Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)–variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH–VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity  Although conventional monoclonal antibodies are now used as drugs to treat a variety of difficult-to-cure diseases including cancers, their use is limited by high production costs due to the requirement for a mammalian expression system and their poor penetration into tumor tissue , 2. In aThe difficulty of producing large amounts of homogenous bsAbs through traditional  techniques, such as hybrid hybridomas and chemical cross-linking, has hindered their wider adoption and development as therapeutic reagents ; howeverSeveral previous reports have suggested that the order or stability of the V domains is important to increase the functionality and applicability of bsAbs regardless of whether they are small recombinant reagents or IgG-like tetravalent ones , 11. Forin vitro and in vivo. Our results show that in addition to rearranging the domain order of bsDbs, increasing affinity may be an ideal strategy for enhancing the cytotoxicity in anti-parallel-type constructs, and that E2x3-LH is an attractive candidate for a next-generation anti-cancer drug.In the present study, we first classified the four possible domain orders of bsDbs as either anti-parallel (the order of the variable heavy (VH) and variable light (VL) domains of both chimeric single-chain components is either VH-VL or VL-VH) or parallel  Figure . We thenKA values of both hEx3-Dbs for each target antigen remained nearly unchanged when hEx3 precomplexed with either of the target antigens was used as a ligand solution  and CD3εγ. Although hEx3-LH is more cytotoxic than hEx3-HL , the binon Table . These rTo further evaluate the binding affinities of hEx3-Dbs with different domain orders, including the parallel-order bsDbs hEx3-O5 and hEx3-5O, we performed kinetic analyses for immobilized sEGFR by surface plasmon resonance (SPR) imaging. The final yields of hEx3-HL, -LH, -O2, and 2O were 3.2, 0.7, 2.1, and 2.2 mg/L culture, respectively. Among the hEx3-Dbs tested, only hEx3-O5 showed low affinity to sEGFR  of each variable fragment (Fv) face outward Figure , consistin vivo testing will be difficult.To evaluate the influence of the domain order on the inhibition of human carcinoma cell growth, we analyzed the four fractionated E2x3 dimers in cell proliferation colorimetric assays. Consistent with previous data , the LH Our previous data showed that the structural superiority of hEx3-LH was associated with increased cytokine production by effector cells, thus enhancing the cancer growth inhibitory effect of the construct . To invein vivo antitumor effects of E2x3-LH and hEx3-LH, we transplanted mixtures of TFK-1 cells and T-LAK cells into SCID mice, which we then treated for four days with the bsDbs. Compared with the vehicle control, treatment with 2 μg of hEx3-LH or 2 μg of E2x3-LH markedly inhibited tumor growth in SCID mice   and enhaStructural modeling well supported these findings. That is, anti-parallel models (hEx3-HL and hEx3-LH) showed that the two CDR-H3s of each Fv face outward and thus are able to bind two targets, whereas parallel models (hEx3-O5 and hEx3-5O) revealed that the N-terminal Fvs cover the CDR-H3s in the C-terminal Fvs to interfere with their binding abilities Figure . In contin vivo. Finally, compared with hEx3-LH, E2x3-LH exerted higher growth inhibitory effects in a tumor-inoculated SCID mouse model  with different domain orders : pRA-hExE. coli strain BL21 Star (DE3)  and purified from bacterial supernatant and periplasmic fractions by using immobilized metal-affinity chromatography. Gel filtration analysis  was used to fractionate the dimers of each bsDb. The column was equilibrated with phosphate-buffered saline (PBS), and then purified bsDb was loaded onto the column at a flow rate of 2.0 mL/min.The hEx3 and E2x3 series of bsAbs, each representing the four different possibilities for domain order, were prepared by using the bacterial expression system we described previously . BrieflyThe interactions of bsDbs with sEGFR or CD3εγ were analyzed thermodynamically by using microtitration calorimetry  . The metThe interactions between sEGFR and bsDbs were analyzed by SPR spectroscopy . The methods for the expression and purification of sEGFR have been described previously [All hEx3-Dbs models for HL-, LH-, O5-, and 5O-type constructs were generated manually by using PyMOL. Although we previously analyzed the X-ray crystal structure of h528 Fv (1WT5) , that of6 cells/mL) were cultured for 48 h in medium supplemented with 100 IU/mL recombinant human IL-2  in a culture flask  that was precoated with OKT3 IgG (10 µg/mL).A human bile duct carcinoma (TFK-1) cell line established in our laboratory  was usedin vitro inhibition of cancer cell growth was evaluated by using a 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay kit  as reported previously [The eviously .4) in the presence or absence of overnight-adhered TFK-1 cells (5 × 103) in 96-well plates. Supernatants were harvested and used in enzyme-linked immunosorbent assays according to the manufacturer’s instructions  after 20 h of co-culture for human IFN-γ or after 16 h of co-culture for human TNF-α.bsDbs  were co-cultured with T-LAK cells  mice . Starting at 1 h after TFK-1 inoculation, mice (n = 5 per group) were injected intravenously with bsDb or PBS at the indicated doses; this treatment was repeated once daily for 3 consecutive days. Tumor size was measured weekly by using calipers, and the approximate tumor volume  was calculated from linear measurements of the width  and length  as follows: V = (A2 × B)/2. Experiments involving mice were reviewed by the Committee on Ethics in Animal Experiments of Tohoku University and were performed under the Guidelines for Animal Experiments of Tohoku University and according to the laws and notifications of the Japanese government.For each mouse, 1.0 × 10


A FLT is a whole-herd test conducted in a herd following the confirmation of bTB in an animal at slaughter. We considered five different inspection scenarios, each based on meat inspection and bacteriology in series, including current meat inspection (CMI) and four visual-only inspection scenarios  with reducing inspection sensitivities. Separately for each inspection scenario, a simulation model was used to estimate the herd-level detection sensitivity and the number of bTB-herds  that would and would not be detected. The simulated mean herd-level detection sensitivity estimates were 0.24 for CMI, and 0.16, 0.12, 0.10 and 0.08 for VOI2-5, assuming a 2-, 3-, 4- and 5-fold decrease, respectively, in the animal-level detection sensitivity of VOI relative to that of CMI. The estimated number of non-detected bTB-herds is substantial with CMI, and increases in the series of VOI scenarios with decreasing herd-level detection sensitivity. If VOI were introduced without alternative surveillance means to compensate for the decrease in animal-level inspection sensitivity, such changes might jeopardise bTB surveillance, control and eradication programmes in cattle herds of non-OTF countries, including Ireland.The European Food Safety Authority (EFSA) has published a series of opinions to assess the impact of changing from the current meat inspection procedures (CMI) to visual-only inspection (VOI) procedures. Concern has been raised that changes from CMI to VOI would adversely affect the effectiveness of surveillance for bovine tuberculosis (bTB) in EU member states, both for countries with and without official status of bTB freedom . This study was conducted to estimate the impact of a change from CMI to VOI in abattoirs on herd-level detection sensitivity in Ireland, a non-OTF country. Using national Irish data, we identified all herds that sold at least one animal to slaughter during 2010–12 whilst unrestricted for bTB. For each of these herds, we calculated the number of cattle sent to slaughter whilst unrestricted, the number of factory lesion tests (FLT) that had been performed, and estimated the apparent within-herd prevalence (AP Meat inspection has the dual purpose of contributing to safeguard public health and food safety as well as to enable surveillance of animal health and welfare , and botIn 2012 and 2013, the European Food Safety Authority (EFSA) published a series of opinions considering changes within the European Union from the current meat inspection (CMI) procedures to visual-only inspection procedures (VOI) . These cIn OTF countries, surveillance for bTB is undertaken in abattoirs for early detection of recurrent infection and to provide evidence of freedom. With respect to the opinion on inspection of meat from bovine animals, the main focus of EFSA’s AHAW Panel EFSA was on the potential detrimental effects of VOI on substantiating OTF country status . The AHAIn non-OTF countries, such as Ireland, both field and abattoir bTB surveillance are conducted. In Ireland, all herds are subjected to field surveillance at least once yearly, but more frequently if considered at higher risk. At this test, all animals more than 6 weeks of age are subjected to the single intradermal comparative tuberculin test (SICTT), and positive animals (noting that test interpretation can vary depending on herd risk status) are deemed “reactors”. In addition, all cattle are subjected to post mortem inspection for bTB at an abattoir  at the time of slaughter . With boMycobacterium bovis infection (the infectious cause of bTB) will be detected subsequently through follow-up laboratory testing)  of cattle  see . As a coThis study was conducted to estimate the impact of a presumed change from CMI to VOI in Irish abattoirs on herd-level detection sensitivity during 2010 to 2012.At the end of 2012, there were 6.2 million cattle in 107,308 herds in Ireland, with an average herd size of 58 animals . LivestoThere has been a national bTB