Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae and swine influenza virus (SIV), as well as the evaluation of pulmonary Mycoplasma-like lung lesions, pleuritis, histopathological lesions and diseases occurrence associated with risk factors, such as management, housing and productive indexes. We conveniently selected 2536 animals for serology testing, and 900 lungs at slaughtering of animals from 30 multisite herds in Goiás State, Brazil.The objective of the study was to evaluate the occurrence and severity of Porcine Respiratory Diseases Complex (PRDC) pathogens in the Goiás State, Brazil. Were assessed the serological antibodies occurrency of M. hyopneumoniae, all herds presented seropositive animals at some stage of production. Even though most herds (29/30) vaccinated against this pathogen, 90.0% (27/30) of the herds presented at least 50.0% of seropositive animals in finishing and slaughter. Overall, antibodies against A. pleuropneumoniae were present in lower occurrence, varying from 22.4% of the animals in the nursery phase to 1.3% of the animals at slaughter. Conversely, SIV circulated in most herds, with 29 seropositive herds without vaccination. The occurrence of anti-SIV antibodies was higher at slaughter  than nursery , and at slaughter, 23 herds (76.7%) presented at least 50.0% of seropositive animals. All herds presented animals with pulmonary Mycoplasma-like lung lesions, and of the 900 lungs evaluated in the slaughterhouse, 665 (73.9%) presented an average Mycoplasma-like lung lesions of 7.3%. Evaluations of the pneumonia index (PI) showed that 73.3% of the herds were strongly affected by a pathology that manifested itself in different presentation forms. Microscopically, there was a predominance of bronchopneumonia lesions (74.6% of affected lungs), with a high occurrence of the chronic form (57.1%), and there was a moderate to marked proliferation of bronchial associated lymphoid tissue (BALT) in 64.1% of the affected lungs. Pleuritis were observed in 13.5% of the animals.For M. hyopneumoniae antibodies in finishing phases and slaughter may be influenced by pathogen circulation in vaccinated herds, leading to respiratory lesions at slaughter. Additionally, swine influenza virus was broadly disseminated in technified herds in Goiás State.Serological tests evidenced that antibodies against App and SIV were present in the Goiás State herds, and high occurrence of  Mycoplasma hyopneumoniae (M. hyopneumoniae) and Actinobacillus pleuropneumoniae (App) are considered the most important primary bacterial pathogens associated with pulmonary lesions [The Porcine Respiratory Disease Complex (PRDC) is still one of the most challenging health issues faced by the pig industry worldwide due to the interaction of viral and bacterial infectious pathogens, environmental conditions and management practices . Several lesions , and swi lesions .M. hyopneumoniae are considered a primary cause of respiratory problems [M. hyopneumoniae and Pasteurella multocida co-infection, highlighting the importance of those pathogens interaction for swine respiratory disease [M. hyopneumoniae infections [M. hyopneumoniae and Pasteurella multocida [In Brazil, the infections caused by problems . Bacteri disease . The prefections , is posiultocida .M. hyopneumoniae, App and SIV in different production phases. We also evaluated Mycoplasma-like lung lesions, pleuritis, histopathological lesions and diseases occurrence associated with risk factors, such as management, housing and productive indexes.In different countries, respiratory lesions are the main reason for lung condemnation as well as a lower slaughter line pace that is attributed to the higher percentage of carcasses that need to be trimmed  and are This study was conducted in 2016 and 2017. We selected by convenience 30 production units with multisite farrow-to-finish herds. Goiás State contains approximately 100.000 sows, and the herds sampled represent 51.0% of Goiás production. Out of the 30 herds, 24 contained nursery and breeding sites at the same farm. In the other six herds, the nursery was located in the same farms as the finishing animals. The average herd size of sampled units ranged from 400 to 4000 sows. Briefly, five herds had between 400 and 1000 sows, 12 herds had between 1001 and 2000 sows, and 13 herds had sow herd between 2001 and 4000 sows.The number of animals to be sampled in each class of production site  was determined using the following formula .\documeN = required number of samples or animals to be investigated;C = degree of confidence of the sampling procedure;D = number of units with disease/infection ;S = sensitivity of the diagnostic test;M = number of units  of the herd to be studied.The expected occurrence of pneumonia in each of the classes  was based on studies previously conducted in the southern region of Brazil –12. By uBlood was collected by jugular vein puncture using disposable sterile needles and syringes, deposited in commercial tubes  with clot activator, and centrifuged at 1500 x g for 10 min. The sera were separated, aliquoted and stored at − 20 °C until analysis. In order to standardize the age of the animal samples, the nursery pigs were sampled at 50 days-age, the growing pigs at 94 days-age, the finishing pigs at 130 days-age, and the slaughter pigs were sampled between 150 and 180 days-age. Vaccination scheme of all herds sampled is presented in Table In another approach, the slaughtering of the pigs from all previously sampled herds was monitored, and at least 30 animals were selected from each farm as commonly practiced in Brazil . In the The mediastinic lymph nodes and lungs presenting Mycoplasma-like lung lesions in the pulmonary parenchyma were selected and collected for histopathological evaluation. For microscopic analysis, all tissue samples were fixed by immersion in formalin for 72 h, transferred to 70% alcohol, and then embedded in paraffin. Samples of tissues were cut and stained with hematoxylin/eosin (HE) in the CEDISA Laboratory .M. hyopneumoniae, App and SIV by the Enzyme Linked Immunosorbent Assay – ELISA with the commercial kits IDEXX  M. hyo. Ab Test, IDEXX APP-ApxIV Ab Test, and IDEXX Influenza A Ab test, respectively, according to manufacturer’s instructions. The relative level of antibody in the sample was determined by calculating the sample to positive (S/P) ratio, and the cut-off values for M. hyopneumoniae, App and SIV were 0.40, 0.50, and 0.40, respectively.A total of 2536 blood serum samples collected from herds and slaughterhouses were tested for the detection of specific antibodies against In every sampled farm, an epidemiologic questionnaire with 114 questions was used to collect data regarding: zootechnical indexes, management practices, application of biosecurity measures, which vaccines were used, the structure of animals facilities, adoption of welfare practices, environmental data and the feeding system used in the farm. Categorical data was obtained through dichotomous questions, which only allowed yes or no as an answer. Continuous data  were obtained by questions which allowed any type of numeric answer, The questionnaire information was filled by authors during an interview with farm owner and technical team. The interviewer was trained in order to not influence the farmer and the technical team answers. The variables are described in Additional file All values of frequencies obtained for each strata (age group) had a 95% confidence interval (CI) calculated using Wilson’s methodology.The categorical data obtained from the 30 sampled herds was further tabulated. The outcomes selected to analyze the explanatory variables were the serology results of each strata and the PI of the herds.p < 0.2) was used. All potential significant associations were then used in univariate logistic regression models (p < 0.05) to assess the association and to obtain the OR value and its 95% confidence interval (CI). For this analysis two explanatory variables were transformed into categorical data, regarding stocking density at weaning, more than 0.3m2/animal was attributed value 1, while less than 0.3m2/animal was given the value 0; regarding stocking density at growing and finishing, more than 1m2/animal was given the value 1 and less than 1m2∕animal was given the value 0. Another converted variable was the number of animals per trough at weaning, growing and finishing phase, more than 35 animals∕trough was given the value 1 and less than 35 animals∕trough was given the value 0. The data analysis was performed using Epi Info™ .In order to investigate potential association between the outcomes and categorical explanatory variables, the Fisher’s Exact Test (p < 0.05) using the software R [p < 0.05) calculation for non-parametric data.Association continuous variables investigated as potential risk factors and the serological data obtained (outcome), were detected by simple linear regression models were performed  . Considering that most of the herds were vaccinated in the nursery phase , we