinhibitory CUB1 domain potentially positioned to interact with the key residues Tyr-661 and Tyr-665 . The positioning of the CUB domains is facilitated by a folded conformation of the TSP2-TS8 domains around the three flexible linker regions . The affinity of this interaction may be sufficient to compete for binding of the CUB domains to the spacer domain, potentially reducing the stability of the closed conformation. It may also serve to position the D4-CK domains to facilitate the higher affinity interaction between VWF and TSP8, shown here to be critical for activation. The dimeric D4-CK domains of VWF have been shown by EM to adopt a rigid stem-like conformation maintained by specific pair-wise interactions between the densely packed domains . Ultimately, this conformational reorientation would fully expose the spacer domain exosite in readiness for VWF A2 domain unfolding.On the basis of the results presented herein we propose a model of the molecular mechanism of ADAMTS13 conformational activation involving multiple interactions between the D4-CK domains of VWF and the C-terminal domains of ADAMTS13 . In the Fig. 6 regions . A moder regions  , WTΔCUB2 (ADAMTS13 residues 1–1298), WTΔTSP8-CUB2 (ADAMTS13 residues 1–1075), GoFΔCUB1-2 and GoFΔCUB2, the in-frame deletion WTΔTSP8 , and the CUB1-2 (ADAMTS13 residues 1192–1427), CUB1 (ADAMTS13 residues 1192–1298), and CUB2 (ADAMTS13 residues 1299–1427) domain fragments were generated by inverse PCR using the full-length ADAMTS13 constructs. All ADAMTS13 variants and fragments were expressed in HEK293S cells, purified, and quantified using in-house ELISAs as described previously  and VWF D4-CK was also determined by equilibrium plate binding in PBS, 0.1% BSA at pH 7.4 as described (m ADAMTS13 (and its variants) in 50 mm sodium carbonate buffer (pH 9.6) were immobilized onto the wells of a 96-well microtiter plate (Nunc). Increasing concentrations of purified VWF D4-CK (0–500 nm) were applied for 2 h at 37 °C. Bound VWF D4-CK was detected using a HRP-conjugated α-VWF pAb (Dako). Binding curves were fitted to the one-binding site model using GraphPad Prism 7 software to determine DK(APP).Binding between ADAMTS13 (and its variants), the isolated CUB domains and the VWF D4-CK domain fragment was determined by surface plasmon resonance using a BIAcore T100 system as previously described . All SPRescribed , 24. BriDomain-specific antibodies were affinity-purified from a preparation of total IgG purified from rabbits immunized with full-length recombinant WT ADAMTS13. Briefly, recombinant MP-Dis and CUB1 fragments were immobilized on activated NHS Hitrap columns  and used to deplete the IgG pool of antibodies against these domains. Once depleted of α-CUB1 antibodies, the same IgG pool was passed over an immobilized recombinant CUB1-2 fragment to purify antibodies specific to CUB2. The antibody preparations were assessed for purity by SDS-PAGE and quantified by Nanodrop. The specificity of each preparation was confirmed by SDS-PAGE and Western blot of ADAMTS13 fragments using the prepared antibodies (1:1000 dilution) followed by HRP-conjugated goat α-rabbit secondary antibody (Sigma).m) in the absence/presence of 40 nm CUB1, CUB2, or CUB1-2 fragment were prepared in PBS, 0.1% BSA, and 0.02% Tween 20 at pH 7.4 and immunoprecipitated using Protein G Dynabeads (Invitrogen) coupled to antibodies against either the MP-Dis, CUB1, or CUB2 domains of ADAMTS13. Beads were washed, and bound protein was eluted in 1× LDS loading buffer at 70 °C for 10 min. Inputs (pre-IP), flow-through (post-IP), and bound protein in eluates were analyzed by SDS-PAGE and Western blot using a HRP-conjugated anti-His6 antibody (Abcam). To exclude any confounding effect of pH on the CUB-MDTCS interaction, a co-IP analysis of binding between MDTCS and CUB1-2 was performed at both pH 7.4 and pH 6; essentially identical results were obtained.WT or GoF MDTCS  in white 96-well plates (Nunc). Purified CUB1, CUB2, or CUB1-2 domain fragments (6 nm) or VWF D4-CK (2–6 nm) was added before a 45-min preincubation at 37 °C. The reaction was initiated by the addition of an equal volume of 4 μm FRETS-VWF73 substrate (Peptanova GMBH). Fluorescence  was measured at 37 °C at 1-min intervals for 1 h using a Fluostar Omega plate reader (BMG Labtech). Results were normalized to WT ADAMTS13 activity. To exclude any confounding effects of pH on the activation of ADAMTS13 by VWF D4-CK, the relative activities of WT ADAMTS13 (in the absence/presence of 6 nm VWF D4-CK) and GoF ADAMTS13 were determined at pH 6, 6.5, 7, and 7.5. Enhanced activity of WT ADAMTS13 induced by VWF D4-CK was observed at each pH.For FRETS-VWF73 assays ADAMTS13, MDTCS, ΔCUB2 or ΔCUB1-2 variants (WT and GoF), and WTΔTSP8 or WTΔTSP8-CUB2 were diluted to 0.5 nK. S. and M. O. F. performed the research. K. S. and D. A. L designed the research, analyzed the data, interpreted the data, generated the figures, and wrote the paper.


Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase (CITase), a member of glycoside hydrolase family 66 (GH66), catalyses the intramolecular transglucosylation of dextran to produce CIs with seven or more degrees of polymerization. To clarify the cyclization reaction and product specificity of the enzyme, we determined the crystal structure of PsCITase. The core structure of PsCITase consists of four structural domains: a catalytic (β/α)8-domain and three β-domains. A family 35 carbohydrate-binding module ) is inserted into and protrudes from the catalytic domain. The ligand complex structure of PsCITase prepared by soaking the crystal with cycloisomaltoheptaose yielded bound sugars at three sites: in the catalytic cleft, at the joint of the PsCBM35-1 domain and at the loop region of PsCBM35-1. In the catalytic site, soaked cycloisomaltoheptaose was observed as a linear isomaltoheptaose, presumably a hydrolysed product from cycloisomaltoheptaose by the enzyme and occupied subsites –7 to –1. Beyond subsite –7, three glucose moieties of another isomaltooiligosaccharide were observed, and these positions are considered to be distal subsites –13 to –11. The third binding site is the canonical sugar-binding site at the loop region of PsCBM35-1, where the soaked cycloisomaltoheptaose is bound. The structure indicated that the concave surface between the catalytic domain and PsCBM35-1 plays a guiding route for the long-chained substrate at the cyclization reaction. CIs a dextran . Among C8 TIM-barrel as a GH66 catalytic domain and several other β-domains [Paenibacillus agaridevorans T-3040 (CITase from P. agaridevorans T-3040 (PaCITase), which was previously known as BcCITase from Bacillus circulans T-3040) revealed that a β-jellyroll domain belonging to the carbohydrate-binding module (CBM) family 35 (CBM35) is inserted in the catalytic domain at the seventh loop of the (β/α)8 TIM-barrel, and this domain protrudes from the catalytic domain and possesses two sugar-binding sites [n: IG with n degrees of polymerization of glucose) in the catalytic site extends to the second sugar-binding site of CBM35, which acts as subsite –8, representing the enzyme–substrate complex when the enzyme produces CI-8 (Protein Data Bank (PDB) ID: 3WNN). This structure indicates that CBM35 functions to determine the size of the product, causing the predominant production of CI-8 by the enzyme.CI-producing bacteria produce CI glucanotransferase , which catalyses the production of CIs from dextran by intramolecular transglucosylation, and also catalyses disproportionation, coupling and hydrolytic reactions . CITases-domains –11. Threng sites . The firPaenibacillus sp. 598K strain showed good CI production and high CITase activity that was as good as the P. agaridevorans T-3040 strain [Paenibacillus sp. 598K uses CITase (CITase from Paenibacillus sp. 598K (PsCITase)) to synthesize CIs from dextran. When the bacterium produces CIs from starch, it uses another enzyme α-1,6-glucosyltransferase (EC 2.4.1.-) to produce α-1,6-glucosyl-α-glucosaccharides from starch, which are used as substrates for PsCITase [cit gene [Paenibacillus sp. 598K CITase (PsCBM35-1) and PsCBM35-2, and the domain arrangement is similar to that of PaCITase. Although these two enzymes show a high sequence identity of 65%, the CI produced in the highest amounts by PaCITase is CI-8, whereas PsCITase produces CI-7 in the highest amount. Since the high CI-8 productivity of PaCITase is attributed to the sugar-binding site in the CBM35-1 domain working as subsite –8, we hypothesize that the structure of PsCBM35-1 might provide clues that clarify the mechanism of product specificity for PsCITase.Among CI-producing bacteria, the 0 strain . PaenibaPsCITase . PsCITasIn the present study, the crystal structure of PsCITase we determined in its apo- and CI-7 complex forms, and structural differences between the CBM35 domains of PaCITase and PsCITase explain the substrate recognition mechanism of these enzymes that yields different product specificities. The elucidated sugar-binding mechanism of CITases would help in creating the functional enzymes to produce CIs with different polymerization degrees for certain purposes in industrial and material applications.cit gene  and constructed from the pET15b system  was transformed into Escherichia coli BL21(DE3) cells [E. coli cells were disrupted by sonication, PsCITase was purified to homogeneity by nickel-chelating chromatography using a His-Trap Fast Flow 5-ml column . The purified sample was buffer exchanged and concentrated to 3.0 mg ml−1 by ultrafiltration using an Amicon Ultra-30000 MW membrane , and the concentration was checked during this process using the molar extinction coefficient (ε280 =1.62 × 105 M−1 cm−1).The expression plasmids coding for the core domain of the The protein solution was filtered through a 0.1-µm membrane (Merck-Millipore). PsCITase was crystallized by the sitting-drop vapour diffusion method at 293 K using a precipitant solution consisting of 25% PEG3350  and 12% Tacsimate (Hampton Research), pH 5.0. Rod-type crystals with maximum dimensions of 0.05 × 0.05 × 0.3 mm appeared within a month using 50 μl of the reservoir solution with a drop consisting of 1 μl of protein solution and 1 μl of reservoir solution.Diffraction experiments for protein crystals were conducted at the beamline BL-NW12 of the Photon Factory Advanced Ring (PF-AR), High Energy Accelerator Research Organization, Tsukuba, Japan . The cryhttp://cuemol.sourceforge.jp/en/).The crystal structure was determined by the molecular replacement method using the structure of PaCITase  as the 6-tagged full-length cit gene without signal sequence cloned into pET-15b as described before [Site-directed mutagenesis was performed by polymerase chain reaction using KOD-plus DNA polymerases . The template was CITase-598K expression plasmid, which contains the N-terminal Hisd before . The priWild-type and mutant PsCITase were expressed, purified and buffer exchanged as described above. CITase assays were performed by measuring the amounts of CIs produced from dextran as described previously . Reactio21–Met40), were not identified because of the lack of electron density. Apart from the sugar and water molecules, one calcium ion and two sodium ions were observed. Some malonate ions were also observed, which were included in the crystallization conditions.The crystal structures of core PsCITase and its complex with CI-7 (PsCITase–CI-7 complex) were determined by the molecular replacement method at 2.20 and 2.60 Å resolutions respectively. The crystallographic parameters are shown in 41–Ser140) composed of seven β-strands forms an Ig fold. The catalytic domain  comprising the (β/α)8-barrel is well conserved in GH66 enzymes. In the catalytic domain, the PsCBM35-1 domain is inserted as domain B (Gly421–Gly547) at the seventh loop of the (β/α)8-barrel. Domain C, positioned at the C-terminal side of the catalytic domain (Pro615–Met738), comprises a β-sandwich structure and is composed of Greek key motifs. After the C-terminal of domain C, one CBM35 domain (CBM35-2) and an extra function – unknown peptide exist in the deduced sequence of the cit gene; however, they have been removed for the stable protein expression [The PsCITase–CI-7 complex structure is shown in Paenibacillus