Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of  Mycobacterium tuberculosis (Mtb) can survive in vivo for prolonged periods of time (decades) in a clinically undetectable latent state called non-replicating persistence. Mtb is metabolically dormant in the latent state and is unresponsive to most antituberculosis drugs. Diverse stimuli, including hypoxia [ hypoxia , exposur hypoxia , nutrien hypoxia , and inc hypoxia  are thou hypoxia .in vivo growth and survival requires oxygen. However, mycobacteria also experience hypoxic conditions in vivo inside macrophages. Thus, Mtb growth is inhibited when the bacilli are engulfed into granulomas, the inside of which is associated with low oxygen levels [Mycobacteria bovis BCG and to result in dormancy [Early evidence linked hypoxia to dormancy. Wayne established that tuberculosis bacteria settle into a non-replicating, persistent state when grown under low oxygen tension or hypoxia and, more importantly, that the bacilli developed resistance to clinical drugs . Tubercun levels . Similardormancy .Mtb cultures grown in vitro under limited oxygen conditions demonstrated the enhanced expression of a 16 KDa α-crystallin homolog protein (Acr) [Mtb in macrophages [Genetic analysis combined with studies of in (Acr) . Acr is rophages . This prdev genes and the region representing the two-component system with the response regulator was named devR-devS [devR-devS loci, Rv3134c, encodes a well-conserved Ala-Val rich protein whose function remains unknown. However RT-PCR analysis of the RNA isolated from the Mtb cultures using the primers for the devR-devS locus suggests that the genes in the region are co-transcribed and that devS-devR-Rv3134c form an operon [In independent work, the gene products encoding a two-component system with a cognate response regulator were identified using extensive genetic analysis combined with subtractive hybridization ,10. Impon operon .Mtb is exposed to reduced oxygen tension [Mtb survival under these conditions [ Mtb to a lower metabolic activity under hypoxia. A similar hypoxic induction of devR-devS has been observed for BCG grown in an in vitro dormancy model [Rv3133c gene failed to regulate the expression of other proteins responsible for its survival under hypoxia, indicating that the DevR protein is essential for dormancy survival and regulation in BCG. Thus DevR was given the alternative name DosR that stands for DOrmancy Survival Regulator protein and the system the name DosS/DosR [dosS and dosT in Mtb yields bacteria that cannot upregulate the expression of dosR activated genes [Mtb dosR knockout show that it is not essential in vitro for entry, survival, or multiplication of Mtb in human monocytes [dosR system in virulence appears to be model dependent [In order to establish the hypoxia-specific expression of the Acr protein, Sherman and coworkers utilized whole-genome microarray analyses to examine the array of genes expressed when  tension . The Acr tension ,14,15. Inditions ,16. The cy model . ImportaosS/DosR . In the onocytes , althougependent ,21,22, bMtb and M. smegmatis reveals the presence in the latter of genes that correspond to dosS and dosR [Mtb and M. smegmatis. However, M. smegmatis does not have a gene that corresponds to dosT [M. smegmatisdosS is more closely related to MtbdosT than dosS [Comparison of the genome sequences of and dosR . The two to dosT ,25, althhan dosS . dosR regulon [194, has been expressed in E. coli and shown to undergo autophosphorylation at a conserved histidine (His392) and to transfer the phosphate group to DosR [2+ in addition to Mg2+ as the divalent ion for autophosphorylation. Phosphate transfer from DosT to DosR was slower and went to a lower extent than from DosS, with only partial transfer after 30 min compared to complete transfer by DosS in 5–10 min [dosT transcription, unlike that of dosS, is not upregulated during hypoxia. DosS and DosT are sensor kinases that autophosphorylate one of their own histidines and subsequently use the phosphorylated histidine to phosphorylate an aspartate residue of DosR, resulting in binding of DosR to DNA upstream of hypoxic response genes and thus activating the  regulon ,26. DosT to DosR . Unlike 5–10 min . These sbacilli under hypoxia, and the contradictory evidence concerning the role of DosS as a gas versus redox sensor.The full-length protein structures of DosS and DosT incorporate two N-terminal GAF domains, termed GAF-A and GAF-B, followed by a histidine kinase domain that includes the conserved histidine residue that is phosphorylated, and finally by an ATPase domain . Here, wdosS