eradication programme in Ireland for many years. All cattle are subjected to both field and abattoir surveillance, as described previously. In recent years, substantial progress is being made. The number of bTB reactors and herd incidence were each lower in 2013 than in any preceding year of the national programme. The role of wildlife is recognised, and progress is being made towards a bTB vaccination for badgers . SeveralThere are some differences in abattoir surveillance for cattle originating from non-restricted and restricted herds. Cattle from non-restricted herds can be slaughtered at any abattoir  in Ireland. When a bTB-like lesion is observed in an animal slaughtered from a non-restricted herd, laboratory confirmation is undertaken, using either histopathology or culture. If bTB is detected, the herd is restricted and a whole-herd “factory lesion test”  is conducted in the herd from which the lesioned animal had most-recently been resident, this being the application of the SICTT on all animals >6 weeks of age on the day of the test . In restWe conducted a narrative review of published literature relating to the sensitivity and specificity of meat inspection. We limited our review to studies with a gold standard for sensitivity and specificity estimates of detailed laboratory examination and infection-free cattle populations, respectively, and to studies using Bayesian (no gold standard) methods. Further, apart from one seminal study , we focuEstimates of the relative sensitivity of meat inspection (when compared with detailed laboratory examination) vary widely, including 9.5% , 28.2% , 29.4% , 36.5%  and 55% The median sensitivity of bacteriology was estimated at 76–79% in the meta-analysis and latent class analysis , althougBased on the information above, we calculated an estimated median sensitivity and specificity for meat inspection of 55% (9.5–87.1%) and 99.3% (97.4–99.9%), respectively, and of bacteriology of 77 and 100%, respectively. Assuming independence between these two diagnostic methodologies, the animal-level detection sensitivity of the meat inspection and bacteriology in series for a bTB-affected carcass will be low , 7–67%) and the animal-level detection specificity very high (approaching 100%). Very similar results would be expected if we were to consider meat inspection and histopathology in series. Therefore, with a 5-fold drop in animal-level inspection sensitivity, the estimated median animal-level detection sensitivity would be 8% .In Ireland, DAFM manages the Animal Herd Computer System (AHCS), which contains a unique record of each bTB-related testing event in each herd nationally. AHCS integrates with a number of other systems, including the Animal Identification and Movement system (AIM), which stores a computerised record of all movements in and out of all herds nationally, including movement to slaughter .Using methods described previously , an episFor each eligible herd, we interrogated these data using SAS v9.3  to identify:The number of animals sent to slaughter whilst the eligible herd was unrestricted, A. The resulting distribution was explored using SAS proc univariate. The observed number of eligible herds by each of the recorded number of cattle slaughtered during 2010–12 were ranked and listed .wh as B/A (as defined above), based on the assumption that each FLT was triggered by a single bTB positive slaughtered animal.Those animals that were detected as bTB-positive based on meat inspection and subsequent laboratory confirmation, have not been directly recorded in the national database. Rather, we recorded the number of FLTs, B, performed for each eligible herd during 2010–12, and for the herds with one or more FLTs we estimated the APData for herds that slaughtered more or less than 2 SD from the mean were excluded. This was undertaken to eliminate the largest suppliers of slaughter cattle, which are likely to be cattle dealers, feedlots, assembly herds, etc., rather than proper cattle herds. Within the Irish programme, a feedlot herd is defined as “a specialist finisher of beef that does not deliberately engage in the active breeding of animals, notwithstanding that an occasional cow/heifer may calve because it was pregnant on arrival to the feedlot”. In contrast to other herds, there can be fewer restrictions on movement of cattle into restricted feedlot herds depending on the epidemiological circumstances, noting that all animals from these herds are destined for slaughter . The numUnless indicated otherwise, all analyses involving simulations and results thereof have reference to these 85,916 study herds, being herds  that sold at least one animal to slaughter during 2010–12 whilst unrestricted.For the purpose of estimating herd-level detection sensitivity (HSe), the distribution of true within-herd bTB prevalence within infected herds is required. To achieve this, herds were first filtered to include only those that had at least one FLT. To avoid inflating within-herd prevalence due to small denominators, we dropped those herds that had sent less than 20 animals to slaughter whilst unrestricted. Next, the apparent within-herd prevalence for each infected herd was calculated as nFLT/nSlaughtered. The true within-herd prevalence for each of these herds was then estimated using Rogan-Gladden estimation  with aniA simulation model was created to estimate HSe see . For easWhere:h), i.e., the probability that a randomly selected herd contains ≥1 infected animal. Initially this value was set at 0.052 as the proportion of herds with ≥1 reactor in the dataset. However, since the animal-level detection specificity of abattoir surveillance is assumed to be 1.0 and the probability of a test-positive in the iterations where the herd is uninfected is 0, these iterations have no impact on HSe and will decrease the efficiency of the simulation. Therefore, in order to aid with computation, µ was set to 1.0.µ is the true herd-level prevalence  in series: CMI, VOI2 , VOI3 (3-fold decrease), VOI4 (4-fold decrease) and VOI5 (5-fold decrease). Similarly, given uncertainty over the ASe of abattoir surveillance, the baseline CMI ASe was varied by ±10% and the analysis repeated for each of the 5 scenarios.M. bovis that sent at least one animal to slaughter when unrestricted during 2010–12) that were detected and those that were not detected for each of the inspection scenarios as follows:From the simulated herd sensitivity values, we estimated the number of bTB-herds  of bTB was estimated as the proportion of study herds with at least one FLT out of all study herds:The apparent herd prevalence (APh) of bTB:Dividing this proportion by the mean herd sensitivity (HSe) estimate for CMI gives an estimate of the true herd prevalence  was randomly assigned an infection status  using a Bernoulli distribution with a probability equal to the TPh (above). Each infected herd was then assigned a test (+ve/−ve) status using a Bernoulli distribution with the probability equal to a HSe sampled at random from distribution of simulated HSe values. For each scenario, the number of infected herds and the number of herds that were detected was summed across all herds.Separately for each inspection scenario, each herd in the overall dataset . It is also noteworthy, that the mode values for the number of cattle slaughtered from herds with at least one FLT and from all herds are both 1. In each case, the herd-size distribution is extremely skewed, since a large proportion of herds provided just a single animal for slaughter during the 3-year-period.Summary statistics on the number of animals sent to slaughter whilst the herds were un-restricted throughout the period 2010–2012 are presented in wh across all herds for the 3 year period was 0.0034 , the herd-level detection sensitivity compared to CMI would drop by 50%, if mean sensitivity estimates are compared, and by 64%, if median sensitivity estimates are compared  and apparent within-herd prevalence (APwh)  as presented in Pwh) see  each refPwh) see . Among nPwh) see . In a rePwh) see . In the Pwh) see  used a pPwh) see  and the Pwh) see , substanPwh) see .There were differences between study herds with at least one FLT and all study herds with respect to the number of cattle sent to slaughter . This coM. bovis will be detected in one or more animals from an infected but non-restricted herd during abattoir surveillance. Each of these terms is defined in the glossary (In the EFSA opinion , animal-glossary .This work highlights the importance of abattoir surveillance in non-OTF countries, to facilitate both the early detection of infected herds and the detection of infection in herds with one or more animals with bTB lesions but without animals positive to the skin test. In OTF countries, abattoir surveillance is also extremely important, forming the basis of national surveillance to substantiate bTB free status . In eachSalmonella (Trichinella testing regime (There have been recent changes to EU legislation relevant to meat inspection of pigs, including the visual inspection of pig carcasses and offal by officials at post-mortem , in assolmonella  and a mog regime . In contIf VOI were introduced in EU cattle abattoirs without alternative surveillance means to compensate for the decrease in the herd-level detection sensitivity of abattoir surveillance, such changes might jeopardise bTB surveillance, control and eradication programmes in non-OTF countries, including Ireland. Based on the results of a recent study , the impPW designed the study and monitored the compilation of the datasets and the estimates from these to be used as basis for the simulations. CM developed the simulation program based on the datasets and prepared and completed the simulation procedures. EH contributed to the analytical design, presentation and interpretation of data for the simulations. IH and TC carried out the preparation, analysis and presentation of the slaughter data and results for further analysis. SM provided access to and interpretation of the Irish slaughterhouse data, and provided literature as the basis for the sensitivity estimates of CMI. PW and SM drafted the initial manuscript, and all co-authors reviewed the work critically, suggested revisions and finally approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest


The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition. The prototypical activators of transcription, such as Gcn4 and Gal4 transcription factors, have been used in molecular biology to study gene regulation in eukaryotes –6, folloThere are four activation domains reported for Gal4 protein. Early Gal4 studies reported one weak N-terminal activation domain AD-I  and one strong C-terminal activation domain AD-II  . SimilarHowever, the results from Melcher and Johnston studies ,27 clearIn the following Gal4 study from Ptashne, a shorter activation domain AD-III (840–857 aa) was declared as the major activation domain in Gal4 protein . Howeverwww.piskacek.org  [e.g. as shown for the Msn2 orthologs [We have reported a new activation domain for the Gal4 transcription factor, the nine amino acids transactivation domain, 9aaTAD . The act RC1-12) ,33. Occarthologs ,34). TheThe position of the Gal4 9aa TAD activation domain did not correspond to any of the previously reported activation domains AD-I, AD-II or AD-III from Ptashne lab. Rather, the position of the activation domain 9aaTAD correlated with the Gal80 binding region and with the region missing in the Gal4 loss-of-function mutant gal4-64 (1–852 aa) ,27.In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains was functional. Our results showed that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein.EcoRI site of the vector pBTM116 . The constructs G1-G45 and H1-H45 were generated by PCR and subcloned into pBTM116 EcoRI and BamHI sites. All construct have a spacer of three amino acids inserted into the EcoRI site; peptide -NNN- . All constructs were sequenced by Eurofins Genomics. Further detailed information about constructs, primer sequences are available on the request.The construct pBTM116-HA was generated by an insertion of the HA cassette into the lacZ reporter driven by the lexA operator. In all hybrid assays, we used 2μ vector pBTM116 for generation of the LexA hybrids. The yeast strain L40, the Saccharomyces cerevisiae Genotype: MATa ade2 his3 leu2 trp1 LYS::lexA-HIS3 URA3::lexA-LacZ, is deposited at ATCC (#MYA-3332). For β-galactosidase assays, overnight cultures propagated in YPD medium  were diluted to an A600 of 0.3 and further cultivated for two hours and collected by centrifugation. The crude extracts were prepared by vortexing with glass beads for 3 minutes. The assay was done with 10 ul crude extract in 1ml of 100 mM phosphate buffer pH7 with 10 mM KCl, 1 mM MgSO4 and 0.2% 2-Mercaptoethanol; reaction was started by 200 ul 0.4% ONPG and stopped by 500 ul 1 M CaCO3. The average value of the β-galactosidase activities from three independent experiments is presented as a percentage of the reference with the standard deviation . We standardized all results to previously reported Gal4 construct HaY including merely the activation domain 9aaTAD with the activity set to 100% [The β-galactosidase activity was determined in the yeast strain L40 ,36. The  to 100% .The crude cell extracts were prepared in a buffer containing 200 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol (v/v), separated by SDS-PAGE, and blotted to nitrocellulose. The immuno-detection of proteins was carried out using mouse anti-HA antibody  or mouse anti-LexA . The secondary antibodies used were anti-mouse IgG antibodies conjugated with horseradish peroxidase . The proteins were visualized using Pierce ECL  according to the manufacturer’s instructions.Figs . The activities of all Gal4 constructs in this study were tested in LexA hybrid assay . The expression of the LexA-Gal4 protein hybrids were proved by western blotting .To we revised function of all reported Gal4 activation domains, we had generated a set of the Gal4 constructs . Surprisingly, the constructs G10, G42, G405 and G50, with activation domains AD-I, AD-II or AD-III, had no substantial activity.The constructs G577, G81, and G49, which all included the activation domain 9aaTAD, have the capacity to activate transcription  and H47  strongly activated transcription . The activities of the corresponding constructs without the artificial peptides  were dramatically reduced or completely diminished. This observation was even more pronounced in the constructs without the HA-tags . The weak activities were observed in two out of four constructs with the HA-tags . All constructs without the HA-tags had none substantial activity, therefore the weak activity of the construct H42 (and even less in H48 construct) was considered as an elevated basal level, which extended to 15% of the Gal4 C-terminal construct G49 and 10% of the 9aaTAD construct H577. The LexA-HA construct Hdd with the HA-tag was inactive and therefore the HA-tag could not activate transcription .We have observed that the three constructs with the artificial peptides including the H41 , H43 . To avoid artefacts, we have inserted the peptide linker of three Asparagines (NNN) between LexA and Gal4 in all our constructs .In order to fully justify the 9aaTAD domain function, we have generated the Gal4 constructs G49 (776–871 aa) and G50 (776–863 aa) that ended before and after the activation domain 9aaTAD. Furthermore, we have generated two different constructs including merely the activation domain 9aaTAD with two different fusion regions (boundaries) to the LexA DNA binding domain—constructs H577 (857–871 aa) and HaY (860–871 aa)  and HaY (860–871 aa) strongly activated transcription comparably with the most active Gal4 C-terminal segments; the constructs G49 (776–871 aa) and G81 (776–881 aa). The activity of the activation domain 9aaTAD is over hundred fold stronger than previously reported the activation domain AD-III as shown for the construct G405 (840–857 aa). The artificial activation domain (construct U39) activated transcription about two hundred fold stronger than the activation domain AD-III (construct G405) and demonstrated a cloning error in the original AD-III construct pRJR200 [As shown in  pRJR200 .Historically, the Gal4 together with Gcn4 and p53 transcription factors represented the acidic activators ,34,37–42In this study, we found that none of the previously reported Gal4 activation domains AD-I, AD-II or AD-III was able to activate transcription. Therefore, the domain 9aaTAD is the exclusive activation domain in the Gal4 protein, which has the decisive competence to activate transcription. Thus the term acidic activation domain reported for Gal4 protein ,25,31 anWe demonstrated that artificial peptides included in the original Gal4 constructs  and fusiThe original Gal4 construct pRJR200  should dLLTGLFVQD-, where hyphen marks fusion site) with the second half of the native Gal4 activation domain 9aaTAD  or with the part of the Gal4 activation domain AD-III .We have previously reported that the fusion of the Gal4 DNA binding domain with random peptides could occasionally generate strong artificial activation domains . The GalLLTGLFVQD—WTDQ). This fusion was a powerful activator of transcription, which was two-fold stronger than the Gal4 activation domain 9aaTAD (construct HaY). The construct pRJR200# demonstrated presence of a strong artificial activation domain 9aaTAD in the original pRJR200 construct [The latter fusion (construct pRJR200#), the Gal4 DNA binding domain and four amino acids of the Gal4 activation domain AD-III were fused together without any suitable peptide spacer  was strong activator of transcription [Similar artificial transactivation domain was generated previously by fusion of the Gal4 DNA binding domain (96–100 aa) and P201 activator . The Galcription .DDVYNYLFD DEDTGMFNTTTM ; the Gal4 activation domain 9aaaTAD was included inside of the peptide: DDVYNYLFD; structural data available at PDB accession code 3E1K) [K.l.Gal9  [Fig 4). Furthermore, recent report has suggested a functional link between the artificial peptides binding to the Gal80 protein and their ability to activate transcription; "Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast" [The position of the activation domain 9aaTAD (857–871 aa) correlates with the Gal4 region 854–875, which is necessary and sufficient for the interaction with inhibitory protein Gal80. The 21 aa long peptide derived from the Gal4 region 854–875 tightly binds to the inhibitory protein Gal80 (peptide: de 3E1K) . Similarde 3BTS)  .n yeast" .The Gal4 protein is known to interact with MED15 , TAF9 4 and Tra1Fig 4) to the previously report [Collectively, we inferred the Gal4 regulation, which involves the activation domain 9aaTAD, the Gal4 mediators of transcription and the inhibitory protein Gal80. We propose a new model of the Gal4 regulation (y report .http://www.uniprot.org/uniprot/?query=9aatad&sort=score) and the 9aaTAD prediction service is available online (www.piskacek.org).The activation domains 9aaTAD are universally recognized by the transcriptional machinery in eukaryotes. Currently, the 9aaTAD family comprises over 40 members including Gal4, Oaf1, Pip2, Pdr1, Pdr3, Leu3, Tea1, Pho4, Gln3, Gcn4, Msn2, Msn4, Rtg3, E2A, MLL, p53-TAD-I, p53-TAD-II, HNF4 / NHR-49, FOXO3, NF-kB, NFAT, CEBPA/E, ESX, ELF3, ETV1, KLF2/4, EBNA2, VP16, HSF1, HSF2, HsfA, Gli3, Sox18, PIF, Dreb2a, MTF1, OREB1, WRKY45, NS1, MKL1, TRP32, VP16, EBNA2, KBP220, ECapLL, P201, AH, and B42 transcription factors. We and others have shown that the activation domains 9aaTAD have competence to activate transcription as small peptides ,36,66–80S1 FigThe protein level produced from the Gal4 constructs in L40 strain were monitored by Westernblotting. The proteins comprise LexA a HA tags with a total size of about 21 kDa.(TIF)Click here for additional data file.


AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD . Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family. The protein family of AAA-ATPases (ATPases associated with a variety of cellular activities) achieves functional diversity by a shared core mechanism combined with pathway-specific adaptors1245Well-studied models like the yeast ribosome biogenesis factor Drg1 or the multifunctional mammalian protein p97 have provided deep insights into the mechanisms of inter- and intramolecular synchronization of ATP binding and hydrolysis781051213141214The N-domain of p97 mediates the direct physical interactions with the majority of co-factors and adaptor proteins that recruit the enzyme to the target pathways7101420211311131824Saccharomyces cerevisiae and is highly related to mammalian p97 and its yeast orthologue Cdc48828Drg1 (Diazaborine resistance gene 1) is an essential type II AAA-ATPase in the yeast The AAA-domains of Drg1 and p97 show an amino acid sequence identity of about 50%, while the N-domains exhibit a significantly smaller degree of sequence conservation . NeverthDRG1 and screening for mutations conferring temperature-sensitive growth phenotypes resulted in isolation of the novel drg1-21 allele. This allele contains two point mutations causing two amino acid exchanges in the N-domain of Drg1; an exchange of threonine 100 for isoleucine (T100I) and of leucine 108 for proline (L108P), which together cause a pronounced growth defect at 37 °C  in combination with the EQ2 exchange  using the shuttling pre-60S maturation factor Arx1 as bait protein . We immobilized GST-Rlp24C and injected purified Drg1 variants to analyse their interaction properties with the substrate. We demonstrated previously that Drg1 requires the presence of nucleotides to form hexamers and that only the hexameric form of the protein is able to interact with Rlp24CIn order to examine whether the dysregulation of the ATPase activity of Drg1-21 is the result of an altered interaction with the substrate protein Rlp24, we performed d(ATP) of about 20 μM for the D1 domain and about 80 μM for the D2 domain (d(ADP) of about 120 μM for the D1 domain, while binding to D2 was below the detection limits of our experimental setup. Together with our previous finding that blockage of ATP hydrolysis in D1 results in more stable interaction with the substrateInterestingly, the wildtype protein purified in the presence of ADP showed lower response units than the protein purified in the absence of nucleotide  but was able to grow at 37 °C  act in physiologically unrelated pathways. While Drg1 is specific for one distinct pre-ribosomal maturation step, p97 acts in a variety of cellular pathways by means of different adaptor proteins731This idea became obvious by comparing both, structural and functional features of the IBMPFD variants of p97 on the one hand and the Drg1-21 variant on the other hand. These include positioning of the exchanges at the N-D1 interface131813For some p97-IBMPFD variants it was demonstrated that they co-precipitate increased levels of ubiquitinated proteinsThe dysregulation of Drg1-21 suggests that binding of the substrate cannot be transmitted to the ATPase domains and the N-domain cannot fulfil its regulatory function. According to our structural models, T100 corresponds to R86 of p97. A mutation to alanine at this position is linked to “IBMPFD-like” structural alterations in p97sup protein explains the suppression of the temperature sensitive phenotype of Drg1-21. In cryo-EM structure of p97 obtained in the presence of ATPγS, where the N-domain adopts an elevated position reminiscent of the IBMPFD variants, residue R65 (which corresponds to N77 in Drg1), is rotated towards the N-D1 linker which undergoes a loop to helix transition upon ADP to ATPγS loading into D126). It is therefore tempting to speculate that the N77K exchange in Drg1 reverses the defects of Drg1-21 by interfering with the N-D1 linker. This could shift the up/down equilibrium of the N-domain towards the down-position and thus rescue the phenotype of the double mutation in Drg1-21. However, in the absence of an experimentally determined structure for Drg1, and given the fact that the region containing the N77 residue is less confidently represented in our Drg1 models, these explanations are largely speculative. Therefore the exact mechanism behind the suppressor exchange N77K has to remain elusive and awaits further investigations.Our screen for suppressor mutants showed that incorporation of a polar sidechain at position 100 of Drg1-21 is sufficient to restore activity of the protein. Polar and hydrophobic interactions at the N-D1 interface seem to be critical determinants for the positioning of the N-domain relative to the D1 domain. In p97, R86 forms multiple polar contacts with residues of the N-D1 linker (D204 and D205) and similar interactions were also predicted for the IBMPFD residues R93 and R95Our findings suggest that the primary defect in the Drg1-21 mutant is the altered interaction between N- and D1-domain which might be counteracted by conformational rearrangements at the N-D1 interface. Taken together, the characterization of the Drg1-21 variant gave new insights into the regulation of ATPase activity of Drg1 as one of the crucial trans-acting enzymes during eukaryotic ribosome biogenesis. Our findings revealed a striking correlation with the pathological IBMPFD variants of p97. This indicates that despite low sequence conservation of the N-domain, the basic mechanisms of regulation of the ATPase activity are conserved among AAA-ATPases. In this context our work also contributes to the understanding of the molecular basis of the deleterious effects caused by dysregulated AAA-ATPases, including IBMPFD variants of p97.All yeast and bacterial strains used in this study are listed in 2+. The mutagenized PCR fragments were cloned into a centromeric vector (pRS315) and transformed into the heterozygous diploid W303 DRG1/drg1-1::URA3 strain. After sporulation, random spore analysis was used to select for cells carrying the disrupted chromosomal copy of DRG1 as well as the mutagenized allele on the plasmid. Temperature sensitive alleles were identified by replica plating and incubation at permissive (25 °C) and restrictive (37 °C) temperatures. Localization of the mutations was identified by sequencing.Temperature sensitive alleles were generated by error prone PCR mutagenesis in the presence of 0.05 mM Mndrg1-21 allele and the alleles with the single mutations were generated by overlap-PCR using primer pairs carrying point mutations followed by intragenic fragment swapping to exchange fragments from pRS315-DRG1 for mutagenized PCR fragmentsThe centromeric plasmids containing the GST-DRG1) plasmid described in ref. CUP1-promoter and an N-terminal GST-tag, which is separated from the DRG1-alleles by a PreScissionTM protease  site to allow removal of the GST-tag. The mutant drg1 alleles were introduced by intragenic fragment-swapping. The expression protocol of the Drg1 variants in this study is a modified version of the protocol for wildtype Drg1 which is described in detail elsewhere8600 of 0.01 in baffled flasks and incubated at 25 °C under constant shaking at 110 rpm. At an OD600 of 0.2, protein expression from the CUP1 promoter was induced by adding 0.1 mM CuSO4. After another 18 hours at 25 °C the cells were harvested by centrifugation, washed with aqua bidest. and stored at −80 °C. Purification of the GST-fusion proteins was performed essentially as described in ref. The pCUP1 expression plasmids in this study were derived from the pAZ7  and overexpression of the GST-Drg1 variants was induced by addition of 0.5 mM CuSO4 for 3 hours. Afterwards, cells were harvested by centrifugation and stored at −80 °C. Cell lysis and pre-60S particle purification was performed as described in ref. Tandem affinity purifications of pre-60S particles were performed as described in ref. 37Protein samples were separated using NuPAGE™ Novex™ 4–12% Bis-Tris gradient Protein Gels (Thermo scientific). Western blot detection of specific proteins after transfer to PVDF membranes (Millipore) was described in ref. 6) and GST-tagged Rlp24C fragments was performed as described in detail in ref. Purification of the Hexahistidine-tagged (HISTM plate reader (GeniusPro). For the measurement of the stimulated activity, saturating concentrations of purified HIS6-Rlp24C (1.68 μM) were added. All values were calculated as specific activity ; for pairs with unequal variances (confirmed by F-test) p-values were calculated by an unpaired t-test with Welsh’s correction. All calculations were performed using the GraphPadTM Prism software (version 3.03).ATPase activity was measured using the Malachite green phosphate assay (Bioassay systems) and was carried out as described previously8TM system as described previouslySPR binding experiments with immobilized GST-Rlp24C were performed on a BiacoreXdrg1-21 allele was performed by mutagenic PCR in the presence of 0.05 mM Mn2+. The mutagenized PCR fragments were cloned into centromeric pRS315-plasmids and used for transformation of the