observed only 22 herds with seropositive animals in this phase. In the growing phase, only four herds were positive, and one of them was negative in the nursery phase. Five herds presented seropositive pigs at the finishing phase, and at slaughter, 14. Farm 1 presented seropositive animals at all stages of production.Only three herds had more than 50.0% of seropositive animals in the nursery phase    of the animals presented lung lesion score higher than 10%. In general, the mean percentages of lung lesions ranged from 0.2 to 17.4%, with an average of 7.3%. All herds contained animals with Mycoplasma-like lung lesions, including Farm 2 , while using protocols of empty period between batches in the growing-finishing pig facilities was labeled as a protective factor .p values of all associations are shown in Table The variable “Cross-fostering” in the maternity facilities, which aimed at redistributing pigs to standardize the size of the litters, was inversely associated with the SIV seropositivity in the growing-finishing pigs . The OR and 2 and p values obtained for each model.The continuous variables were submitted to a linear regression model in order to detect potential associations. Table M. hyopneumoniae antibodies at weaning was also inversely associated with ADWG. At the growing-finishing phase, it was also inversely associated with the number of days of empty period between batches and the adoption of cleaning and disinfection protocols at the finishing-growing facilities (Table M. hyopneumoniae seropositivity and the presence of other nearby pig herds.The presence of es Table . A posit2 = 0.43 and p < 0.05). Despite the relatively low R2, the model was significant, indicating a potential association between the variables.The SIV seropositivity in the growing/finishing phases was positively associated with mortality in the phase. Since the mortality in the finishing pigs also presented a positive association with higher PI and medium size pulmonary lesions, all aforementioned variables were used to create a multiple linear regression model , although 13 herds had less than 50.0% of seropositive animals in finishing and at slaughter phases. Lower occurrence of antibodies were observed in nursery and growing animals .A high seropositivity of Mycoplasma hyopneumoniae shedding did not show significant differences between vaccinated and non-vaccinated populations [M. hyopneumoniae. However, since this study was cross-sectional and did not provide direct evidence of pathogen circulation, it is not possible to confirm that hypothesis.Taking into account the presence of Mycoplasma-like lung lesions at slaughter and the high occurrence of seropositive animals in finishing phases, it is possible to infer that vaccination was not preventing pathogen circulation in later phases in these herds. In addition, consequently, the high number of seropositive could have been be influenced by new infection. In an experimental study, ulations . Thus, iM. hyopneumoniae and SIV ELISA tests, the serological test used in this study did not detect vaccinal antibodies, and detected both pathogenic and non-pathogenic strains. The prevalence found in the nurseries may be explained by maternal derived antibodies, which are detectable until up to 12 weeks of age [The serological results for App showed a low occurrence of the ApxIV toxin antibodies, with the highest occurrence found in the nursery animals 20.5%), followed by 5.7% at slaughter, and the lowest in the growing and finishing animals . Unlike the .5%, folls of age .Despite the non-vaccination against SIV, a high seropositivity in herds was observed along all stages of production , a fact that was expected since enzootic SIV circulation in Brazilian herds has been reported . ConsideM. hyopneumoniae and SIV was higher in the latter phases than in the first ones, presenting 100% of seropositive pigs at slaughter. It is evident that there are herd risk factors that favor the occurrence of pneumonia and, if uncorrected, may intensify losses, mainly due to the association with secondary pathogens [To evaluate the degree of the occurrence of pneumonia in the herds, the PI of each farm was calculated. The mean value found among the 30 herds was 1.15, ranging from 0.07 to 2.03, confirming the presence of pneumonia in the herds. Considering this information, the state of Goiás appears to be in accordance with the prevalence data from other Brazilian states, with values varying from 0.86 to 1.54 and a mean of 1.03 . Herds wathogens .M. hyopneumoniae, the farm presented animals seropositive to SIV in the nursery and growing phases.This fact leads us to believe that the occurrence of pleuritis was strongly associated with other pathogens. As observed in studies in slaughterhouses, 33.3% of the rejected carcasses presented pleuritis lesions . The onlM. hyopneumoniae infection since other respiratory pathogens may lead to similar lesions [P. multocida, should be considered within the most likely differential diagnoses [Most of the histological lesions observed in this study (74.6%) were described as bronchopneumonia prevailing in the chronic form (57.1%), with characteristics of BALT hyperplasia ranging from moderate to severe (64.2%). These findings were suggestive, but not exclusive, of an  lesions . Other piagnoses . The occM. hyopneumoniae, and additional confirmatory tests, such as PCR, are required [The injuries characterized as interstitial pneumonia (8.1%) bronchiolitis either chronic or necrotizing (4.4%), are generally induced by viral infection. However, animals in remission may present pulmonary lesions similar to those caused by required .M. hyopneumoniae and Pasteurella multocida, mainly associated with pneumonia, and co-infection of App and P. multocida, mainly associated with pleuritis [The microscopic observation of chronic and fibrinous pleuritis 13.5%), the macroscopic observation of pleuritis (mean of 10.3%), and the occurrence of eight individual lung samples with abscesses are compatible with App infection. These data corroborate a previous study  in which%, the maRegarding APP, a positive association was discovered between anti-ApxIV antibodies and more than 30 animals per trough (OR: 12.24). App transmission can occur by the air or by direct contact, specifically nose-to-nose contact. A previous study showed that nose-to-nose transmission was up to 10 times more frequent than air transmission . Higher Protective factors were also detected in this study (inverse associations), such as having protocols of empty period between batches in the facilities and the App seropositivity in growers (OR: 0.10). Not performing an empty period between batches was positively associated with pleuritis and APP seropositivity in the swine herds, reinforcing the idea that all-in/all-out management is an important procedure in the prevention of respiratory diseases .M. hyopneumoniae antibodies in the growers. This association could be explained by the production flow of the sampled herds since most of the herds were multisite and the arrival of susceptible animals from the maternity sites was constant.The linear regression detected an inverse association between the number of days of empty period between batches and the occurrence of M. hyopneumoniae in the pork producing chain, corroborating the inverse association found in this study between the ADWG and M. hyopneumoniae antibodies found in the growers and finishers. A previous study showed that losses due to M. hyopneumoniae infection could reach US$2.80 for each infected animal with pulmonary lesions [A reduction in the daily weight gain is the main production impact caused by  lesions .Regarding SIV, the adoption of cross-fostering practices improve colostrum ingestion, resulting in a more robust protection against infectious diseases .M. hyopneumoniae, Actinobacillus pleuropneumoniae and SIV, and lung lesions suggest that PRDC could be a problem in the pig production systems in the state of Goiás. Thus, associated risk and protective factors were related with management, reinforcing the importance of good management practices for pig health.This study showed that the seropositivity against Additional file 1. Description of the occurrence of the variables investigated as risk factors and their respective frequencies in the herds sampled in the state of Goiás, Brazil.Additional file 2. Mycoplasma hyopneumoniae seroprevalence in the weaners, growers, finishers and pigs at slaughter from the 30 sampled herds from the state of Goiás, Brazil, and the respective 95% confidence interval (CI 95%).Additional file 3. Actinobacillus pleuropneumoniae seroprevalence in the weaners, growers, finishers and pigs at slaughter from the 30 sampled herds from the state of Goiás, Brazil, and the respective 95% confidence interval (CI 95%).Additional file 4. SIV seroprevalence in the weaners, growers, finishers and pigs at slaughter from the 30 sampled herds from the state of Goiás, Brazil, and the respective 95% confidence interval (CI 95%).


L. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-x L, Bcl-2, Bcl-w, Bfl-1, Mcl-1) that inhibit apoptosis by directly engaging their pro-apoptotic counterparts, the effector proteins Bax, Bak (and possibly Bok), or the upstream initiators of the pathway, the BH3-only proteins  [L, have perhaps provided the greatest insights into the molecular mechanisms by which the intrinsic apoptosis pathway is regulated. Indeed, the first three-dimensional structural data for any family member came from studies on Bcl-xL  , subsequrk, Bik) . Howevere clinic ,7,8. In BCL-X gene was first cloned in 1993 based on its similarity to BCL-2 due to the capacity of a murine bcl-2 cDNA to hybridise to a related chicken gene [BCL-X ortholog. In the same year, the genes for MCL1, A1 and BAX were also cloned [Bcl-2 Homology (BH) domains 1 to 4, as well as a hydrophobic C-terminal region predicted to act as a transmembrane anchor [BCL-X gene was identified, another shorter isoform (termed Bcl-xS) was also isolated that encodes a protein lacking the BH1 and BH2 domains, but possessing both the BH3 domain and hydrophobic tail [The ken gene . This seo cloned ,54,55, ao cloned ,57,58,59e anchor ,61. Whenbic tail . The thiL and Bcl-2 have the longest sequences in the region that spans the BH domains . This is due to both Bcl-xL and Bcl-2 possessing a longer sequence connecting the N-terminal region containing the BH4 domain and the BH3 domain, compared to the other family members. Bcl-xL shares the highest sequence identity with Bcl-w (51%) and Bcl-2 (45%), with all the other multi-BH domain proteins being just 18–25% identical. Shortly after the cloning of all of these genes, the first three dimensional structure of any Bcl-2 family member was determined. This structure provided the first insights into the roles of each of the conserved BH domains, and more importantly, provided the first clues on how Bcl-2 family proteins interact with each other to regulate apoptosis.Among the multi-BH domain proteins, Bcl-xL [L were also reported [The first three-dimensional structure of a Bcl-2 family protein was that of human Bcl-xL . This wareported ,11. In eL structure consists of eight alpha helical regions (α1-α8) (The Bcl-x (α1-α8) a  compared to in other structures  where the helices adopt more of a V-shape, resulting in a more open groove  makes an electrostatic interaction with Arg-139 on Bcl-xL, a residue within the BH1 domain that is also conserved on all pro-survival proteins. Mutation of any of these residues on Bak reduced affinity for Bcl-xL, whilst other contacts between the Bak peptide and Bcl-xL made smaller contributions to the overall affinity of the interaction [L, together with that of a Bak BH3 mutant (Q75L) provided a rationale for the slightly weaker affinity conferred by this mutation  that also has profound physiological effects when introduced into mice [Using NMR, the complex of Bcl-xtermined . The BH3eraction . Neverthnto mice .L and natural pro-apoptotic BH3 domain peptide ligands  that is required for high affinity binding [L not seen in other structures [Over the years there have been multiple NMR and X-ray crystal structures reported for complexes between Bcl-x ligands  includin binding , whilst ructures .L upon BH3 peptide binding. Interactions with BH3 peptides induces an opening of the hydrophobic groove where α3 shifts away from the peptide and in most cases becomes less helical and disordered, though to different degrees depending on the peptide  [Another notable difference is the disposition of Phe-105 which varies somewhat between structures. In some complexes  it is orientated out of the groove, whereas in other complexes it lines the h2 pocket, though in different orientations d,e. The  peptide a. The α4 peptide b whereas binding   compared to sequences such as Bim and Bad. These structures showed a similar orientation of Phe-105 and Tyr-101 as in the complex with Bak BH3, likely due to both BH3 domains having a hydrophobic residue at the h3-1 position rather than a hydrophilic residue seen in most other BH3 domains  due to the presence of a long side-chain (lysine) at the h1+1 position that is typically a smaller residue in pro-apoptotic BH3 domains [The shorter isoform of Bcl-x domains ,79. Bioc domains   that can induce transcription-independent apoptosis [L for binding to diverse ligands, the physiological relevance of these complexes still requires further investigation.A highly divergent BH3 domain  and their susceptibility to proteolysis limits such applications. Nevertheless, several approaches have been used to improve these properties of BH3 peptides to make them more “drug-like”. In this context, Bcl-xL  have shown remarkable mimicry of the canonical interactions seen with native BH3 domain peptides despite the addition of approximately one extra carbon atom per helical turn [A second approach to increasing resistance to proteolysis is to incorporate β-amino acids into a peptide sequence. These amino acid residues possess one extra carbon atom in their backbone compared to α-amino acids and are not readily recognised by proteases, rendering peptides in which they are incorporated more resistant to enzymatic degradation. The BH3 domain has been extensively used as a model system to understand the optimal arrangement of such residues for maximal binding affinity ,35,84,85cal turn ,33,34,35L bound to one of these peptides was important to facilitate design of further sequences to reduce Bcl-xL binding and thereby enhance their anti-apoptotic effect. Another peptide based on Bak BH3 was modified to incorporate a photoactivatable “switch” that increases affinity for Bcl-xL by stabilising the helix [L showed that the peptide-stabilising crosslinker induces a slight shift in the register of the peptide helix along the groove relative to wild-type Bak BH3, with concomitant remodelling of the groove to retain favourable interactions with the peptide.Other BH3 peptides have also been designed with non-natural amino acids to promote different functionalities. For example, Bim BH3 peptides with a novel extended side-chain at the h3 position were generated to create a Bak inhibitor . A struche helix . A strucL described above lack the C-terminal transmembrane domain due to difficulties producing the full-length protein in a soluble form at concentrations required for structural studies. This technical issue has, therefore, precluded structural studies on the membrane-bound form of Bcl-xL. However, two recent structural studies have begun to address the important question of how membranes affect the structure and function of pro-survival proteins given this is their natural cellular milieu [L with its transmembrane domain (though lacking part of its unstructured loop) was purified directly from bacteria in a soluble form [L, this appears to be more dynamic. The binding affinity of the full-length Bcl-xL to a BH3 peptide from Bid was reduced compared to just the soluble domain without the transmembrane helix, indicative of the need to displace the transmembrane helix, and this was confirmed by NMR chemical shift data. Surprisingly, NMR studies on the full-length Bcl-xL integrated into nanodisc lipid bilayers showed that the overall structure of Bcl-xL is very similar to that of the soluble form without its tail region, contrary to earlier NMR studies using detergent micelles as membrane surrogates [L soluble domain was ligated to the transmembrane domain embedded within the nanodisc lipid bilayer [L closely resembles the soluble form, and retains high affinity binding to a BH3 peptide (from Puma). This approach enabled a structure of the membrane-embedded transmembrane domain to be determined and showed the expected helical conformation transversing the membrane. Detailed NMR analyses enabled construction of a model in which the Bcl-xL soluble domain is loosely attached to the membrane surface with the BH3 binding groove oriented towards the lipid bilayer membrane surface, as might be expected for engagement of BH3 domains from membrane-bound pro-apoptotic binding partners such as Bax and Bak. Future studies could also apply this approach to other pro-survival proteins that have been difficult to produce with their transmembrane regions to see if they interact with membranes similarly to Bcl-xL, and ultimately, to establish how pro-survival proteins engage with Bax and Bak in the context of the mitochondrial membrane to inhibit apoptosis.All structures of Bcl-xr milieu ,87,88. Brrogates , and it  bilayer . This stbone fide’ BH3-mimetic was ABT-737 developed by Abbott Laboratories (now AbbVie) [L were instrumental in the discovery of ABT-737, as well as multiple other subsequent BH3-mimetics.The development of small organic compounds with the capacity to interact with pro-survival Bcl-2 proteins and inhibit their activity was a landmark in the apoptosis field, and in the targeting of protein-protein interactions using small molecules, in general. These compounds are referred to as "BH3-mimetics" due to their capacity to mimic the action of native BH3 domain ligands. Although many compounds have been reported to be able to antagonise Bcl-2 proteins, the first ‘ AbbVie) . StructuL [L as well as Bcl-2 and Bcl-w, but not Mcl-1 or Bfl-1. Accordingly, ABT-737 was shown to have activity on Bcl-xL-dependent tumour cell lines and in xenograft models. Eventually ABT-737 was modified to generate an orally bioavailable analogue (ABT-263 / Navitoclax) that has been used in several past and on-going clinical trials [ABT-737 was discovered using “SAR by NMR”, a high-throughput NMR-based technique to screen for compounds that bound to Bcl-xL . Two fral trials . FurtherL used in the development of ABT-737 [L was reported later [Although there were multiple (NMR) structures of fragments bound to Bcl-x ABT-737 , the fired later   had the same effect [L inhibited dimerisation [L dimers is still unclear, there is evidence that dimerisation increases pore-forming capacity in liposomes [Recent structural studies have shown that “activation” of Bax and Bak involves a significant conformational change whereby the α5-α6 helix hairpin is lost, and instead these helices form a single extended helix, releasing helices α6-α8 (referred to as the "latch" domain) that reciprocally swap with α1-α5 (“core” domain) of a second Bax or Bak molecule, creating a domain-swapped dimer ,91. Thisaline pH . Subsequaline pH  , particularly within a membrane. Structural studies with complexes of full-length BH3-only proteins are challenging due to most (except Bid) being intrinsically disordered, though such structures could inform novel mechanisms of apoptotic regulation that do not involve the canonical BH3-in-groove interaction. For example, a recent study demonstrated that residues within the hydrophobic tail of Bim engage a (currently unknown) site outside of the BH3 binding groove on Bcl-xL, thereby increasing the affinity of the interaction [Relevant to this review would be structures of Bcl-xeraction . This “dL with full-length Bax and/or Bak. These would provide important insights into how the BH3 domain of the pro-apoptotic protein is exposed to enable interaction with the Bcl-xL hydrophobic groove, something that is not immediately apparent from the currently available structures. This in turn would potentially also provide insights into how Bax and Bak oligomers are assembled, as they would likely follow a topologically similar BH3-in-groove arrangement [L could potentially also provide similar information, given that Bid adopts the canonical Bcl-2 protein three-dimensional topology (unlike other BH3-only proteins).Perhaps even more interesting would be structures of Bcl-xangement . In thisL binding partners that are not core members of the Bcl-2 protein family. These include proteins such as voltage-dependent anion channel 1 and ryanodine receptors, both of which influence intracellular calcium flux [L BH4 domain (outside of the hydrophobic groove), the precise mechanism by which Bcl-xL regulates these proteins is unclear, hence, structures of these complexes could also be useful to better understand the molecular details of these processes.Finally, there are a number of Bcl-xium flux ,100,101.Although determination of many, if not all, of these structures is likely to prove challenging, recent advances in structural biology techniques particularly in (cryo-)electron microscopy, could be of significant benefit for providing a complete high-resolution view of how the intrinsic apoptotic pathway is regulated and executed.