gene exhibits homology with histidine kinases and sequence analysis initially suggested that DosS might have three transmembrane domains [Early efforts to clone and express the full-length DosS protein were not successful due to difficulties in solubilizing the protein . The dos domains , a predi201, the truncated C-terminal histidine kinase domain of DosS consisting of 201 amino acids was first expressed by Saini et al. in E. coli as a His(6)-tagged protein that formed inclusion bodies [Dosn bodies ,29. The n bodies , establiMtb DosS were cloned by Sardiwal et al. [E. coli cells as a mixture of soluble and insoluble proteins, from which some soluble protein could be purified. UV-vis spectroscopic analysis of these truncated proteins provided the first evidence that heme is bound to the GAF-A domain [In a more extensive study, five poly-His tagged constructs of l et al. , includiA domain . E. coli in a pET23a + vector [d = 3.1 ± 2.0 μM. A similar expression system was used to produce a construct consisting of the GAF-A plus GAF-B domains, as well as full-length DosS [Subsequently, the N-terminal GAF-A domain of DosS consisting of residues 63 to 210 plus a six-His tag was expressed in + vector . To imprgth DosS ,31. Pyrigth DosS .et al. expressed full-length DosS and DosT, as well as the DosS GAF-A (residues 1–210) and DosT GAF-A (residues 1–208) domains in a pUC-19 vector [Sousa 9 vector . The exp9 vector . dosS and dosT genes in-frame with either a His-tag or an 11-kDa small ubiquitin related modifier (SUMO) tag in a pET15b vector [E. coli cells and the soluble fractions of the proteins, purified by nickel affinity chromatography, were confirmed by pyridine hemochromogen assays to bind heme with a 1:1 heme:protein stoichiometry [Steyn and coworkers cloned the full-length b vector . The conhiometry . Finally, the Kang laboratory in Korea expressed the DosS GAF-A domain from Asp63 to Arg210 with a poly-His tag separated from the desired protein by a TEV protease site. After expression and TEV cleavage, this yielded a purified protein with five additional residues (GAMDP) at the N-terminus . Mtb proteins, full-length M. smegmatis DosS has been expressed in E. coli and purified in its ferrous state [In addition to the work with the us state . Mtb DosS by mutating the individual histidines of the truncated GAF-A domain to alanines [His bond in which the histidine is neutral and not strongly hydrogen bonded [13CO. The crystal structure of the DosS GAF-A domain showed that the His149-iron Fe-N bond distance was 2.1–2.2 Å  and azide (Kd = 8.81 μM), but does not detectably bind large azole agents such as econazole and ketoconazole [UV-visible and resonance Raman studies of the truncated GAF-A domain and full-length protein showed that DosS reversibly binds Oconazole . Resonanconazole . Upon anconazole . The resconazole . Mtb DosT GAF-A domain has been determined at 2.3 Å resolution in both the deoxy- and oxygen-bound states  have been reported, but so far do not clarify the role of GAF-B in the full-length protein [The crystal coordinates of the  protein . The cryM. smegmatis DosS was shown to not bind cGMP or cAMP, in accord with similar findings for the Mtb GAF-B domain [The GAF-B domain of B domain , and cauThe crystal structures of the kinase cores of both DosS  and DosT  provide information on the structure of the kinase domain and identify residues that are important for ATP binding, phosphorylation and communication with the N-terminal signaling core of the protein . The kinCrystal structures of other known histidine kinases have conserved N, F and G box elements essential for ATP binding and a long characteristic loop called the ATP lid motif in their ABDs that covers the nucleotide and protects it from unwanted reactions ,41. InteThe crystal structure of the kinase domain of DosS determined at 1.8 Å resolution revealed the presence of two identical ABDs, each of them containing 3 α helices and 5 stranded β-sheets that together form a sandwich fold . A short201, which lacks the GAF-A and GAF-B domains, was found to have autophosphorylation activity and site-specific mutagenesis identified His395 as the DosS autophosphorylation site [2, results in inhibition of this activity. It is also relevant that the M. smegmatis H150A DosS protein, which binds no heme, lacked autokinase activity, but a construct consisting of the GAF-B and kinase domains was active under all conditions—i.e., the protein without the heme domain was active but no longer responsive to gas binding [An early attempt to express full-length DosS and DosT yielded the proteins as inclusion