drg1-21 shuffle strain [pRS316-drg1-21]. To identify mutations that suppressed the temperature sensitivity caused by the drg1-21 allele, the transformants were plated on SD-leu agar plates and incubated at 37 °C. Plasmid shuffling was performed on SD-agar plates containing 1 g/L 5-FOA (5-Fluorootic acid). Plasmids of transformants with restored growth at 37 °C were isolated and the location of the mutation was identified by sequencing.Generation of random mutations of the For structural analysis of the mutants, we re-used the model built previously with YASARA29How to cite this article: Prattes, M. et al. A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1. Sci. Rep.7, 44751; doi: 10.1038/srep44751 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI. But despite this, and despite the fact that purified PDI is highly soluble and stable in solution, no high-resolution structure of PDI was published for many years. The first X-ray crystal structure of a full-length PDI (from the yeast S. cerevisiae) appeared in 2006 (2B5E), an alternative structure of yeast PDI was reported subsequently (3B0A) and structures of truncated human PDI were published in 2013 (4EKZ and id: 4EL1).PDI is abundant in mammalian secretory tissues such as liver, pancreas and placenta and a simple high-yielding purification procedure from such sources was developed in the early 1980s This long delay before structures were obtained from crystals of PDI strongly suggests that it is intrinsically flexible. This idea is also implied by two further key PDI properties, namely its multi-domain architecture and its remarkable diversity of biological roles and interactions. These properties are discussed in more detail in the next sections. The review then pulls together a large body of evidence indicating that PDI shows extensive flexibility in solution, introduces the results of simulating this flexibility by a new rapid method, and compares the predictions from these simulations to current experimental data. Finally, the review considers how the observed and predicted flexibility of PDI in solution could impact on its biological functions and discusses experimental methods that could now be applied to provide further detailed descriptions of PDI flexibility.Homo sapiens (abbreviated hPDI) and from Saccharomyces cerevisiae (abbreviated yPDI). Where data on PDI from another species are cited, the species is specified. The general term ‘PDI’ is used where a statement is true across species. Both H. sapiens and S. cerevisiae express several homologs of PDI that are generally regarded as members of the ‘PDI family’ The review focusses on PDI from 2a-b-b′-a′), two of which probably resembled the simpler protein thioredoxin (termed a and a′) and two of which were of unknown structural type (b and b′). NMR studies on recombinant fragments by Creighton and collaborators then established the domain boundaries  was recognised as lying beyond the thioredoxin-like domains, and it was also shown that there is a linker region, termed x, lying between the trx-folds of the b′ and a′ domains a-b-b′-x-a′-c.Repetitive features in the amino acid sequence of rat PDI undaries a and shoundaries b, even ta and a′ domains in catalysis of thiol/disulfide chemistry b′ domain of PDI, and that they competed with the binding of unfolded proteins, but that all domains played a part in the binding of larger ligands a and a′ domains and a promiscuous ligand binding site whose core was located in the b′ domain b′ domain that interact directly with ligands Distinct roles for the PDI domains were also described. The key enzymatic action of PDI is its capacity to catalyse thiol:disulfide oxidoreductions dependent on a dithiol/disulfide active site. So the identification of trx-fold domains containing the sequence –CGHC- immediately suggested roles for the 3in vitro were known PDI was initially identified as a ‘ribonuclease reactivating enzyme’ but its discoverers soon demonstrated its action in catalysing the oxidative re-folding and re-activation of several other small disulfide-bonded proteins. They proposed ‘… the hypothesis that the enzyme is a general and non-specific catalyst for disulfide interchange in proteins containing disulfide bonds’ in vitro experiments can be a convenient small disulfide, such as oxidized glutathione (GSSG) or dissolved molecular oxygen, or hydrogen peroxide In these reactions PDI catalyses net formation of disulfide bonds and the source of oxidizing equivalents in these These substrates are proteins which have been unfolded and reduced, with each free thiol then being reacted to generate a mixed disulfide with glutathione The proteins identified as substrates for PDI-catalysed thiol-disulfide interchange vary widely in size, charge, number of chains and domains, and in the fold of those domains. For example, basic pancreatic trypsin inhibitor (BPTI) is very small, comprising 58 amino acids linked by 3 disulfides into a compact domain roughly 3 nm × 2 nm × 2 nm, while human serum albumin (HSA) comprises 585 amino acids, with 17 disulfide bonds and is organised as 3 domains forming a solid equilateral triangle with triangular edges of 8 nm and a depth of 3 nm. For comparison, an immunoglobulin G (IgG) molecule comprising 4 chains and a total of 12 immunoglobulin-fold (Ig-fold) domains, contains 12 intra-domain disulfides, 4 or more inter-chain disulfides and has dimensions 10 nm × 10–15 nm × 2.5 nm. BPTI is basic (pI = 10.5) and contains very little helical backbone whereas HSA is acidic (pI = 5.5), 67% α-helical and all the disulfides link helices 4Studies on cultured cells reveal a similar diversity and range of protein substrates for PDI action. The pioneering study was that of Roth & Pierce Subsequently PDI has been shown to be directly involved in the biosynthetic folding of a wide range of other proteins. Molinari & Helenius in cellulo is ‘substrate-trapping’ using mutants of PDI in which active sites have been mutated to –CxxA- so that a permanent mixed-disulfide is formed between this mutant active-site and its protein substrate in celluloin cellulo when cells are depleted of PDI in cellulo with a specific trans-membrane member of the PDI family A popular systematic approach to identifying PDI substrates E. coli allows the host bacterium to be more productive in generating folded recombinant human proteins (such as antibody fragments) in these heterologous environments Nevertheless, the specific examples quoted above imply that the oxidative folding of a very wide range of significant secreted and membrane proteins is directly facilitated by PDI. The range of PDI action in living cells is also shown by its widespread biotechnological exploitation in the expression of high-value recombinant proteins. Over-expression of PDI in yeast is a successful strategy for increasing production of valuable recombinant human proteins such as serum albumin and transferrin in vivo is clearly very wide but it is not limitless. Toxins from marine snails such as cone snails (Conus) comprise a vast diversity of disulfide-bonded proteins which include unique structural domains expressed only in conoidean venoms The substrate range of PDI 5In vitro, net oxidation is usually enabled by provision of oxidized glutathione (GSSG) to reoxidize reduced PDI. But the situation within the ER is different and more complex. To enable the continuous operation of PDI in oxidative protein folding, the re-oxidation of reduced PDI is performed primarily by specific flavoproteins that directly couple disulfide bond formation to the reduction of molecular oxygen. These ER-located oxidoreductins  transfer oxidizing equivalents from O2 to a flavin cofactor and then internally via a series of dithiol/disulfide groups to directly oxidize reduced active sites in PDI (for reviews see 2O2 and in higher organisms (but not in yeast) mechanisms exist to use this as an alternative oxidant to reoxidize reduced PDI. The mammalian ER contains several enzymes capable of catalysing this process The previous section has highlighted PDI acting with very broad specificity on unfolded or partly folded protein substrates. In these actions, PDI functions as an oxidant and/or isomerase. 