Intra-chain domain interactions are known to play a significant role in the function and stability of multidomain proteins. These interactions are mediated through a physical interaction at domain-domain interfaces (DDIs). With a motivation to understand evolution of interfaces, we have investigated similarities among DDIs. Even though interfaces of protein-protein interactions (PPIs) have been previously studied by structurally aligning interfaces, similar analyses have not yet been performed on DDIs of either multidomain proteins or PPIs. For studying the structural landscape of DDIs, we have used iAlign to structurally align intra-chain domain interfaces of domains. The interface alignment of spatially constrained domains (due to inter-domain linkers) showed that ~88% of these could identify a structural matching interface having similar C-alpha geometry and contact pattern despite that aligned domain pairs are not structurally related. Moreover, the mean interface similarity score (IS-score) is 0.307, which is higher compared to the average random IS-score (0.207) suggesting domain interfaces are not random. The structural space of DDIs is highly connected as ~84% of all possible directed edges among interfaces are found to have at most path length of 8 when 0.26 is IS-score threshold. At this threshold, ~83% of interfaces form the largest strongly connected component. Thus, suggesting that structural space of intra-chain domain interfaces is degenerate and highly connected, as has been found in PPI interfaces. Interestingly, searching for structural neighbors of inter-chain interfaces among intra-chain interfaces showed that ~86% could find a statistically significant match to intra-chain interface with a mean IS-score of 0.311. This implies that domain interfaces are degenerate whether formed within a protein or between proteins. The interface degeneracy is most likely due to limited possible ways of packing secondary structures. In principle, interface similarities can be exploited to accurately model domain interfaces in structure prediction of multidomain proteins. Proteins domains can be defined as an evolutionarily conserved region of sequence or a compact region of protein structure that are usually considered as evolutionary and/or functional units of proteins. Traditionally, sequence domains are classified based on sequence conservation that are documented in databases such as Pfam  and SMARPrevious analyses on multidomain architectures have shown that only a limited repertoire of domain combinations are observed among all possible combinations of domains and some domains are known to associate with specific domains whereas others are versatile and combine with diverse domains ,12–15. FThe domain-domain interaction interfaces can play an important role in allosteric regulation, substrate recognition of enzymes –27, and In past years, many analyses have focused on understanding the nature of protein structural space, which can be broadly viewed as a collection of known tertiary structures, mostly from the perspective of evolution as well as prediction and design of protein structures –33. ThesAs protein-protein interaction interfaces has been shown to be structurally degenerate , we askeSince interacting domains could possibly be spatially constrained depending on whether inter-domain linker region/s and domain/s linearly separate them, we evaluated structural degeneracy of DDIs on four non-redundant datasets, which represent a varying degree of constraints on DDIs. The linked domain pairs are: a) fully constrained system is when domains are separated by only one inter-domain linker as in consecutive continuous domains (single sequence segment forming a domain); b) consecutive and discontinuous domains (constituting of one or more sequence segments) pairs are taken as less constrained because at least two inter-domain linkers separate domains, and c) least constrained system when non-consecutive continuous domains, which are separated by more than one domain. Finally, inter-chain domain interfaces are taken as having no constraints for domain interactions. The comparison of intra-chain with inter-chain domain interfaces could also potentially give a basis to construct a combined interface template library to improve structure prediction of interfaces. As has been mentioned before, the similarity of interfaces is evaluated using IS-score after non-sequential structural alignment of the interfaces using iAlign . Hence, The structural domains and their classification were obtained from CATH v 4.1.0) database .1.0 data from EBIi.e. domain consisting of only one single segment. The continuous domains separated only by inter-domain linker  are referred to as consecutive continuous domains. Since it is possible to have structures with missing residues lying in the intervening domains, we imposed a criterion on the length of inter-domain linkers of such structures to consider them as consecutive domains. Based on the observed minimum CATH domain length, we set the length cut-off for inter-domain linker to a maximum of 13 residues for structures with missing residues between domains. This strict criterion would ensure that at least there is no intervening CATH domain in structures with missing residues. This process resulted in a redundant set of 2411 proteins. From this, we prepared a list of 1511 non-redundant (at 40% sequence identity at domain level) consecutive continuous domain dataset (domain-CC-2) using the procedure described later in the methodology section that ensures that at least one domain is non-redundant at 40% sequence identity.From set A (consists of 2 classified domain proteins), we selected PDB entries having only continuous domains i.e. a domain is composed of more than one segment. Since the discontinuous domain consists of non-contiguous sequences, it will pose a problem in the sequence alignment while constructing a non-redundant dataset. To address this issue, we analyzed coverage and length distribution of segments in discontinuous domains. This showed that many discontinuous domains (63%) have a major segment (more than 70%), which contributes to the domain structure. Thus, we chose to represent discontinuous domain with one major segment. For this, we empirically imposed following conditions to select the major segment of a domain: a) it should be the longest segment with a minimum length of 100 residues and ≥ 50% coverage of total discontinuous domain length, b) it should contribute at least 20 residues for domain interaction interface . From this, we constructued two sub-datasets: a) consecutive continuous domain pairs and b) all combinations of consecutive and non-consecutive continuous domain pairs. The non-consecutive domain pairs have at least one intervening domain between two domains. We followed the same procedure to construct consecutive domain pairs, as described before, that resulted in a set of 1113 domain pairs. These were used to construct non-redundant dataset at 40% sequence identity at domain level that resulted in 759 domain pairs (domain-CC-M). For the second set, we made all possible combinations of interacting domain pairs for a given PDB entry that resulted in a total of 1553 domain pairs . These wFull-length protein sequences are usually used for generating non-redundant DDI datasets. However, such non-redundant dataset does not have information of non-redundancy available at the level of domains because domain boundaries are not used as an input for alignment or extracting words as in case of CD-HIT . Moreovei = (1 to N) and j = (1 to N) such that cluster numbers i < j (i and j are cluster numbers). For each such combination of clusters, first the common PDB entries having different domain numbers between two clusters are identified. Then, depending on the number of structures  identified in previous step following is performed: a) if there are zero common cluster members (structures), then no domain pair structure is selected; b) if only one common entry exists, then it is taken as representative structure; and c) if there is more than one common PDB entry, then a representative non-redundant structure is selected that has the highest (best) resolution with the longest length. The overview of this procedure is shown in supporting Figure A in i.e. if the order of domains for two common PDB entries is in reversed order, both domain pairs are to be considered in the non-redundant dataset.In this procedure, first CD-HIT  is used To prepare protein-protein interaction dataset or inter-chain domain interactions, we took 17659 heteromers protein-protein interaction dataset from previous work . From thiAlign  program was developed for aligning protein-protein interfaces  and the DDI of ospA [outer surface protein A with two domains having antiparallel β-sheet topology], which consists of similar antiparallel β-strands, that is detected in the interface alignment . AnotherSince intra- and inter- chain domain interfaces are degenerate, we investigated whether merging inter-chain domain interfaces data with intra-chain domain interfaces in the template library could increase the number of significant structural matches for DDIs. For this, the closest structural neighbour search was conducted for consecutive