agaridevorans T-3040 CITase (PaCBM35-1), formerly BcCBM35-1) [The overall structure is conserved with that of GH66 enzymes  and 3. TCBM35-1) . Domain In the complex structure with CI-7, CI-7 and IGs were observed at three sites  and 4, i309 and Glu381, and the sugar chain elongated into the gap between the catalytic domain and the CBM35-1 domain with a zigzag chain structure owing to the α-1,6 glucosidic linkages. According to the subsite nomenclature for GHs [515 and hydrogen-bonded by the main chain of the sixth β-strand of CBM35-1 domain. Glc–7 formed a hydrogen bond with the main chain of Phe208 of the catalytic domain. Glc–2 to Glc–7 had the normal 4C1 chair conformation, but the electron density for the Glc–1 was rather ambiguous and Glc–1 was refined to have an unusual E3 conformation [At the catalytic site, the bound CI-7 was observed as the cleaved linear form like isomaltoheptaose (IG-7) in the catalytic cleft A. Electr for GHs , the pos for GHs . Subsiteormation .159, Glu573 Asp574 and Arg586 side chains of the catalytic domain and the side chain of Tyr470 of the CBM35-1 domain. The non-reducing end glucose was stacked on the side chain of Tyr519. This sugar chain may form part of the hydrolysed product of CI-7 by the active enzymes.A short IG was found between the catalytic and CBM35-1 domains, besides the aforementioned IG-7 B. Three 507 from both molecules stacked two glucose moieties of the bound CI-7. However, the CI-7 molecule did not have two-fold symmetry in its structure; the bound structure took multimodal conformations. The electron density was averaged by the symmetry between the multimodal conformations and this resulted in indistinct density. The Trp507 side chain functioned as a platform for stacking interactions and the surrounding residues formed hydrogen-bonding interactions. These interactions provided the binding site specific to the mid-point glucose of the α-1,6-glucosyl linkage.The third bound ligand was observed in the canonical sugar-binding site of the CBM35-1 domain C, as obsTo investigate the role of the sugar-binding site of PsCITase, four mutants that changed the aromatic side chains at the sugar-binding sites of PsCBM35-1 were prepared. The mutants were W515A (subsite –6), Y470A, Y519A  and W507A . As shown in The obtained structure of the protein–sugar complex was prepared by soaking CI-7 into the crystal drop. The expected structure of the complex should have contained the docked cyclic IGs in the catalytic site. However, the hydrolysed linear IGs were present in the structure, enabling us to identify the subsites from the catalytic site to the CBM35-1. The identified subsites indicated that the concave region formed by the catalytic domain and CBM35-1 supply the substrate-binding site for dextran, the long chain substrate.515 in PsCITase and Trp514 in PaCITase, in the CBM35-1 domain differed (515 side chain provides the platform for parallel stacking on the glucose moiety as subsite −6 and recognizes the mid-point glucose moiety of the α-1,6-glucan. The C1-and C6-hydroxyl groups of the bound glucose moiety were positioned at both sides of the platform to form α-1,6-glucosyl bonds with the adjacent glucose moieties (514 side chain participates by forming the side wall of the binding pocket, which functions as subsite –8, along with the PaCITase Tyr499 and Phe501 residues, and the C6-hydroxyl group of the bound glucose moiety is buried at the bottom of the pocket, concomitantly expressing the terminal-binding mode (500, Gln502, Ile497 and His516, of PsCITase contribute to the glucose-binding mode.The PsCITase–CI-7 complex structure and the PaCITase–IG-8 complex structure  both shodiffered . In PsCImoieties A. On theing mode B. The su502. In PaCITase, the bulky Phe501 side chain in PaCITase forms the sugar-binding pocket and creates steric hindrance. Thus, the IG-8 sugar chain binds in a manner that circumvents Phe501. On the other hand, the Gln502 side chain of PsCITase adopts a conformation that enables a shortcut for the substrate to bind between the catalytic site and the binding site at Trp515. This different conformation of Gln502 means that the conserved Trp515 of PsCITase forms the proximal subsite −6, whereas PaCITase Trp514 is involved in subsite −8. Thus, the different product specificity of these two CITases could be attributed to the structural difference at the second sugar-binding site of CBM35-1. Since PsCITase does not have the terminal-binding subsite –8, PsCITase does not produce CI-8 predominantly, but produces CIs with degrees of polymerization of seven or higher, with the polymerization degree seven being the main product by PsCITase and higher polymerization degrees being produced at lower amounts as the polymerization length increases. The alanine mutation on Trp515 did not affect the product specificity of the enzyme but reduced the overall production of CIs, and Trp515 appeared to function as a component of substrate-binding cleft for the long substrate.Comparison of the IG structures docked in these two enzymes also showed that the subsite location from –1 to –4 are conserved in these enzymes, but the subsite positions from subsite –5 to −8 differ . The difIn the direction beyond the subsite −7, three glucose moieties, which appeared to be a part of IGs produced by hydrolysis of soaked CI-7, were observed. The α-anomeric hydroxyl group of the reducing-end glucose that is located at the inner position of the concave is directed towards Glc–7 of the docked IG-7. No electron density was observed between the two IG sugar chains, but the concave seemed wide enough for IGs to cover both docked sugar chains. The distance between the Glc–7-O6 atom and the reducing end O1 atom of the docked sugar chains was ∼12 Å, and at least three glucose molecules might be necessary to link the two docked sugar chains, indicating that the positions of the newly found three glucose moieties corresponded subsites –11 to –13. Although these sites did not affect CI productivity and size specificity of the enzyme, the concave region between the catalytic and PsCBM35-1 domains appears to be a good guide route for a substrate with a long sugar chain, such as dextran, to access the catalytic site.In our previous study on PaCITase, we succeeded in producing engineered PaCITase mutants that produce large amount of CIs with higher degrees of polymerization by introducing mutations around the IG-binding subsites . The sucCBMs are usually small domains and more than 30 CBM families adopt a β-sandwich fold. In the CBMs comprising the β-sandwich structure, sugar-binding sites are located mainly at two sites: the major site is termed the variable loop site and is located on the tip of the β-turn, whereas the other binding site is located on the surface of the concave β-sheet and is termed the concaved face site . CBM35s We have determined the crystal structure of PsCITase, elucidating IG-binding mode at the catalytic site and PsCBM35-1. Two sugar-binding sites in PsCBM35-1 did not affect size specificity of the produced CIs, but enhanced the total CI production of the enzyme. The concave between the catalytic domain and PsCBM35-1 appeared to be a guiding route for the long-chained substrate. The information obtained here provides the structural basis for enzymatic improvement of PsCITase.


Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor. Published 12 October 2017In one of our figures and its supplement , we compared new unpublished data with data that had previously been published. Although reference is made to this fact in the text and in the legends, differences between the current manuscript and Li, et al. 2012, regarding the nomenclature used to describe identical samples, requires additional clarification. As such, we have provided additional text for the corresponding legends.The following text has been added to the legend for Figure 2: ‘We note that data for the R-F and F domains in panels (a) and (b) are the same as presented in Li et al., 2012 to allow for direct comparison of the opposing effects of the DBD and the R-domain on the F-domain.’Similarly, the following text has been added to the legend of Figure 2—Figure supplement 1: ‘We note that the data in (b) are the same as presented in Li et al. 2012 , according to the current nomenclature, wherein the R-F construct is equivalent to the A isoform of NTD and the F construct is equivalent to the C3 isoform of NTD.’The original Figure 2 legend is shown here for reference:Coupling of the R-domain and DBD to the functional F-domain in GR.(a) TMAO-induced folding for the F-domain alone and with either the R-domain or the DBD. (b) Protease sensitivity assay: comparing F-domain and F-domain with R-domain (left) performed at a protein (1 mg/ml): trypsin mass ratio of 1000:1 ; comparing F-domain and F-domain with DBD (right) performed at a protein (1 mg/ml): trypsin mass ratio of 100:1. Note: each protein pair (i.e. F vs. R-F and F vs. F-DBD) was run on the same gel, with the intervening lanes removed in the figure for clarity. (c) Luciferase assay for C3 isoform (GR F-DBD) versus chimeric construct .The corrected Figure 2 legend follows:Coupling of the R-domain and DBD to the functional F-domain in GR.(a) TMAO-induced folding for the F-domain alone and with either the R-domain or the DBD. (b) Protease sensitivity assay: comparing F-domain and F-domain with R-domain (left) performed at a protein (1 mg/ml): trypsin mass ratio of 1000:1 ; comparing F-domain and F-domain with DBD (right) performed at a protein (1 mg/ml): trypsin mass ratio of 100:1. Note: each protein pair (i.e. F vs. R-F and F vs. F-DBD) was run on the same gel, with the intervening lanes removed in the figure for clarity. (c) Luciferase assay for C3 isoform (GR F-DBD) versus chimeric construct . We note that data for the R-F and F domains in panels and (b) are the same as presented in Li et al., 2012 to allow for direct comparison of the opposing effects of the DBD and the R-domain on the F-domain.’The original Figure 2—Figure supplement 1 legend is shown here for reference:Domain stabilities determined by TMAO-induced protein folding transitions.b) Comparison of the m-values fitted from TMAO induced folding of F-domain and R-F with the published m-values for known representative globular proteins  for details). The m-value comparison suggests that the F-domain and R-domain can adopt folded conformations similar to globular proteins in terms of surface area buried upon folding.(a) Thermodynamic parameters obtained from TMAO-induced folding experiments demonstrate that DBD stabilizes a folded conformation of the F-domain while the R-domain destabilizes that conformation. Parameter values obtained from the fits of the TMAO-induced folding data for the constructs shown in Figure 2a. See previous publication  for details. Because the thermodynamic analyses report on the free energy differences and not the mechanistic bases of the energy differences, the reported free energies of folding (ΔG0F−UΔGF−U0) may not necessarily reflect the stability of unique conformations. Instead, they may be reporting on the stability of a conformationally heterogeneous ensemble. Toward this end, it has been reported that ID proteins may adopt poly-beta sheet formations  as part of their functional states. As the effect of TMAO on structure is almost entirely defined by the influence on backbone atoms (i.e. excluding backbone hydrogen bond donors and acceptors from solvent) , any state or ensemble of states that facilitates removal of backbone atoms from solvent will be stabilized by TMAO. As such, the current analysis provides evidence that a functional state exists whose probability is proportional to transcriptional activation. However, the structural properties of that state are not known.  Thermodynamic parameters obtained from TMAO-induced folding experiments demonstrate that DBD stabilizes a folded conformation of the F-domain while the R-domain destabilizes that conformation.m-values fitted from TMAO induced folding of F-domain and R-F with the published m-values for known representative globular proteins  for details). The m-value comparison suggests that the F-domain and R-domain can adopt folded conformations similar to globular proteins in terms of surface area buried upon folding. We note that the data in (b) are the same as presented in Li et al. 2012 , according to the current nomenclature, wherein the R-F construct is equivalent to the A isoform of NTD and the F construct is equivalent to the C3 isoform of NTD.Parameter values obtained from the fits of the TMAO-induced folding data for the constructs shown in Figure 2a. See previous publication  for details. Because the thermodynamic analyses report on the free energy differences and not the mechanistic bases of the energy differences, the reported free energies of folding (ΔG0F−UΔGF−U0) may not necessarily reflect the stability of unique conformations. Instead, they may be reporting on the stability of a conformationally heterogeneous ensemble. Toward this end, it has been reported that ID proteins may adopt poly-beta sheet formations  as part of their functional states. As the effect of TMAO on structure is almost entirely defined by the influence on backbone atoms (i.e. excluding backbone hydrogen bond donors and acceptors from solvent) , any state or ensemble of states that facilitates removal of backbone atoms from solvent will be stabilized by TMAO. As such, the current analysis provides evidence that a functional state exists whose probability is proportional to transcriptional activation. However, the structural properties of that state are not known. (b) Comparison of the The article has been corrected accordingly.