bodies that, after solubilization, purification, and refolding, afforded proteins with autophosphorylation activity. It is virtually certain that these proteins lacked the heme prosthetic group due to the solubilization and refolding steps , which iion site ,29. Thes binding .2 bound, confirms this inference [versus O2 to the heme. Thus, whereas two populations of CO and NO complexes are observed with the isolated GAF-A domain, only one conformer is significantly present in the GAF-A/GAF-B or full-length proteins. The binding of O2, however, is essentially the same in all the constructs and is consistent with preservation of a hydrogen-bonded network that stabilizes the O2 in a specific conformation [Resonance Raman comparison of full-length DosS and its truncated GAF-A domain indicates that two conformers exist of the Fe-CO complex in the truncated protein, whereas only a single conformer is detected for the full-length protein. This finding indicates that interactions between GAF-A and the other two domains in full-length DosS alter the GAF-A heme environment ,38. A monference . Fusion ormation .1/2 values of 16.5, 26.0, and 73.6 h, respectively [The rate of autooxidation of the DosS ferrous dioxygen complex decreases in going from the isolated GAF-A domain, to the GAF-A/GAF-B fusion, and finally to the full-length protein, with tectively . Interaconk increase in the order GAFA (0.18 ± 0.007 μM−1·s−1) < GAF-A/GAF-B (0.26 ± 0.024 μM−1·s−1) < full-length DosS (0.31 ± 0.031 μM−1·s−1) [offk values are virtually unchanged (2.57–2.74 s−1) for the three proteins  . In contproteins . In vitro, the endogenous Mtb ferredoxin reductase/ferredoxin pair encoded by the genes Rv0688 and Rv0763c rapidly reduces full-length ferric DosS  to the ferrous state [ΔdosTversusΔdosS knockout strains indicated that fdxA was induced 82-fold in a dosS-specific manner [us state . Howeverus state ,42 and ac manner . FdxA madosR regulon is induced by ascorbic acid under both hypoxic and normoxic conditions [Mtb and must be reduced to the ferrous state before it can respond to gaseous stimuli. As mentioned, the nditions ,44. One nditions . In thisnditions . A smallin vitro. Ascorbate can reduce DosS in vitro, although ascorbate reduces the DosS ferric-NO complex to the ferrous-NO complex more rapidly than it does the unliganded ferric protein [dosS-dependent manner, but ascorbate probably acts in this instance by reducing cytochrome c and thus increasing the availability of reducing equivalents [In vitro, flavin nucleotides in the presence of dithionite accelerate the reduction of ferric DosS compared to the reduction mediated by dithionite alone, but NADH does not directly reduce the protein [Non-enzymatic agents have been used to directly reduce DosS  protein . As noteivalents . In vitr protein .In this view, the ON state of the protein is characterized by the absence of a strong hydrogen bonding interaction of the distal ligand with Tyr171. This may allow the tyrosine to partner with other hydrogen bond donors in the distal pocket, which directly or through a conformational change may be responsible for communication with the phosphorylation domain. For example, the tyrosine was shown in the crystal structure of the DosS GAFA domain to be hydrogen-bonded to Glu87, which in turn is hydrogen-bonded to His89 . Alterna2-bound state.The activity of the ligand-free Fe(II)-deoxy state may arise from the release of steric or hydrogen-bonding constraints in the distal pocket. The hydrogen-bonding interactions of the tyrosine to other distal residues, as seen in the CO and NO bound forms, are clearly not critical for activity, as the Y171F mutant that has no hydroxyl group retains its activity in the deoxy form. It is likely that in the deoxy state, the protein is able to sample both active and inactive conformations, giving rise to an autophosphorylation activity that is intermediate between the high activities of the CO or NO-bound states and that of the inactive ORecent crystal structures of the DosS and DosT kinase domains  reveal ain vivo, although it doesn’t seem to perturb the protein-protein interaction between DosR and DosS [On the response regulator side, the Asp54 that is phosphorylated in DosR to initiate the signaling cascade was shown to be involved in a protein-protein interaction with the DosS to complete the phosphotransfer reaction. Accordingly, a D54A mutant suppressed interaction of the DosR and DosS proteins, as revealed by a pull down assay. Furthermore, a conserved residue, Lys104, is expected to sense the phosphorylation of DosR and the conformational changes