continuous domain interfaces (1511) against three different template libraries. These three different template libraries were: a) inter-chain (1464 interfaces of PPI), b) intra-chain (1511 interfaces of DDI), and c) mixed set of both inter- and intra-chain domains (2975 interfaces of PPI and DDI). The closest match of 1511 interfaces against 1464 PPI resulted in mean (SD) IS-score of 0.304 (0.027) having ~85% of significant matches, where as, in a combined dataset of both intra-chain and inter-chain domain interfaces resulted in average (SD) IS-score of 0.314 (0.027). This is statistically significantly  different than the mean IS-score of 0.304 and 0.307 obtained from searching against inter-chain and intra-chain domain interfaces respectively. Interestingly, the number of significant IS-score matched interfaces also increased from ~85 (88) for inter (intra)-chain to 93%. Therefore, it becomes quite evident that enriching the intra-chain template library with inter-chain domain interfaces can certainly assist in identifying more structurally similar interfaces for intra-chain domain interfaces that would not be possible while using only DDI template library derived from multidomain proteins.A is said to be kth neighbor of IB, if the minimum path length from node IA to IB is ≤ k. Since domain-CC-2 dataset is the largest among DDIs, we performed network analysis on digrah only for this dataset. The fraction of all possible directed pairs at a given kth neighbor for varying IS-score is shown in supporting Figure E1 in kth neighbor consists of ~83% of interfaces at a threshold of 0.26 and k = 8  interfaces drawn based on a predefined IS-score threshold with a path length of one. Here, the directed edge was considered because IS-score is not transitive and it is not same for two interfaces when target interface is changed. For instance, IS-score for A-B is not same as B-A, where B and A are target interfaces respectively. An interface Ik as a function of IS-score and LSCC at given IS-score as a function of k are shown in supporting Figures E3 and E4 respectively in As we have found that including PPIs improve overall closely related matches for intra-chain domain interfaces, we examined whether connectivity of structural space can be improved by including inter-chain domain interfaces. For this, we utilized search results of domain-CC-2 against PPI and vice-versa to include only edges between interfaces (nodes) from PPI and DDI. The summary of all possible directed pairs at given In the present study, we explored the structural landscape of domain-domain interfaces, by discretizing proteins as domains and structurally comparing domain-domain interfaces among structurally unrelated domains. For this, we compared intra-chain domain interfaces among consecutive/non-consecutive domains. In our analyses of domain interfaces, we found that domain-domain interfaces are structurally degenerate and are not random interfaces. The same property of interfaces has been shown for PPI interfaces. Importantly, a possible constraint imposed due to linker region between domains does not affect the general features of interfaces and during evolution of mutlidomain proteins linkers probably adapt to accommodate appropriate intra-chain domain interactions to either facilitate protein function and/or stability. The detailed investigation into understanding the basis of similar interfaces showed that similar packing of secondary structures and flat nature of domain interfaces are primarily responsible for similarity among domain interfaces. Essentially, flat interfaces can allow different interfacial secondary structural elements to be aligned in case of purely geometric matches. Thus, this study provides insight into evolution of domain interfaces.Further, we searched for similar inter-chains interfaces within DDI interfaces and found the best structural matches between them with mean IS-score of 0.311. These show that degeneracy among domain interfaces are observed even when domains are from the same or different protein. This is most likely because of limited ways of packing secondary structure elements. The network analysis of interfaces at significant IS-score threshold showed that domain-domain interface is highly connected and continuous, which increases upon including PPI interfaces. Importantly, combining the inter-chain with intra-chain domain interfaces could enrich the interface template library that could be used for modeling either PPI or domain interfaces of multidomain proteins. The interface structural similarity among DDIs suggests toward a possibility that domain-domain interaction interface evolved from non-specific to specific interaction depending on the functional/structural significance of interfaces as it has been previously speculated for PPI interfaces .S1 Table(PDF)Click here for additional data file.S1 FileFigure A: Overview of non-redundant dataset construction. A Flow chart showing steps of non-redundant domain pairs dataset generation with an example. Figure B: Distribution of scores for the best structural interface matches of domain-CC-M. Scatter plot of interfacial RMSD versus (1) fraction of aligned residues (fres) and (2) fraction of aligned contacts (fcon) for the closest match of 759 consecutive continuous domain-domain interfaces extracted from proteins with >2 CATH structural domains. Each point is colored based on IS-score. Distribution of IS-score is shown as histogram. The same scheme is used in Figure D. Figure C: Comparison of interface planarity and IS-score. Scatter plot showing relationship between planarity of domain-domain interface to the best IS-score of interface obtained for each representative 2270 consecutive domains. Planarity is measured using PRINCIP program in SURFNET  suit of programs that is a root-mean square deviation between interface Cα-atoms and the best fit of plane through the interface Cα-atoms. Figure D: Distribution of scores for the best structural interface matches of domain-CU-2. Scatter plot of interfacial RMSD versus (1) fraction of aligned residues (fres) and (2) fraction of aligned contacts (fcon) for the closest match of 512 consecutive and non-continuous domains extracted from proteins with only two CATH structural domains. Each point is colored based on IS-score. Distribution of IS-score is shown as histogram. Figure E: Network connectivity of DDI in structural space. The fraction of directed pairs of nodes (interfaces), which are connected with at most kth neighbor are plotted as a function of IS-score. This is shown for DDI and a combined DDI+PPI interfaces in panels (1) and (3) respectively. Here, fraction is computed as nk/(N x (N-1)), where nk is the number of kth neighbor pairs and N is total number of interfaces in a graph. The relative size of LSCC at various k for graphs generated at a given IS-score thresholds are shown in panels (2) and (4) for DDI and a combined DDI+PPI interfaces respectively.(PDF)Click here for additional data file.


Elucidating the role played by the nanoscale-material architecture on the amyloid fibril stability is a crucial breakthrough to better understand the pathological nature of amyloid structures and to support the rational design of bio-inspired materials. The computational study here presented highlights the superior mechanical behavior of the S-architecture, characterized by a Young's modulus markedly higher than the U-shaped architecture. The S-architecture showed a higher mechanical resistance to the enforced deformation along the fibril axis, consequence of a better interchain hydrogen bonds' distribution. In conclusion, this study, focusing the attention on the pivotal multiscale relationship between molecular phenomena and material properties, suggests the S-shaped Aβ1−42 species as a target of election in computational screen/design/optimization of effective aggregation modulators.Alzheimer's Disease (AD) is related with the abnormal aggregation of amyloid β-peptides Aβ Whereas, the ignored protein domain is known to be unstructured and not present in the U-shaped pdb file (2BEG [12]), the S-shaped pdb model (2MXU [13]) contains an additional beta-sheet domain between residues E11-K16. The presence of the E11-K16 structured domain is reasonably expected to strengthen the interchain hydrogen bonds, further increasing the stability of the S-shaped fibril assembly. This evidence has been already demonstrated in recent literature [11].Further investigations are needed in order to better describe U-shaped and S-shaped fibrils rupture dynamics, when a consistent part of hydrogen bonds starts to be broken. An interesting avenue for future studies could be the application of the computational platform developed in the present work to study the destabilization of U-shaped and S-shaped complexes with novel compounds, since despite extensive studies, drug after drug aimed at targeting Aβ has failed the clinical trials.MD and GG conceived the research. GG and MR did the molecular dynamics simulations. MD, GG, JT, UM, and AD analyzed and rationalized the data. All authors wrote the paper and critically commented to the manuscript for important intellectual content, read and approved the final manuscript.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.