2β2 tetramer in which the α subunits are members of a wider Fe2 +- and α-keto-glutarate-dependent hydroxylase family, while the β subunits are PDI molecules In addition to these redox partners, PDI also participates in surprising cysteine-independent long-term interactions in the ER, as a component of specific functional complexes. Prolyl-4-hydroxylase (P4H), a key enzyme in the post-translational modification of collagens, exists in mammals as an αin vitro. CC Wang's group showed that PDI acts as a classic molecular chaperone suppressing aggregation and increasing folding yield during the refolding of several model proteins and suppressing aggregation during thermal denaturation in vivo, but there is good evidence that PDI does act as a chaperone in vivo during the folding and assembly of procollagen within the ER The suggestion that PDI can act as a molecular chaperone is also supported by extensive work Hence the number of proteins with which PDI interacts in living cells extends well beyond its wide range of oxidative folding substrates, to include redox partners, long-term functional partners and short-term chaperone substrates.6S. cerevisiae) PDI determined by Schindelin, Lennarz and colleagues 2B5E) and the first clear picture of the overall conformation. It confirmed many deductions drawn from earlier work — the presence of four trx-fold domains plus a distinct x-region linking the b′ and a′ domains, and a distinct, non-trx-fold C-terminal region  obtained from crystals generated at higher temperature a domain relative to the remainder of the molecule so that the two active sites no longer face each other across the tips of the horseshoe. Protease-sensitivity was used to show that this twisting at the a-b domain interface .However, no structures of abb′xa′ fragment  of human PDI were generated in presence and absence of DTT and structures were solved to resolutions of < 3 Å allowing reduced (PDB id: 4EKZ) and predominantly oxidized hPDI (PDB id: 4EL1) to be compared. Analysis of these structures and their comparison with each other and with the higher resolution structure of yPDI (2B5E) provides interesting insights. The three structures are very similar and notably there is little difference between the three structures in the relative orientation of the b-b′ domains, but the a and a′ domains show different orientations relative to this base. The distance between the α-carbons of the exposed active site Cys residues in the a and a′ domains is 26.7 Å in yPDI, 27.6 Å in reduced hPDI and 40.3 Å in oxidized hPDI. The differences between reduced and oxidized crystal forms of hPDI are primarily found in the b′xa′ region — the a-b domains can be superimposed very easily with an rmsd (root mean square difference) value of 1.9 Å. However, in the oxidized structure, there is significant rotation of the b′xa′ region relative to ab and significant rotation of a′ around the x-linker, leading to the increase in distance between the active sites noted above and to greater exposure of the ligand-binding region within b′. Wang et al. a domain appears to be oxidized while that in the a′ domains is reduced. It is also worth noting that in the reduced hPDI structure (4EKZ) a molecule of DTT is present bound to the b′ domain, so it is not clear if the differences between this and the structure observed in absence of DTT are exclusively the result of a difference in redox state.Relatively recently, the first crystal structures of mammalian PDI have been published a′ and ligand binding site in b′ are buried. There is good evidence for low-affinity dimerization of yPDI in solution, but it is not clear if this involves the interactions observed in the crystal state b domain of one molecule is inserted into the ‘horseshoe’ of an adjacent molecule, interacting extensively with the a domain. Similarly, in the hPDI crystal structures, the a domain of one molecule is inserted into the hypothesized ‘binding cleft’ of the other, extensively in the case of oxidized hPDI (4EL1) and to a limited extent for reduced hPDI (4EKZ). These interactions are unlikely to be significant in solution since dimerization of hPDI in solution involves either mutual contact of active sites b′ domains b′ domain, the core of the PDI ligand-binding site.As with all crystal-derived structures, the results raise two questions. Are these PDI structures significantly constrained by their specific crystal environments and contacts? And how representative are they of the ensemble of structures that are likely to occur in solution? In the ‘high temperature’ yPDI structure (3B0A) there is extensive contact between neighbouring molecules to form a crystallographic dimer in which the active site in H. insolens and it is considered in a later section.The publication of a high-resolution structure of a complex between a PDI fragment and a peptide ligand 7There is extensive biophysical evidence that mammalian PDI is highly dynamic in solution. The evidence comes from chemical cross-linking, proteolytic susceptibility and from a range of physical methods including intrinsic and extrinsic fluorescence, high-field NMR and small angle X-ray scattering (SAXS). The flexible nature of PDI is indicated in these studies i) by the presence of structural features in solution that cannot be reconciled with the structures determined by X-ray crystallography, ii) by the co-existence of alternative conformations (in absence of redox change) and iii) by the presence of alternative redox-dependent conformations.a and a′ domains (of bovine PDI) could be cross-linked by reagents capable of spanning 16 Å and by longer homologs a and b′ domains and also links between Lys450(467) (in a′) and both the N-terminus and Lys86(103) (in a domain). All these cross-linked residues are > 50 Å apart in the hPDI crystal structures and hence the formation of these cross-links in solution implies extensive deviation from the crystal structures and compaction of the protein. The implication that the active sites in PDI a and a′ domains approach much more closely than is observed in crystal structures is also supported by functional evidence. Araki and Nagata in vitro and their observations are consistent with a model in which there is direct intramolecular transfer of electrons between active sites within a PDI molecule from the a to the a′ domain.In very early studies, cross-linking with bifunctional thiol-directed reagents showed that the active sites in x-linker region, W347(364). In the b′x fragment of hPDI, this unique tryptophan gives rise to two distinct 15N-1H HSQC NMR signals  mutant) or a very hydrophobic buried environment (in the triple mutant). Intrinsic fluorescence studies and the high resolution X-ray structure of the b′x I272(289)A mutant (PDB id: 3BJ5) confirm these interpretations, showing that W347(364) in the x-linker can move freely in and out of the major binding site in the b′ domain. Most importantly, the NMR signal from W347(364) in full-length wild-type PDI is in a position intermediate between the two environments and shows asymmetric line broadening, demonstrating that it is in conformational exchange between the two forms x-linker moves between an exposed hydrophilic environment  and a hydrophobic environment buried within the binding site . These results imply that PDI can access in solution a structure in which the x-linker obstructs access to the ligand binding site on b′; such a structure is observed in crystals of the b′x fragment Considerable insight into hPDI flexibility has come from exploiting the distinctive fluorescence and NMR signals from the tryptophan residue located in the  signals , implyinbb′x fragment of hPDI interpreted by ModelFree analysis demonstrate the dynamic nature of the b′x region  dominating throughout the b′x regions. Conformational flexibility in b′ extends beyond its core ligand-binding site, encompassing much of the domain and the associated x-linker , but enhance activity involving a small molecule disulfide (di-eosin-GSSG). The researchers inferred that there is allosteric communication between the ligand-binding site and the catalytic sites and showed, by a combination of solution techniques, including small-angle X-ray scattering, that binding of bepristats shifts the active-sites to a more reduced state, induces movement of the x-linker region, and leads to compaction of the PDI molecule.Further exploration of the flexibility of human PDI in solution occurs in a study of novel specific PDI inhibitors 8S. cerevisiae PDI in reduced and oxidized state, but valuable insight comes from extensive studies of PDI from a thermophilic fungus Humicola insolens. In this case, NMR data indicated that substrates bind to a hydrophobic surface spanning the b′ and a′ domains which becomes more solvent-exposed on oxidation, and also that the redox state of a′ influences the dynamic properties of the b′ domain (based on H-D exchange and NMR relaxation parameters) a′c relative to abb′ domains and a reduction in contact between b′ and a′ domains on oxidation b′xa′ fragment of this PDI were determined by X-ray crystallography in the reduced (PDB id:3WT1) and oxidized (PDB id:3WT2) states No structural comparisons have been published for b′xa′ fragment and the unfolded protein, α-synuclein, was characterised by high-field NMR, indicating that the oxidized PDI fragment interacts preferentially, and identifying a region of α-synuclein that specifically interacts with the ligand-binding site. Based on this, a peptide fragment of α-synuclein was synthesized and co-crystallized with the oxidized PDI b′xa′ fragment (residues 208–449) and the structure solved to 1.6 Å resolution (PDB id:5CRW). Two alternative modes of binding were observed in the crystals, but a major binding site on the b′ domain was defined and it was shown that ligand binding led to a change in relative orientation of the b′ and a′ domains. Yagi-Utsumi et al. b′ domain of human PDI determined previously H. insolens PDI show an extensive redox-dependent conformational change focussed around the x-linker and the interface between b′ and a′ domains, consistent with that observed in X-ray crystal structures of reduced and oxidized hPDI.Recently, work on PDI from this fungus has provided the first high resolution structure of a PDI:ligand complex 9a-b and b′-a′ domain interfaces. However, the recognition that flexibility is highly likely does not provide any useful insight into the precise nature of PDI dynamics, and the extensive indications of PDI flexibility in solution do not provide high-resolution structural data. A rapid method for simulating realistic protein motion at residue or preferably atomic resolution is required.The construction of PDI as a linear sequence of trx-fold domains provides the potential for inter-domain flexibility and such flexibility would provide some rationale for the extraordinary versatility of PDI functions and the diversity of its interactions. This flexibility would be entirely consistent with the X-ray-derived structures, which show that the inter-domain contact areas are relatively small, especially at the Modelling of protein internal motion is conventionally performed by molecular dynamics (MD) simulations, but for a protein of the size of PDI (c. 500 residues) this is extraordinarily demanding in terms of computational resources. Furthermore, most MD simulations are limited to motion on < 1 μs timescales, whereas functional conformational changes involving relative motions of domains may occur many orders of magnitude more slowly 4EL1) and reduced (4EKZ) hPDI structures 4EL1 (oxidized) is missing residues (250–254) and (320 − 323), whereas 4EKZ (reduced) is missing (240–244) and (322 − 323)). We did not model or constrain these missing regions, but the flexibility simulations did not move the ‘free’ chain ends to unfeasible distances. The results of these simulations are striking and are broadly similar for human and yeast PDI. The basic geometry of the core β-sheet within each trx-fold domain is fully maintained through the simulations so that the protein motion can be represented, to a first approximation, as relative motion of domains  and modes 8, 9, 10 etc. are the next lowest frequency modes of internal flexibility. Flexibility analysis has focussed on modes 7–11 (m7–m11) since these five most facile modes of internal motion encompass the great majority of the capability the protein has for internal flexible motion 7 involves the hinge-bending motion of the outer domains a and a′ relative to the b-b′ base (8) involves the rotation of a and a′ relative to the b-b′ base. Higher frequency modes m9–m11 combine hinge-bending and rotational motion, so that it is convenient to characterise the flexible motion in terms of changes in orientation of adjacent domains – a ‘tilt’ angle representing hinge-bending, and a ‘twist’ angle representing relative rotation.In normal mode analysis of protein motion, modes 1–6 represent rigid translational motion of the molecule in three-dimensional space and rigid rotation of the molecule around three perpendicular rotational axes. So the lowest frequency (most facile) mode of internal motion is termed mode 7  mode of motion, allows an opening and closing of the PDI ‘horseshoe’ such that the volume enclosed within it varies very considerably and the distance between the Cα atoms of the active-site residues in the a and a′ domains ranges from close contact to a great distance  show similarity in potential inter-domain motion and so have similar scope to open and close and to ‘twist’ to take up conformations different from the starting crystal conformation.The net effect of the five lowest frequency modes of motion ma-b interface in m10, directly converts the inter-domain orientation of the starting structure (2B5E) into that observed in the alternative crystal form (3B0A). In every case, the simulation is not provided with information on alternative structures, so this ‘interconversion of structures’ by our flexibility simulations strongly validates the plausibility of the trajectories.Most strikingly, the range of flexible motion in low frequency modes, starting from either of the two hPDI structures includes the domain orientations found in the other structure , so thatb′ domain, suggesting that this domain, which forms the core of the PDI ligand-binding site, can flex to optimize its interactions with ligands  was used as the initial state for an MD simulation, it was found to be relatively stable and led only to minor local changes in conformation To validate the simulation approach, the motions of yPDI predicted in the flexibility simulations were checked for consistency with those found in a short (30 ns) MD simulation. There is good consistency between the approaches see e.g. , but thea and a′ domains are much closer than in the original structures and new interactions are formed between non-adjacent domains. Desmond et al. a and a′ domain mid-points approach to within 30 Å. Most strikingly, the MD simulations a and a′ domains approaching to within 5.4 Å. This distance is consistent with direct covalent bonding between the groups and Yang et al. It is also interesting to compare the results of simulations of human PDI flexibility with those of a recent molecular dynamics simulation based on the same hPDI structures 3F8U), ERp27 (PDB id: 4F9Z) 2R2J)  see .b′ and a′ domains, consistent with biophysical data for human and H. insolens PDI. But the modelling is not only consistent with experimental data; it goes beyond it by providing plausible trajectories for motion of the protein, fully defined at the atomic level in terms of PDB co-ordinates. We conclude that the method provides a valuable basis for exploring the full scope for motion of PDI and similar large multi-domain proteins and for making predictions that can be tested experimentally.In summary, modelling of PDI flexibility produced results consistent with experimental data on both fungal and mammalian PDI. Specifically, the simulations predict i) the close approach of the active sites consistent with cross-linking and functional data on mammalian PDIs, ii) the domain re-orientations that interconvert the reduced and oxidized crystal conformations of hPDI and that interconvert the high and low temperature crystal structures of yeast PDI, and iii) the extensive relative motion of the The full dataset from simulations of flexible motion of human and yeast PDI, including intermediate structure files, is available at Desmond et al. 10a and a′ domains relative to b′b′. Whereas the MD simulation of human PDI motion highlighted a transition to a more compact structure in every case, flexibility simulations show the potential for both ‘opening’ and ‘closing’ motions. This flexibility permits a vast change in the volume included between the domains, in the distances between key sites,  and in the relative orientation of these sites. Flexibility of this kind provides a rationale for the extreme diversity of PDI interactions with folding substrates and protein partners. Whereas PDI alone in solution may be more compact than observed in the crystal structures, it is very probable that its complexes with large protein ligands cover a wide range of structures, both more ‘open’ and more ‘closed’ than observed in crystals.The most striking aspect of the flexibility of PDI revealed by a range of solution data, by the few X-ray structures and by the flexibility simulations, is that there is extraordinary potential for change in the relative orientation of the b′ domain, implying that the promiscuous ligand-binding site in this domain can flex to optimize its interactions with ligands. Since all information on PDI/ligand interactions highlights the same site on the b′ domain as the core of the ligand-binding site, it appears that PDI redox function in transferring electrons from a reduced protein substrate to an oxidant partner such as Ero1 or Gpx7 requires dissociation of the first ligand before the second can bind. Facile flexible motion of PDI is likely to enable this ligand exchange. Differences in conformation between oxidized and reduced states of PDI imply that flexible motion between such conformations could be a key aspect of the PDI functional cycle in oxidative protein folding.Furthermore, the flexibility simulations and the NMR data  indicateHowever, in the absence of high-resolution structural and dynamic data on PDI/ligand complexes, such conclusions remain general rather than specific. While it is tempting to conclude that overall conformational change in PDI is driven by changes in redox state, leading to changes in ligand-binding properties, it is equally logical to suggest that changes are primarily driven by ligand-binding. In fact, it appears that redox state and ligand-binding are linked functions in PDI, but as yet no detailed thermodynamic analysis of this linkage has been undertaken. In its absence, it is risky to provide models linking PDI conformational change causally to functional transitions. PDI is clearly a complex machine, but the increasing weight of evidence shows that it is not a hard machine that switches sharply between precisely-defined structures. Rather it is a soft machine that can access a