Dear Editor,DEAD-box proteins, which are named after the strictly conserved amino acid sequence Asp-Glu-Ala-Asp, were first identified as a distinct family in the late 1980s when alignments based on eight homologues of the yeast eIF4A highlighted the presence of several conserved motifs  of pathogens by pattern recognition receptors (PRRs) is important for the induction of type I interferons (IFN)  is shown targeting hDDX41 protein to the nucleus. Our study provides basic structural information for further researches on hDDX41 biological function and valuable insights for the treatment of DDX41-related diseases in the future.Here, we report the crystal structure of human DDX41 (hDDX41) DEAD domain complexed with an SOfree = 0.23). Details of data collection and refinement statistics are listed in Table S1. The crystal used for data collection belonged to space group P21. One asymmetric unit consists of two molecules of the protein based on the calculated solvent content of 44.15%. The hDDX41 DEAD domain consists of an α/β fold, which is similar to those observed for other members of the DEAD-box proteins for which structures are available. The overall structure consists of ten α-helices (α1–α10) and a β-sheet formed by eight β-strands (β1–β8). Helices α1–α5 are located on one side of the β-sheet, while helices α6–α10 are located on the other side  predicted a monomeric biological assembly. In agreement with this prediction, results of the static light scattering analysis indicated that the DEAD domain exists as a monomer in solution  as the search model and refined to 2.26 Å resolution with an R factor of 0.19 , the SO42− overlaps with the position of α-phosphate of ADP , Prp28 , DDX3X with AMP , DDX5 with ADP , VASA with ANP . It seems that the P-loop is not restricted to one open or closed conformation but has a flexibility that allows it to adapt to different conformations depending on the binding ligands. The most closed conformation is found in Prp28, which leaves no room for any ligand. The most open conformation is found in VASA, which has enough space for ANP. The P-loop of DDX3X with AMP and DDX5 with ADP adopt the same half-open conformation compared with Prp28 and VASA. Although there is no nucleotide in the solved hDDX41 structure, the SO42− bound P-loop adopts a half-open conformation. The P-loop has a shift in Cα-atom positions by up to 2.7 Å between the open and half-open states, and by up to 1.7 Å between the half-open and closed states  and Isothermal Titration Calorimetry (ITC)  Fig. A. The thhttp://consurf.tau.ac.il/) and found out that the mutated amino acids associated with MDS/AML are conserved . Of the nine mutations identified, seven mutations  are locate in the DEAD domain, suggesting hDDX41 function is more sensitive to mutations in DEAD domain than Helicase domain. MDS/AML is now the only reported disease related to hDDX41 protein. However, the relationship between the mutations and disease is still unknown. hDDX41 could serve as a drug target and our study provides a structural basis for disease treatment.hDDX41 is frequently mutated in familial MDS/AML  reveals that the N-terminal region (aa 1–160) is disordered. In addition, the role of this region is unclear. Interestingly, the N-terminal 1–194 amino acids of a homologue of DDX41, Abstrkt, from in vitro. Different lengths of dsDNA and c-di-GMP were tested by TSA, ITC and Surface Plasmon Resonance (SPR) assay, but no binding was detected. As intracellular condition is much more complicated, DDX41’s functions in innate immunity may need other components or depends on both domains. Besides c-di-GMP, STING is also a direct sensor of c-di-GMP Below is the link to the electronic supplementary material.


Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed. Arenaviruses occur worldwide and can cause severe, often fatal hemorrhagic fever in humans. Vaccines and effective treatments are not available. Arenaviruses replicate in the cytoplasm of infected cells and since they cannot synthesize cap-structures they use a mechanism called cap-snatching to steal cap structures from host mRNAs for viral transcription. This mechanism is an attractive drug target, as it is essential for virus replication and virus specific. However, the arenaviral components of this mechanism are poorly defined compared to influenza virus, the prototypic cap-snatching virus. We present the first crystal structures of two putative components of the California Academy of Sciences arenavirus cap-snatching machinery, namely the isolated N- and C-termini of the viral RNA polymerase (L protein). The N-terminus harbors what looks like a functional cap-snatching endonuclease. The L protein C-terminus, despite complete sequence divergence, shows overall structural similarity to the C-terminal region of influenza virus polymerase PB2 subunit, suggesting a common evolutionary origin. A domain clearly related to the PB2 cap-binding domain is present, although cap-binding could not be biochemically demonstrated. The determined structures provide the basis for future research to unravel the details of the arenavirus cap-snatching mechanism and its potential as a target for drug development. The family of arenaviruses is divided in two genera: mammarenaviruses and reptarenaviruses. With the notable exception of Tacaribe virus, rodents are described as the natural reservoirs for mammarenaviruses. Reptarenaviruses have only been found in captive snakes . Some arArenaviruses are enveloped particles that contain two single stranded negative sense RNA segments. The two genome segments code for four viral proteins, the nucleoprotein (NP), the glycoprotein-precursor, the small matrix protein Z and the large > 200 kDa L protein which harbors the viral RNA-dependent RNA polymerase. The minimal viral components for genome replication and transcription are the viral RNA, NP, and the L protein . The L pTo further characterize the cap-snatching machinery of arenaviruses, we attempted to solve the structure of N- and C-terminal domains of L proteins of various arenaviruses. Eventually, we have been successful with the L protein of the California Academy of Sciences virus (CASV), which is a reptarenavirus. Here we present the crystal structures of the two terminal domains of the CASV L protein: the cap-snatching endonuclease in the N-terminus and the 326 C-terminal residues, which, by analogy to LASV, might play a role in transcription . The actEscherichia coli  crystallized in space group P2n of 2 Å . Except n of 2 Å , S1A Fig2 between the monomers. The most intensive intermolecular contacts are between the very C-terminal 40 residues of each chain (buried surface area 1100 Å2).In the crystallized dimer the two U-shaped monomers interlock with each other to form a ring with a hole in the middle with a buried surface area of approximately 3000 ÅTo identify known structural homologs of our structure we used the DALI program for protein structure comparison  and perfInterestingly, for L-Cterm domain 2 the list contained the cap-binding domain of influenza virus PB2, which was also found when using the full monomer of CASV L-Cterm as search model. Other hits for domain 2 were acetyltransferases, sulfatases, methyltransferases, β-lactamases, and TATA-box binding proteins, again with relatively low Z-scores (< 5.0).Despite a complete lack of sequence homology CASV L-Cterm and influenza PB2 show a