Entamoeba histolytica, is one of the major enteric infections in humans, resulting in 40–100 thousand deaths annually. This protist has undergone remarkable alterations in the content and function of its sub-cellular compartments as well represented by its unique diversification of mitochondrion-related organelle, mitosome. We conducted domain-based search on AmoebaDB coupled with bioinformatics analyses and identified 22 potential lipid transfer protein homologs in E. histolytica, which are grouped into several sub-classes. Such in silico analyses have demonstrated the existence of well-organized lipid transport machinery in this parasite. We summarized and discussed the conservation and unique features of the whole repertoire of lipid transport proteins in E. histolytica.Eukaryotic cells are organized into separate membrane-bound compartments that have specialized biochemical signature and function. Maintenance and regulation of distinct identity of each compartment is governed by the uneven distribution and intra-cellular movement of two essential biomolecules, lipids, and proteins. Non-vesicular lipid transport mediated by lipid transfer proteins plays a pivotal role in intra-cellular lipid trafficking and homeostasis whereas vesicular transport plays a central role in protein trafficking. Comparative study of lipid transport machinery in protist helps to better understand the pathogenesis and parasitism, and provides insight into eukaryotic evolution. Amebiasis, which is caused by  Eukaryotic cells are organized into distinct membrane-enclosed organelles or compartments. Each organelle has distinctive lipid and protein signature and dedicated function , called mitosomes, which have drastically modified morphological, structural, and functional features compared to those of the aerobic mitochondria  analysis on AmoebaDB in order to identify the potential LTP candidates in E. histolytica. We also discussed the conservation and unique features of LTPs, and their possible mechanisms and roles in biology and pathophysiology of amebiasis.Lipids can either directly affect the membrane charge and the curvature by their altered local distribution or act as precursors to accelerate the local lipid synthesis by lipid metabolizing enzymes. Furthermore, it has also been established that in the nucleus, distribution and metabolism of lipids and lipid-mediated signaling play essential roles in cell proliferation, differentiation, and stress adaptation in higher eukaryotes  was found in PKC, cytosolic phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD) and phosphoinositide  3-Kinase  localized to the perinuclear region in response to Ca+2 import  that can form a lipid binding cavity, which binds and accommodate the hydrophobic moieties of lipid molecules from the aqueous environment  domain containing proteins is mostly studied. Mammalian START domain containing proteins are sub-divided into broad sub-families based on their domain organization (membrane targeted and soluble START) and lipid specificities (cholesterol/oxysterol binding proteins and phospholipid/ sphingolipid binding proteins). STARD1 sub-family (contain of STARD1 and STARD3/MLN64) and STARD4 sub-family  transport cholesterol to various cell organelles , residing in various cell organelles  metabolizing enzymes, their lipid substrates, byproducts and downstream effectors, involved in various aspects of transcription, chromatin remodeling, mRNA maturation, cell proliferation, differentiation, and stress management  homologs  Saccharomyces sp., and grouped based on their domain organization Plasmodium sp. . A summary table indicates the repertoire of LTP homologs will be analyzed in E. histolyticaWe conducted an InterPro domain search (PFAM) analysis on AmoebaDB version 38  in order to identify the potential LTP candidates in E. histolytica possess four ORP homologs , which could mediate sterol transport between the ER and other organelles E. histolytica. Low mutual similarity among E. histolytica START protein homologs (Data not shown in this review) indicate their diverse ligand specificities and functions, as observed in higher eukaryotes  contain only START domain, which likely indicate their cytosolic localization E. histolytica has two potential Sec14 homologs (EHI_146930 and EHI_158090), which also possess Ras GTPase activating protein (Ras-GAP) or Rho-GAP, respectively in vivo a complex with Rab5 and Rab11 on endosomal membranes through its Sec14 domain. Thus, Sec14 domain, which was previously known as a phospholipid binding module, mediates protein-protein interactions with Rab and Rho-GTPases and regulates receptor-mediated endocytosis  could potentially play a similar function in endocytic and trogo/phagocytic processes.E. histolytica genome contains a single PRELI-like domain containing protein (EHI_143630) E. histolytica possesses a highly divergent form of the mitochondrion called mitosome, which is unique in its content and function to Entamoeba  is evolutionary conserved or it has a distinct cellular function unique to this protozoan parasite.The E. histolytica lacks some of LTP homologs known to be present and functional in other eukaryotes E. histolytica has no homologs for human PITPs, similar as Saccharomyces sp.  containing proteins  were investigated using data available at AmoebaDB  Entamoeba species, E. invadens, has two EHI_155260 homologs . Similar patterns of upregulation of these two gene transcripts were observed during encystation (data not shown) , ceramide aminoethyl phosphonate (CAEP) was also reported , and lysine glutamic acid rich protein 1 (KERP1)  - and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors  in its PM irrespective of serum concentration  and named it as E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L). They identified EhPCTP-L by virtue of interaction with anti-chicken embryo caveolin-1 monoclonal antibody, which indicates their potential role in caveola-mediated endocytosis. EhPCTP-L mainly binds to anionic phospholipids phosphatidylserine (PS) and PA, and is localized to the PM and the cytosol  mediated silencing of EHI_178560 causes growth retardation of E. histolytica trophozoites  and EhNPC2 proteins responsible for the trafficking of exogenous cholesterol in E. histolytica trophozoites and also influence the phagocytosis process  and PtdIns4P 5-kinases , and a panel of PI phosphatases . Identification of a repertoire of PI kinases and phosphatases enforces the notion that E. histolytica LTPs also participate in the constant replenishment of PIs at all cellular compartments at various physiological conditions. E. histolytica also possesses 5 putative DAG kinases (data not shown), thus is able to produce PA from DAG. This is consistent with the hypothesis that the reciprocal transport of PtdIns and PA between the ER and the PM  1  contain putative NLS and NES, indicating their potential roles in nuclear lipid homeostasis by nuclear-cytoplasmic shuttling, as well described in higher eukaryotes  finger-containing protein (EHI_138970), which also contains NLS. Thus, it is conceivable that a panel of these proteins coordinately function in the nucleus. As mentioned above (3.2), E. invadens apparently possesses two homologs  of the BUD13/NLS/NES-containing START domain containing protein . These E. invadens genes showed upregulation of gene expression during encystation , which possess BUD13 domain and potential NLS and NES, is involved in nuclear lipid transport, more specifically shuttling between the nucleus and the cytoplasm, and signaling, and thus may be important for growth, stage conversion, and/or evasion from various stresses. In addition, some of the PI and PA metabolizing enzymes in E. histolytica also contains four ORP , 15 START domain homologs other than Sec14 E. histolytica trophozoites  are involved in cytoskeletal reorganization associated with cell motility and adherence to the host cells and microorganisms. The genome of E. histolytica by domain-based survey of LTP homologs in AmoebaDB. The E. histolytica genome possess single PRELI domain containing protein (EHI_143630). It is worth investigating whether this PRELI domain containing protein is involved in mitosomal transport. E. histolytica possess a single START domain containing protein (EHI_155260) with potential NLS and NES, indicating its potential role as a nuclear lipid transporter and its ability of nucleocytoplasmic shuttling depending upon physiological conditions. Upregulation of gene expression of its two homologs  in E. invadens during encystation (De Cádiz et al., E. histolytica genome possess a repertoire of START domain containing proteins. Some of them can function as potential PITP similarly as in Plasmodium sp. (Van Ooij et al., E. histolytica, which depends greatly on receptor-ligand mediated signaling processes. On the other hand, E. histolytica lacks canonical PITP homologs from higher eukaryotes. Furthermore, E. histolytica lacks any homologs of SMP containing proteins, which are involved in the organization of various MCSs and ERMES complex (Helle et al., E. histolytica ensures the complexity and biological significance of LTPs in a variety of cellular processes: lipid transport between cellular compartments, adherence, phagocytosis, trogocytosis, endocytosis, signal transduction, vesicular traffic, cytoskeletal reorganization, nuclear regulation, lipid presentation, and sensing. Further studies are needed to elucidate the role of individual LTPs on the biology and pathogenesis of this parasite. Once E. histolytica-specific LTPs and lipid transfer mechanisms are identified, they may potentially provide a novel drug target against this medically important parasite. Since most of existent chemical interventions against lipid signaling pathways target particular lipid metabolizing enzymes (Nile et al., We have discovered a panel of conserved and lineage-specific LTPs in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