wide ensemble of states from which particular sub-ensembles are selected on the basis of various factors including redox state and ligand-binding and dissociation.This review has surveyed the experimental data that indicate flexibility in PDI and has shown that rapid simulation of PDI flexibility provides models of motion consistent with these data but with far greater structural detail. But at the same time, the review highlights the paucity of high-resolution structural data that could provide a real model of how flexibility underpins PDI's functional interactions with its ligands and partner proteins. Further experimental data are urgently required.11Currently models for PDI function are being built based on a very small number of high resolution X-ray derived structures. A greater number and variety of high-resolution X-ray structures and  of PDI/ligand complexes would provide hugely valuable additional insights (though possibly at the cost of complicating the picture). In particular it would be helpful to have structures of PDI in complex with a long-term protein binding partner, either with a ‘stalled’ incompletely folded protein substrate or in one of the functional complexes  in which PDI acts as a long-term chaperone.But other methods need to be pushed forward in order to provide dynamic high-resolution data for the protein in solution. A range of NMR approaches are available Cross-linking combined with mass spectrometric detection of cross-linked peptides has demonstrated its potential Alternatively, Förster resonance energy transfer (FRET) methods could be applied. The technology for single-molecule FRET is now well-developed and time-resolved approaches are available that can monitor over time the change in distance between labelled sites Whether these or other experimental methods come to the fore, it will be essential to use rapid simulation of flexibility in order to have productive interplay between modelling and experiment. The challenge of understanding the functional significance of flexibility in the multiple roles of PDI is such that all available tools will need to be applied.Transparency document.Image 1


Sequestosome1 (p62/SQSTM 1) is a multidomain protein that interacts with the autophagy machinery as a key adaptor of target cargo. It interacts with phagophores through the LC3-interacting (LIR) domain and with the ubiquitinated protein aggregates through the ubiquitin-associated domain (UBA) domain. It sequesters the target cargo into inclusion bodies by its PB1 domain. This protein is further the central hub that interacts with several key signaling proteins. Emerging evidence implicates p62 in the induction of multiple cellular oncogenic transformations. Indeed, p62 upregulation and/or reduced degradation have been implicated in tumor formation, cancer promotion as well as in resistance to therapy. It has been established that the process of autophagy regulates the levels of p62. Autophagy-dependent apoptotic activity of p62 is recently being reported. It is evident that p62 plays a critical role in both autophagy and apoptosis. Therefore in this review we discuss the role of p62 in autophagy, apoptosis and cancer through its different domains and outline the importance of modulating cellular levels of p62 in cancer therapeutics. P62, also well known as sequestosome 1 (SQSTM1) in humans, is a scaffold protein , tumor nIt has been established that one of six domains that p62 contains is an N-terminal Phox/Bem1p (PB1) domain, which mediates the efficient formation of p62 polymers, where the acidic surface residue Asp 69 (D69) of one p62 PB1 domain binds to a basic surface residue Lys 7 (K7) in the next PB1 domain by hydrogen bonding . The PB1MEK5 has been reported as the only MAP2K that expresses the PB1 domain. Nakamura K and colleagues have thereby further demonstrated the functional form of a MEKK2-MEK5-ERK5 complex through their PB1 domains . LinaresThe ERK5 pathway plays a crucial role in cardiac development , angiogeThe ZZ zinc finger domain of p62 is known to be the binding site for RING finger protein tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). It is also responsible for binding to receptor-interacting protein (RIP), a TNFα signaling adaptor B 30]..30].For example, Yu and colleagues showed that this complex can also activate the NF-ĸB and p38MAPK signaling pathway. Using a combined stable isotope labeling with amino acid in cell culture proteomics methodology, it was indeed observed that the p62 ZZ-type zinc finger domain is the innate defense regulator 1 (IDR-1) binding site. Therefore it is a potential therapeutic target for inflammatory and infectious diseases . Sanz etLC3-interacting region (LIR) is required for the autophagy degradation of the mammalian protein . P62 intThe KIR domain of p62 is located next to the LIR domain and resembles the ETGE motif utilized by Nrf2 for its interaction with Keap1 . Under nThe UBA domain of p62 has a significant role in the function of p62 . UBA domThere is currently a general consensus that p62 is an effector of selective autophagy as well as a substrate to autophagy . The p62P62 and NBR1 are bound by their PB1 domain to induce p62 oligomerization that allows binding to LC3 to form p62-LC3 through LIR domain of p62. LIR domain of p62 induces autophagy by binding with LC3 and UBA domain binds with ubiquitinated protein by ubiquitin. These binding can regulate autophagosome formation, consequently which processes autolysosome by the presence of lysosome to regulate proteins aggregation cargo for selective autophagy.Oligomerization of p62 serves as a new indication of the exposure of LC3-binding proteins to autophagic degradation of ubiquitinated diseases-related protein aggregation for liver diseases and neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) ,16,72. ICancer arises from uncontrolled proliferation and sustained defective cell survival with a potential to invade or spread to other parts of the body . The uprThe rise in p62 levels due to its elevated transcription or low levels of autophagy has further been demonstrated in oncogenesis and resistance to cancer therapy as well as in numerous other diseases. Tumor promotion activity of p62 was observed in hepatitis B virus (HBV)-associated hepatocarcinoma. HBV is involved in the reprogramming of the glucose metabolism in hepatocytes leading to hepatocarcinoma . This isNot only are the high levels of p62 been observed in tumor promotion but they have strongly further been implicated in causing resistance to cancer therapy especially platinum-based therapeutic strategies . Over-exVarious studies have proven the activity of cancer stem cells (CSCs) within tumours. CSCs are implicated to be the cause for most cancers therapeutic resistance, relapse and metastasis. Indeed, in breast cancer cells, there was an additional layer of heterogeneity that was proven to be behind the self-renewing tendencies of the cancer cells. P62 was further demonstrated to be highly expressed in this layer and its depletion by siRNA robustly inhibited this resistance in both in vivo and in vitro studies ,89. ThisThe human cholangiocarcinoma cell line with cisplatin resistance was used to investigate the role of autophagy in chemotherapeutic drug resistance. The pentose phosphate (PPP) pathway activity was demonstrated to be promoted more after cisplatin treatment and this pathway is observed to be the one behind drug resistance in cholangiocarcinoma cells. The detected p62 and LC3 autophagy substrates proved a higher flow of autophagy in cholangiocarcinoma cells . AutophaApoptosis is a well-known mechanism of programmed cell death (PCD) and it is mainly induced by activation of a family of cysteine proteases known as caspases. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into cytoplasm to engage executioner caspases . There iWe have previously demonstrated that among the most efficient cancer therapeutic strategies, especially against gastric cancer cells, are those that use the autophagy-dependent apoptosis to induce cell death ,97. SeveIt is apparent that p62 is one protein that cannot by any means be neglected when designing cancer therapeutic strategies. Recently there have emerged numerous studies suggesting a combination of autophagy inhibitors with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in cancers resistant to the latter therapy. These cancers were discovered to upregulate autophagy as a cytoprotective means. However, with this evidence that autophagy-deficient cells accumulate p62, thereby posing another tumorigenic potential, this combinatory strategy may not be the best for most cancers. The possibility of inhibition of both autophagy and p62 has indeed been strongly suggested. We therefore further press on the immediate need for in-depth research on p62 inhibitors if the current autophagy inhibition strategies under clinical trials are to be more successful. Further studies on the pathways that p62 modulate to effect its tumorigenic effects needs to be undertaken as well as studies that shed more insight into the protein’s function in physiologically relevant models.