remarkable similarity in overall domain architecture and sub-domain topology Figs  and 3. SMost importantly, the highest degree of similarity was seen between the L-Cterm domain 2 and the PB2 cap-binding domain . Both arBesides the structural organization of the isolated domains, their arrangement in the primary structure is conserved between influenza virus and CASV Figs  and 3: i7GTP. First, the cap-analogue was soaked into the CASV L-Cterm crystals. However, electron density did not appear in the cavity formed by Y1872, F1806, and W1818, i.e. in the position expected by comparison to PB2 , a eukaryotic cap-binding protein, bound to m7GTP-agarose, we could not detect binding of CASV L-Cterm . These mostly negatively charged residues interact with a positively charged patch on the second molecule  nor a dimeric form with a different dimer interface (domain 2) binds mThe cap-snatching mechanism has been proposed and characterized so far only for mammarenaviruses based on (i) sequencing results showing 4–5 non-templated nucleotides at the 5' end of viral mRNAs and (ii) structural and functional data demonstrating the existence of an endonuclease in the N-terminus of the L protein . Therefo12121 with four molecules per asymmetric unit. The structures of the four molecules are very similar with the only difference in the C-terminal 15 residues, which are not visible in all molecules (RMSD between 0.227 and 0.317 Å). The CASV endonuclease has basically the same fold as endonucleases from LASV, Pichinde virus (PICV), and lymphocytic choriomeningitis virus (LCMV)   even thos (LCMV) . Slight s (LCMV) . The eles (LCMV) . We alsos (LCMV) .Cap-snatching was first discovered in influenza virus . The strWe solved the structure of the 326 C-terminal residues of a reptarenavirus L protein. Despite the lack of any significant sequence homology, the domains of this 37-kDa fragment are structurally similar to the cap-binding and adjacent domains of influenza virus PB2 . Both pr2+ are strong arguments for the presence of a functional endonuclease in the L protein of reptarenaviruses, even though, identical to the isolated endonuclease domain of LASV, nuclease activity was undetectable biochemically  as a percentage of the wild-type in ≥3 independent transfection experiments). Synthesis of the antigenome and Ren-Luc mRNA was evaluated by Northern blotting using a radiolabeled riboprobe hybridizing to the Ren-Luc gene. A defective L protein with a mutation in the catalytic site of the RNA-dependent RNA polymerase served as a negative control (neg). Signals on Northern blots were quantified via intensity profiles. The data are also presented numerically in (TIF)Click here for additional data file.S5 FigA) front sight and B) back sight. Residues that were found to be important for viral transcription in LASV minireplicon system in this and a previous study . 32P. 32P17].(TIF)Click here for additional data file.S10 FigAcidic and basic amino acid patches from the C-terminus, which interlock with each other in the protein dimer, are marked with dashed circles. The electrostatic surface potential is shown from -5 KT/e in red to +5 KT/e in blue and was calculated using PDB2PQR and the APBS-tool of PyMOL.(TIF)Click here for additional data file.S11 FigA) Identity matrix and B) similarity matrix of N-terminal sequences. Matrices were calculated based on the presented alignment of N-termini (http://imed.med.ucm.es/Tools/sias.html) and values are given in percent relative to the mean length of sequences compared. Abbreviations: Full virus names are given in legend to -termini  using th(TIF)Click here for additional data file.S12 FigA) Identity matrix and B) similarity matrix of C-terminal sequences. Matrices were calculated based on the presented alignment of C-termini (http://imed.med.ucm.es/Tools/sias.html) and values are given in percent relative to the mean length of sequences compared. Abbreviations: Full virus names are given in legend to -termini  using th(TIF)Click here for additional data file.S13 Fighttp://espript.ibcp.fr) [The alignment was generated using ClustalOmega  and inclibcp.fr) .(TIF)Click here for additional data file.S14 Fighttp://espript.ibcp.fr) [The alignment was generated using ClustalOmega  and inclibcp.fr)  with man(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 TableThis list contains tested fragments that were either insoluble, not suitable for crystallization trials or could not be crystallized successfully.(DOC)Click here for additional data file.S1 MethodsThe methods of LASV minireplicon system and the endonuclease assay are described in detail.(DOC)Click here for additional data file.


TM reference number JN104480) and characterized its domains. We suggest that the enzyme consists of a shorter than previously described catalytic domain, a newly identified substrate binding domain, and a C-terminal type 9 secretion system signal peptide. By separately expressing the two domains in E. coli, we confirmed that the binding domain is ulvan specific, having higher affinity for ulvan than most lectins for their ligands (affinity constant: 105 M−1). To our knowledge, this is the first description of an ulvan-binding domain. Overall, identifying this new binding domain is one step towards engineering ulvan enzymes that can be used to characterize ulvan, e.g. through enzymatic/mass spectrometric fingerprinting analyses, and help unlock its full potential.Biomass waste products from green algae have recently been given new life, as these polysaccharides have potential applications in industry, agriculture, and medicine. One such polysaccharide group called ulvans displays many different, potentially useful properties that arise from their structural versatility. Hence, performing structural analyses on ulvan is crucial for future applications. However, chemical reaction–based analysis methods cannot fully characterize ulvan and tend to alter its structure. Thus, better methods require well-characterized ulvan-degrading enzymes. Therefore, we analysed a previously sequenced ulvan lyase (Genebank The green algal biopolymer ulvan is an underexploited biomass in spite of its promising applications in the food industry, e.g. as a dietary fibre or as an antioxidant10Ulva9Ulvans are sulphated hetero-polysaccharides derived from green macro algae of the genus Because their properties strongly depend on molecular weight17AlteromonadalesE. coli, we gained insight into the domain structure of the enzyme and discovered, among other things, the first ulvan binding domain. To prove the functionality of this domain, we expressed it separately from the catalytic lyase domain and investigated its binding specificity and affinities in different assays.A major breakthrough in ulvan structure analysis occurred with the discovery of the first endo-ulvan lyase2021Nonlabens ulvanivorans (previously Percicivirga ulvanivorans)21E. coli, we carefully re-examined its domain structure. Previously, the enzyme was described as having two domains: a catalytic domain with an N-terminal signal peptide and a second domain of unknown function, separated by a low complexity regionTo overcome previously describedTo validate this structure and to confirm the function of the binding domain, we established two deletion constructs containing either only the binding domain or only the catalytic domain, without the N- and C-terminal signal sequences . We wereWe