In genetic disease, multidomain proteins are often affected by numerous mutations, but the effects of these mutations on protein stability and their roles in genetic disease are not well understood. Here, we analyzed protein globular domains to understand how genetic mutations affect the stability of multidomain proteins in inherited disease. In total, 291 domain atomic structures from nine multidomain proteins were modeled by homology, equilibrated using molecular dynamics in water, and subjected to global computational mutagenesis. The domains were separated into 7 groups based on protein fold homology. Mutation propensities within each group of domains were then averaged to select residues critical for domain fold stability. The consensus derived from the sequence alignment shows that the critical residues determined by global mutagenesis are conserved within each group. From this analysis, we concluded that 80% of known disease-related genetic variants are associated with critical residues and are expected to have significant destabilizing effects on domain structure. Our work provides an  The polypeptide chain undergoes a stochastic folding process through the many possible conformations accessible until the most thermodynamically stable structure is achieved. The native state of proteins generally corresponds to this structure. When the energy surface or ‘landscape’ has the correct shape, only a small number of the available conformations must be sampled2. The energy landscape, encoded by the amino acid sequence, can consequently change in response to changes in the sequence. These disturbances in the energy landscape often result in an unfolded or partially folded protein structure, and it follows that these structural changes translate into functional changes3.The amino acid sequence of a protein encodes its three-dimensional structure and its biological function4. Multiple domains provide structural and functional plasticity in single-domain proteins, and multidomain proteins are thought to have higher stability within the proteome5. Genome analyses show that over 70% of eukaryotic proteins consist of multiple domains7, but large proteins are difficult to study experimentally. Determination of 3D structure for multidomain proteins presents a number of challenges due to their large molecular size8, which can span 1000–5000 amino acid residues each4.Protein domains, described as compact globular units usually containing 50–150 amino acids, are individually folded to maintain proper structure and function9. Many inherited diseases are linked to protein misfolding resulting from missense mutations, which are believed to disrupt the physicochemical properties of proteins, such as charge, hydrophobicity, and geometry10. Although a precise link between genetic mutations and disease phenotype is difficult to establish, it has been shown computationally that the perturbations caused by genetic mutations modeled at the atomic level of protein structure were associated with patients’ b-wave electroretinogram (ERG) amplitudes, as demonstrated for X-linked retinoschisis12.In human inherited disease, genetic mutations may affect the stability of domain structures. Although the exact role of missense mutations in inherited disease is not well understood, these genetic mutations can disrupt the energy landscape and result in protein misfolding and loss of protein stabilityin silico global mutation screen was developed to evaluate the effect of missense mutations on protein stability15. This approach calculates an unfolding propensity for each missense mutation, which is derived from the changes in Gibbs free energy between the wild-type and mutant protein structures, to describe the effect of the mutation on protein stability. The ‘foldability’ parameter is a new parameter that identifies residues in the protein structure that could play a role in protein destabilization13. Residues with high foldabilities lead to protein destabilization, indicating the essential role of each residue in maintaining proper protein folding and stability. In contrast, residues with low foldabilities will typically maintain the proper protein fold despite the presence of a mutation. Residues with the highest foldabilities are referred to as “critical” residues for the critical role they play in proper protein folding15. Following the principle that many inherited diseases may be caused by misfolding due to missense mutations, residues described as critical are expected to coincide with residues associated with inherited disease. Hence, the unfolding mutation screen (UMS) serves as a tool to analyze the complex relationship among missense mutations, protein folding, and disease. The results of this study can be found at the Ocular Proteome website (https://neicommons.nei.nih.gov/#/proteome).An 7, the effect of mutations in a particular domain could be considered as a perturbation of protein stability and could be evaluated for each individual domain. Therefore, due to the lack of defined multidomain protein structure, an algorithm was developed to evaluate the stability of each protein domain.Currently, the analysis of genetic mutation effects on multidomain proteins is hampered by the absence of information on the complete 3-dimensional structures of these proteins. However, as protein domains behave as autonomous folding unitsHere, 9 multidomain proteins associated with different inherited eye diseases were studied. In total, 291 protein domains were individually subjected to UMS, and structural alignments of homologous domains were used to average the unfolding data into a single unfolding matrix describing each domain type. The averaged unfolding matrix was used to calculate foldability, and critical residues were identified at high-foldability positions, which occurred across all domains. The analysis showed that 83% of disease-causing mutations  that resulted in large magnitudes of destabilization (those with predicted unfolding propensities above 0.9) occurred in critical residues with high foldabilities, suggesting that residues critical for maintaining proper domain folding could also be critical for stability of the multidomain protein. Additionally, the sequence alignment of each domain set studied reveals conservation of residues described as critical for protein stability across the domains of various proteins, highlighting the importance of these residues for protein structure.Protein domain stability was analyzed for nine proteins, eyes shut homolog (EYS), Fibrillin-1 (FBN1), Fibrillin-2 (FBN2), complement factor H (CFH), protocadherin-15 (PCDH15), protocadherin fat 1 (FAT1), protocadherin fat 4 (FAT4), roundabout homolog 3 (ROBO3), and cadherin-23 (CDH23) .The proteins were split into individual domains and then divided into 7 groups by homology. These domains were epidermal growth factor-like (EGF-like), laminin globular (laminin-G), sushi, immunoglobulin-like C2-type (Ig-like C2-type), fibronectin type-III, cadherin, and transforming growth factor-beta (TB). In total, the 291 protein domain structures were each individually homology-modeled , consistent with the literature13, and Ramachandran plots were produced to verify plausible dihedral angles17.Domain structures were modeled as described in the Methods section to ensure viable, stable structures with proper stereochemistry. To assess the quality of the improved models, they were subjected to an internal control designed to verify the quality of the side-chain rotamers of the model−2). Supplemental Table An internal control was used to create self-mutated structures for each residue in the protein sequence. Quality models subjected to the internal control were expected to produce confidence intervals centered on 0.5 with small p-values  for each of the nine multidomain proteins . The total number of disease-related mutations with predicted severe destabilization effects on domain structure ranged from 13% (FAT4) to 57% (FBN1) and was on average 33%.The data show that 82% of all severe-destabilization mutations occur in residues considered critical for protein structure. The percent of severe mutations mapping to critical residues ranged from 64% to 100%, revealing a correlation between the residues implicated in disease and those believed to be critical for the structure of individual domains and the whole protein. Moreover, on average, critical residues associated with disease-causing mutations had foldability scores of approximately 14.In Fig. The identification of protein missense mutations associated with disease phenotypes showcases the relationship between protein residues associated with disease and individual domain residues that are believed to be critical for domain stability. The results above suggest that stabilizing protein structure residues are critical for maintaining proper folding in individual protein domains.The foldability of identically conserved and similarly conserved residues was examined to assess the relationship between residue conservation and foldability. Supplemental Table In Fig. Bar graphs highlighting identically conserved residues and similarly conserved residues over the length of the domain sequence are shown for each domain in Fig. in silico methods, we developed an add-on to the previously published UMS14. Nearly 300 domains from 9 multidomain proteins associated with inherited eye disease were homology-modeled and subjected to global computational mutagenesis. This mutagenesis produced unfolding propensity data for all domains studied and a measure of protein stability derived from domain stability for proteins that have otherwise been largely unstudied.In this work, we sought to find determinants of multidomain protein stability. Multidomain protein structure estimation has remained elusive due to the shortage of suitable experimental and computational methods designed to handle such large proteins. The lack of defined structures for multidomain proteins hinders our ability to understand the relationship between protein stability and disease. Using The multidomain UMS identifies the effects of all possible missense mutations on globular domain structures and defining a foldability parameter allows us to identify residues that provide critical stability for proper domain folding. The method developed here uses free energy measures to quantify domain stability (as an unfolding propensity) in response to genetic perturbations, and these descriptors of domain stability are integrated to describe multidomain protein stability. Each domain was subjected to the UMS, which produces unfolding propensity data and stores it in a 2D matrix. Unfolding data matrices for sets of homologous domains of each protein were averaged to obtain consensus patterns of domain stability and filter noise associated with structural dissimilarities and modeling errors. A foldability parameter was derived from the unfolding propensities as a measure of the sensitivity of a residue to missense mutations; a high foldability parameter would correspond to a residue that, when mutated, causes significant destabilizing effects on the domain structure. Residues that are consistently critical for domain structure across all homologous domains were identified. Amino acid residues with high foldabilities across most of each homologous protein domain were determined to be residues critical for protein stability.The determination of protein stability allowed us to investigate the relationship between protein stability and disease-causing mutations. Disease-related mutations were described by the associated unfolding values to gain an understanding of the effect of disease-related missense mutations on the stability of the domain and subsequently, the entire protein. The proteins described were found to have missense mutations, which resulted, on average, in a destabilizing effect measured as 0.7 unfolding. The distribution of unfolding propensities shows that only ~30% of disease-causing mutations have ‘severe’ destabilizing effects on domain structure, while the remaining 70% of mutations had primarily destabilizing effects.18. However, a comparison of average foldabilities of conserved and nonconserved residues of each domain type as shown in a Table The sequence alignment produced for each set of domains reveals that a high percentage of conserved residues