associated with it by forming a hydrogen bond with the phosphorylated Asp54. Mutation of Lys104 to alanine results in no phosphorylation of DosR by DosS and renders it functionally inactive and DosS .Mtb dosR regulon were examined by comparative microarray analysis of ΔdosS, ΔdosT, and the double mutant in the Wayne anaerobic model [dosR regulon. DosS is part of that regulon, so it is itself induced, eventually to a level as much as 100-fold greater than that of DosT. DosS then becomes the primary receptor that maintains the induction of the dosR regulon. Both DosT and DosS are required for full induction and survival of Mtb under hypoxia [dosR knockout showed that DosR is required for survival of Mtb under hypoxic conditions and for its transition from hypoxic to normoxic metabolism [The relative roles of DosS and DosT in inducing the ic model . The res hypoxia . A dosR tabolism . M. smegmatis has a DosS but not a DosT sensor, its DosS is closer in sequence to Mtb DosT than DosS [Mtb DosT, the smegmatis DosS is purified in the ferrous state that binds oxygen and is resistant to autooxidation [MtbdosT, but not dosS, was found to substitute functionally for dosS in an M. smegmatisdosS knockout [dosR regulon is induced mostly during the early phase of the transition from aerobic to hypoxic conditions, consistent with the fact that DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS  [2. The ferrous dioxy form of DosT has been found to be stable in the hands of all the investigators who have purified and characterized this protein. Its rate of autooxidation is very low (< 0.01 h−1) . It is tIn contrast, there is disagreement about the stability of the ferrous dioxygen form of DosS, and consequently about whether it functions as a gas or redox sensor. Steyn and coworkers reported that DosS was purified from their expression system as the ferric protein. This protein could be reduced anaerobically to the ferrous state, but on exposure to oxygen reverted to the ferric state within seconds . Formatiet al., in preparing the DosS GAF-A domain for crystallographic studies, similarly found that in their hands exposure of the ferrous protein to oxygen yielded the ferric protein with little evidence for the ferrous dioxy complex [Cho  complex . Restric complex . Convers−1 and half-lives greater than 24 h in the presence of K+, Na+, Mg2+, or Ca2+  [obs = 36 and 1.2 s−1 at pH 7.5 with 2 μM NO. Ferric DosS does not have autokinase activity but binds NO with high affinity (Kd ~ 5 μM) to give the ferric-NO complex that is readily reduced by ascorbate to the active ferrous-NO complex. Thus, exposure to low levels of NO can convert the inactive DosS ferrous-oxygen complex to the active ferrous-NO complex by a sequence involving oxidation followed by reduction in the reducing environment of Mtb cells [The ferrous dioxygen complexes of DosS and DosT have little kinase activity, but autokinase activity is observed with the ferrous-NO complexes. Stopped flow UV-vis and freeze-quench resonance Raman have been used to show that the ferrous dioxygen complex reacts with NO in a reaction that oxidizes the heme group to the ferric state with concomitant production of nitrate (NO3−) . The reatb cells . 2, CO, and NO from the truncated GAF-A domains of DosS and DosT indicates that the heme pocket in both proteins is relatively closed [2 escapes from the DosT heme pocket on the picosecond time scale. This value, which is similar to that for other hemoprotein oxygen sensors, shows that it is an effective oxygen trap. In contrast, 18% of the oxygen escapes under similar conditions from the DosS GAF-A domain. The heme environment in DosT thus prevents oxygen escape to a much greater extent than that of DosS. Distal Tyr169 in DosT plays a role in constraining the oxygen, as its mutation to a phenylalanine results in a much higher (19%) oxygen escape. In contrast, the fraction of CO that rebinds in the heme pocket is higher for DosS than for DosT. NO rebinding is similar for both proteins, but is substantially slower for the ferric than ferrous states. These differential effects involved the distal tyrosine, but not exclusively, as mutation of other nearby residues in some instances also affected dissociation and rebinding of ligands. The data are consistent with a weaker hydrogen-bond between Tyr171 and the dioxygen ligand in DosS than that from Tyr169 in DosT [Ultrafast time-resolved absorption spectroscopic analysis of the dissociation of Oy closed . Upon ph in DosT . The resMycobacterium smegmatis displayed significant autokinase activity in presence of the ubiquinone/ubiquinol