tested all three constructs for catalytic activity towards polymeric ulvan by measuring the appearance of double bonds at 235 nm, which arises due to the enzyme’s lytic rather than hydrolytic mode of action . We obseWe then tested the binding domain for its ulvan-binding ability and ligand specificity in a dot blot assay . While t5 M−1, and a stoichiometry of seven mono-sulphated ulvan units per binding protein , enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy change (ΔG) - of the molecular interaction3233The inflection point of the enthalpogram shows an N value of 7 so one protein binds to seven monosulphated units of ulvan. According to the classic interpretation, this would mean that one protein has seven equal binding sites. As this is unlikely, we suggest an approach according to Zhao and co-workers, where the inflection point in the ITC profiles does not represent the N value in a known 1:1 binding processUpon re-examination of the domain structure of the first ulvan lyase describedUlva fasciata as previously described® Suprema columns  coupled to a refractive index detector (Agilent series 1200 RID). The molecular weight was determined by calibration using a series of pullulans . The validity of calibration for negatively-charged polysaccharides was confirmed using dextran sulphates .Ulvan was extracted from For investigation of the domain structure, InterProEscherichia coli DH5α, and for recombinant protein expression, E. coli Rosetta 2(DE3)(pLysSRARE2) was used . Plasmids were sequenced at MWG-Biotech AG . The pET-22b(+) vector (Merck) was used for all constructs. Both E. coli stains were grown in LB at 37 °C. For the selection of transformants 100 μg/ml ampicillin (E. coli DH5α) or 100 μg/ml ampicillin plus 34 μg/ml chloramphenicol (E. coli Rosetta 2(DE3)(pLysSRARE2)) was added. For optimal protein expression autoinduction was performed according to ref. 2HPO4, 1.25 M KH2PO4, 2.5 M NH4Cl, 0.25 M Na2SO4), 5052  and 2 mM MgSO4 induced the cells for expression of the target protein. To obtain sufficient amounts of soluble protein, it was obligatory to grow the cultures for 4 h at 37 °C (180 rpm), 18 h at 20 °C (180 rpm) and 4 h at 20 °C (120 rpm). After addition of IPTG to increase protein yield  cells were grown for 3.5 h before harvest. Stocks were prepared for long-time storage with 30% (v/v) glycerol and kept at −70 °C.Recombinant plasmids were kept in N. ulvanivorans ulvan lyase coding gene sequence (JN104480.1 (GenBank)) were used to generate an artificial gene sequence that was codon optimized for expression in E. coli . Due to its 5 prime sequence truncation, the encoded protein does not contain the N-terminal signal peptide identified by Nyvalle-Collén and co-workers(exp.) vector. The BD- and CD-construct coding plasmids pET22b(+)::BD-StrepIIC and pET22b(+)::CD-StrepIIC(exp.) were derived from these vectors by PCR. The necessary deletion was performed by using 5′ phosphorylated “back to back” primer pairs (Base pairs 142 to 1602 of the er pairs .E. coli Rosetta 2(DE3)(pLysSRARE2). Harvested cells were resuspended in 40 mM triethanolamine (pH 8) buffer containing 400 mM NaCl and frozen at −20 °C for at least 24 h. Afterwards, cells suspensions were incubated for 30 min at 37 °C. Simultaneously, 30 mM triethanolamine (pH 8), 30 mM NaCl, 6 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 10 U/ml benzonase were added together with approximately 4000 U/mL lysozyme. The cells were disrupted by brief sonication and the crude extracts were obtained by centrifugation. The Strep-tag II containing target proteins were purified from the crude extract by affinity chromatography using Strep-Tactin columns  according to supplier’s instruction and concentrated in ultrafiltration devices  with a molecular mass cut-off at 10 kDa. For storage at 4 °C, 10% (v/v) glycerol was added. The protein concentration was determined after BradfordBoth domains were synthesized in ® M2 Multi-Mode Microplate Reader  set at 30 °C. The assay was performed with 198 μg ulvan . Two μl of enzyme (10 pmol) was added and the measurement started immediately. Absorbance was recorded for 5 min every 30 seconds.The formation of double bonds was measured at 235 nm to follow the lyase activity as described beforeStrep-Tactin–horseradish peroxidase (HRP) conjugate  and the signal was detected by chemiluminescence.The binding assay was performed as described beforeInteraction of polymer and protein leads to complex formation that visibly influences behaviour in native PAGE . Consecutive injections of 5 μL aliquots of ulvan solution were added with the help of a rotator stirrer-syringe into the calorimeter cell of 1.445 ml filled with ulvan-binding protein in a 0.1 M TRIS buffer at pH 8.5 containing 200 mM NaCl at 25 °C. To minimize the contribution of heat of dilution to the measured heat change, the protein and ulvan solutions were prepared in the same buffer. Injections were made at intervals of 4 min for all titrations. Control experiments were performed by injecting ulvan solution into the buffer solution in an identical manner, and the resulting heat changes were subtracted from the measured heats of binding. Since the first injection is often inaccurate, a 1 or 2 μl injection was added first and the resultant point was deleted before the remaining data were analysed. Integration of the injection peaks and construction of binding isotherms were performed by using the high-precision automated peak shape analysis software NITPICHow to cite this article: Melcher, R. et al. Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain. Sci. Rep.7, 44115; doi: 10.1038/srep44115 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The c-domain sample was found to have a higher coercive field Ec and higher dielectric losses than the other which presents approximately 60% of c-domains and 40% of a-domains as observed by piezo force microscopy (PFM) characterization. Hyperbolic law measurements reveal that the higher coercive field is due to domain wall pinning in deeper defects and hence a higher field Eth is required for unpinning. The dissipation factors due to domain wall motion, however, are similar in both samples since the domain wall density is low and there is almost no interaction between domain walls. The higher dielectric losses in the c-domain oriented sample are a result of a greater contribution from the lattice and seem to be due to strain from the substrate, which is not relieved in a thin sample. PFM and dielectric characterization are complementary methods which provide a better understanding of the domain wall motion.Two Pb(Zr In order to study those domain wall movements, the Rayleigh law has been employed at low field (E < EC/2) to maintain the domain wall density (Rayleigh region)8. This law is composed of two coefficients where one corresponds to the domain wall pinning/unpinning (jump) phenomena. Recently, however a hyperbolic law which is more complete has been developed9. This law permits the contribution from domain wall vibration and the lattice to be distinguished. The dielectric permittivity is then described as a function of the amplitude of the exciting electric field9:The dielectric response of domain wall motions have been already investigated for several years in polycrystalline PbOE0 is the amplitude of the electric field E(t) = E0 sin ωt. εrl is the intrinsic lattice contribution which has its origin from