are considered critical. The data show that nearly 50% of conserved residues are critical for each domain, while nonconserved residues are described as critical in only 30% of cases. These results suggest an apparent relationship between conserved residues and those acting as determinants of protein stability. In part, this observation agrees with our previous results on strong correlation between the protein sequence conservation index and foldability determined for 9 eye disease-related proteins19, but simulations of protein folding for such lengths are computationally expensive. At 2 ns, only very early events of protein destabilization can be modeled. Although these very short simulations may not represent a model for the folding-unfolding process, we assessed the proteins using an internal control designed as a measure of protein model quality. The distribution of p-values from each domain internal control, shown in Fig. −2. Although a well-refined model should have a p-value with a magnitude of 10−5 or better, domains with p-values smaller than 0.05 were still considered attainable structures in our application. In addition, Ramachandran plots of homologous domains showed dihedral angles primarily in energetically allowed regions, which allowed us to appraise these structures as representative models of domain structures.Protein polypeptides fold to produce a native protein structure within approximately 1 to 30 msProtein domain structures and functions are, to some extent, conserved between homologous domains in different proteins. The seven homologous domains from the 9 selected proteins were studied to inspect which residues are mostly conserved across proteins, as well as the relationship between conserved residues and critical residues. For each domain, conserved residues were also classified as critical residues. Over 600 disease-causing mutations were associated with severe destabilization effects on domain structures, and approximately 30% of the disease-causing mutations occurred in conserved residues that were considered critical across all proteins studied. These results highlight the importance of residues described as critical for proper protein folding across many different protein families and establishes their importance for healthy phenotypes.5. However, since it is also known that only a fraction of all domain residues are involved in interactions with other domains7, our approach remains valid. In the future, we hope to develop a multidomain UMS application that incorporates stability derived from domain assembly, since correctly arranged domain structures are often crucial for a full understanding of the functions of multidomain proteins20.An evident restriction of our current approach is the lack of domain-domain interactions that may favor stability. It is known that in multidomain proteins, domain-domain interactions aid in stabilizing domains21. Multi-domain proteins vary significantly in molecular weight. For example, proteins analyzed in this work . The availability of multi-domain protein atomic model is the only limitation for the global mutagenesis analysis. The analysis is standard for our method and numerous protein structures were already analyzed using this technique (https://neicommons.nei.nih.gov/#/proteomeData).Protein databases contains structures of many small domains, which are building blocks of multi-domain proteins. This information could be used for the computational analysis of the domain protein stability. Here we assume that multi-domain protein is a combination of domains, each of these is an independent folding unit. The assumption about the domain independence could be in question if there is a wide-range interaction between neighbor domains within a chain. Indeed, recently it was shown that domain folding for some proteins might depend on inter-domain interface or linker length and flexibility22, each is composed of 47 EGF-like domains interrupted by 9 TB domains , Cadherin domains , and Sushi domain (PDB file: 2QFG). Two domains, Ig-like type and fibronectin type III domains, are independent folding units7.Two large proteins, FBN1 and FBN2, form fiber-like multi-domain structuresin vivo by ribosome. Protein biosysthesis starts from protein N-terminus by a mechanism known as co-translational folding25. During protein synthesis, the nascent peptide travels through the polypeptide exit tunnel of the ribosome, which has a length of about 100 Å and covers about 30–40 amino acids of the nascent peptide in a fully-extended conformation25. Smaller structures such as secondary structure elements can form within the tunnel. However, larger structural domains can be formed when the protein domain appears from the peptide exit tunnel of the ribosome26. Synthesis of whole multi-domain protein might require a significantly longer time . The domain will be relatively ‘isolated’ from other parts of the multi-domain protein and the perturbation caused by mutation could be analyzed computationally.https://neicommons.nei.nih.gov/#/proteome.The automation of the multidomain UMS reduces the need for human labor and has the potential for cloud-based applications. If the domains and unfolding mutation data are prepared in advance, stability information about a protein can be retrieved virtually instantaneously. The data produced in this work are freely available at In conclusion, the multidomain UMS pipeline developed in this work allows us to predict residues and regions in multidomain proteins that are critical for protein structure and function. In the future, a knowledge of critical residues will allow us to examine the relationship among mutations in multidomain proteins, effects on protein stability, and disease phenotypes.http://www.uniprot.org/) using the accession ID numbers and a python script. The domain sequence for each protein can be found in Fig. Protein domain, sequence, and mutation information for 9 multidomain proteins are shown in Table 29. A homology modeling experiment in YASARA uses the target protein sequence to identify possible structural templates by running 3 PSI-BLAST iterations to extract a position specific scoring matrix (PSSM) from UniRef90. The Protein Data Bank (PDB) is then searched for a match, and YASARA builds models for each matched template. For each template, if the alignment is unambiguous, a single model is built. If the alignment is ambiguous, several alternative models may be built. A ‘quality Z-score’ is calculated from the molecular dynamics force field energies, capturing the correctness of the backbone- and side-chain dihedrals as well as packing interactions. The models are ranked by their overall quality Z-scores, and the best parts of each model are combined to obtain a hybrid model, in hopes of synergistically increasing the accuracy.Each protein domain was generated by homology using the molecular graphics, modeling and simulation program YASARA15. The simulation aims to reach a water density of 0.997 g/l and will adjust the pressure accordingly to obtain the previously stated parameters. The simulation frames are saved every 250 ps. The domain structure is subjected to 2 ns of fast molecular dynamics (‘md_fast.mcr’) until the structure is determined to be feasible. A total of 291 domains were modeled. Supplementary Table The obtained 3D atomic structure for each domain was equilibrated using molecular dynamics in water using the standard macro ‘md_runfast’ in YASARA. The macro is optimized with the LINCS algorithm to run accurate molecular dynamics with a ‘fast’ speed setting at a 2 × 2.5 fs time step. Structures are placed in a cubic simulation cell extending 20 Å around the domain structure. The AMBER14 forcefield is used with a periodic cell boundary, and long-range electrostatics used a particle mesh Ewald algorithm with an 8.0 Å distance cutoff. The macro is preset to achieve pH 7.4 and 0.9% NaCl (153 mM) concentration at 25 °C. Sodium and chlorine ions are placed at the locations of the lowest and highest electrostatic potentials until the cell is neutralized. The location of the counter ions does not matter in practice, as they will randomly diffuse away through the simulationhttp://www.hgmd.cf.ac.uk/).Disease-related missense mutations were retrieved from the HGMD .The domains were then subjected to the UMS to generate unfolding propensities for all possible missense mutations, which were organized into a 2-dimensional matrix12; this parameter is used to describe the sensitivity of each location to missense mutations. Foldability is calculated for each alignment position to evaluate the frequency of severe mutations for each location through summation of all unfolding propensities greater than 0.9. The foldability scale ranges from 0 to 19, where a foldability of 0 represents an alignment position at which residues can be mutated without significant effects on structure, and 19 represents a residue that results in severe destabilization when mutated.Foldability is a parameter first described by McCafferty and Sergeev14 as the residues with the highest foldabilities. In our work, foldability is calculated from an aligned ensemble of residues. At each alignment position in the aligned FASTA file (AFASTA), a p-value is calculated using analysis of variance (ANOVA). This p-value quantifies the variability in the unfolding propensities of all residues averaged. Here, critical positions are described as the AFASTA alignment positions with the highest foldability values (>10) and p-values below 0.05. High foldability values identify positions that result in severe protein destabilization, while the accompanying p-value describes the variability at that position. AFASTA alignment positions that invariably result in high destabilization  are determined to be critical across all domains, creating a critical-residue framework that provides stability across all domains.Critical residues for each protein were first described by McCafferty and SergeevA python/bash script was developed to automate the processing of protein domain data. The code is available on request, and the method design is shown in Fig. 30, and the sequence alignment was output as an AFASTA file based on the structural superposition. To increase the accuracy of critical-residue identification, the unfolding propensities of the aligned residues of each domain were averaged for each domain set. At each AFASTA alignment position, a p-value was calculated from the distinct unfolding propensities of each residue averaged using ANOVA.The structures of the homologous domains of each protein were superimposed using UCSF Chimera31, a server that constructs alignments for multiple protein sequences. The alignment of each domain set provides an alignment consensus that identifies the aligned residues identical within each domain , as well as positions in the domain sequence where similar residues are conserved (similarly conserved residues). The averaging of domain structures removes the noise introduced by each structure and illuminates residues with consistent unfolding effects across domain structures.Homologous protein domains was aligned using PROMALS3DSupplemental Material