the deformation of the unit cell6, and εr−rev corresponds to domain wall vibration which is a reversible process and can be a measure of the domain wall mobility5. The sum of these two parameters corresponds to the initial permittivity (when E0 = 0). The parameter αr represents the linear dependence of the permittivity on the exciting electric field. It is associated with domain wall pinning and is related to an irreversible modification of the local polarization10. All parameters depend on the crystal structure but αr is especially sensitive to the presence of impurities, dopants or defects11. The parameter αr is generally high in polycrystalline PZT thin films (~1 × 10−5 m/V)13 because the jump distance is important due to the material’s columnar structure and the low degree of domain wall pinning. As a consequence, the threshold field Eth necessary for unpinning is low and the Rayleigh region (linear variation of the permittivity) is clearly visible. In the present study we address the contribution of the ferroelastic 90 degree domain walls by studying epitaxial PZT thin films, one is mainly c-domain oriented and the other consists of c and a-domains. These two distinct structures allow us to correlate the polarization mechanisms to the respective domain wall motions.This relation is valid for real and imaginary part of the permittivity. 0.2Ti0.8)O3 (PZT) films were synthesized by pulsed-laser deposition (PLD) onto (001)-oriented SrTiO3 (STO) substrates coated with a SrRuO3 (SRO) layer which is used as a bottom electrode. The PZT composition was chosen because it has a lattice parameter of 3.935 Å which is close to that of SRO (3.928 Å)14. Two (001) oriented samples of thickness of 60 nm and 100 nm have been fabricated using similar process as in ref. 2 on vicinal STO obtained by standard etching and annealing process of nominal 0.1° miscut crystals. Nominal 25 nm thick SRO epitaxial layers were firstly deposited at 700 °C in 150 mbar O2 pressure followed by PZT deposited at 600 °C in 250 mbar O2 pressure. The thickness of the PZT layer was chosen below and above the critical thickness at which misfit strain relaxes in order to obtain one fully strained PZT film showing only c-domains and one partly relaxed film showing mixed a/c-domains. Detailed microstructural investigations have been done in ref. The Pb) but with different domain configurations.The surface morphology of the two samples was investigated by piezoelectric force microscopy (PFM) using a Park XE-100 AFM with ac voltage of 2 V and frequency of 22.36 kHz. ATEC EFM 20 tips were used for the P-E loops have been measured using ferroelectric analyzer (aixaCCt TF2000E) at 300 K and 25 kHz to avoid dielectric losses at low frequencies. The capacitance and the dielectric losses tan δ have been determined with an Agilent 4294 A impedance meter at an AC-field amplitude of E0 = 5 kV/cm. The real and imaginary parts of the permittivity have been calculated from the capacitance and the dielectric loss values. The variation of the permittivity has been measured at 10 kHz as a function the AC field amplitude . All the measurements were performed at room temperature (20 °C).Circular platinum electrodes of 160 µm radius were deposited by RF sputtering on both samples in order to realize a Metal-Insulator-Metal (MIM) capacitor. The c-domain sample has a square hysteresis loop with a high remnant polarization (Pr ≈ 95 ± 5 µC/cm²) which is almost equal to the saturation polarization PS. This value is close to the value reported for PZT epitaxial films of the same composition16. The polarization hysteresis curve of the a/c-domain sample has also a square shape, typical of hard ferroelectric. The remnant polarization PR is similar to the c-domain sample, the coercive field, however, is considerably lower (Ec ≈ 280 ± 10 kV/cm for the a/c-domain sample whereas Ec ≈ 780 ± 10 kV/cm in the case of the c-domain sample). As a consequence, the c-domain sample is less saturated than the a/c-domain sample which is easier to polarize due to the strain relieved with the presence of a-domains. The polydomain sample hence can be more easily polarized. The c-domain sample presents also a larger distribution of coercive field which indicates the presence of a higher dc conductivity of fully strained high quality PZT films. This high conductivity is due to a higher mobility related to a low density of extended defects (mainly threading dislocations) and to an influence of polarization on the Schottky barrier. Both effects lead to a higher conductance as a direct influence on the Schottky–Simmons conduction mechanism, as shown in previous work on similar films17.Ferroelectric polarization and switching current hysteresis loops are shown in Fig. δ were measured as a function of frequency  relieved in the case of the c-domain sample.The dielectric constant ncy Fig. . The perc-domain sample, the hyperbolic law has been used for decomposing the different components of the real and imaginary parts of the permittivity. The two parts of the permittivity  and the domain walls are hence blocked. Switching the polarization becomes more difficult and consequently the coercive field is higher. The more important value of the threshold field in the c-domain sample is in agreement with the higher value of the coercive field observed in Fig. In order to get a better understanding of the mechanisms responsible of the higher dielectric losses in the ity Fig.  can be pity Fig. ). The evmentclass2pt{minimεr−rev and αr), the ratio between the two samples is quasi-identical. a/c-domain sample. Consequently, the dissipation factors of the domain wall vibration mrev and of the pinning/unpinning mα of the two samples are of the same order. These factors correspond to the ratio between the imaginary and the real parts of the respective contribution and are reported in Table mrev dissipation factors are equal to 0.34 and 0.35 and are similar to those that reported in the literature for BST thin films18, indicating that the vibration of domain walls is a very dissipative phenomenon11. No information on the mrev values for PZT has been found in the literature. The dissipation parameters mα are lower than those reported for PZT thin films (mα ≈ 0.42)22. This might be due to the lower domain wall density in the two samples studied, as a higher density favors domain wall interaction and hence a higher dissipation factor18. The similar dissipation factor retrieved by our study indicates that the dissipation due to domain wall motion is not sensible to the domain wall density. According to Boser20, the ratio between the coercive field and the threshold field is equal to:L3 is the distance between two domain walls and L0 is the distance between two zero positions of the function which describes the distance between two defects. In our study the ratio is equal to 18 and 34 for the c-domain sample and the a/c-domain sample respectively, which is high compared to what is obtained by Boser (EC/Eth ≈ 2.2)20. The distance between domain walls is apparently so large that there is no interaction between them and the domain walls motions are not influenced by the domain wall density. As a consequence, the dissipation factors are identical for the two samples.Concerning the imaginary part of the domain wall motion coefficients  oriented PZT samples of a different thickness have been studied. PFM characterization shows a majority of