pool, indicating that the redox state of the quinone/quinol did not affect the kinase activity [in vivo studies in which mutations to cytochrome bc1-aa3, a critical component in an electron transport pathway involving the menaquinone pool, led to inhibition of the growth of the M. smegmatis cultured under aerobic conditions, but failed to induce the DosR regulon [Finally, the purified DosS from activity . This wa regulon . These rPhosphorylation of DosR by DosS is required for activation of the regulon. The phosphorylation involves formation of a complex of DosS with DosR, as shown by experiments in which subunits of dihydrofolate reductase were attached to these two proteins and reconstitution of dihydrofolate reductase activity was used as a marker of complex formation .dosR/dosS in M. smegmatis under aerobic conditions showed that the DosR protein binds to a conserved DNA motif located upstream of the acr gene of Mtb [dosRS operon, the sites of binding of DosR to DNA, and the need for recruitment of a second molecule of phosphorylated DosR for gene activation has been reported [A study of the regulation of e of Mtb . Evidencreported . Phosphoreported .2+ ion [dosRMtb strain fails to induce the dosR regulon. Thr82 thus appears to be essential despite is somewhat anomalous topological position [DosR, a member of the NarL subfamily of response regulators, has an N-terminal domain that includes Asp54, the phosphorylation site, linked via a connecting sequence to a C-terminal DNA-binding domain. Asp54 was identified as the DosR phosphorylation site by mutagenesis . Mutagen2+ ion . The equposition .A comparison of the truncated C-terminal DNA-binding domain of DosR with the binding of the full-length protein shows that the isolated C-terminal domain can bind to DNA at the same high affinity target promoter site . One rolMtb DosR and of its C-terminal domain at 2.2 Å and 1.7 Å resolution, respectively, have suggested that a helix rearrangement following phosphorylation may be responsible for DosR activation [The crystal structures of the full-length, unphosphorylated tivation . Analysitivation . The C-ttivation .Mtb to drugs, forcing it to remain in the active state may constitute an approach for more effective suppression of the persistent, latent state of the mycobacteria. In principle, inhibition of the DosS-DosT/DosR sensor system would hinder entrance into the latent state and might result in reversion from the latent to the active state. Given the much higher sensitivity of the active form of Although little work has yet been done in this direction, a homology-based model of DosR was used to identify a phenylcoumarin derivative  that inh2-bound state and the ON NO- or CO-bound states therefore remain unknown. Furthermore, although crystal structures are also available of the truncated GAF-B and kinase domains of DosS, no structure exists of the entire protein. A consequence of these gaps in our knowledge is that the mechanism by which the protein discriminates between the ligands and transmits the binding of activating ligands to the kinase domain remains obscure. The second question derives from the contradictory findings on the stability of the ferrous DosS oxygen complex. Detailed studies of the DosS and DosT GAF-A domains have shown that the ferrous dioxygen complex is not only isolable, but in fact quite stable. The stabilities of the ferrous dioxygen complexes are even greater in the full-length protein, with half-lives in the order of tens of hours. However, in some laboratories, similar constructs have been reported to undergo almost instantaneous autooxidation of the ferrous to the ferric state on exposure to oxygen. Although detailed kinetics for these autooxidation reactions have not been reported, it is clear that critical differences exist in the proteins and the conditions used for their study in different laboratories. The ambiguities introduced by these differences have raised the possibility that DosS is primarily a redox state sensor, unlike DosT, which is generally agreed to be a gas sensor. It is clear that DosS, at least some of the time, acts as a gas sensor, as it functions normally in the ferrous state and when either CO or NO is the bound ligand. If DosS were a redox sensor, one would expect that the ability of the mycobacteria to reduce the ferric to the ferrous state would be a critical determinant of the response, a possibility that requires further exploration. However, in view of the demonstrated aerobic stability of the DosS oxygen complex in at least some hands, it is simplest to view both DosT and DosS as gas sensors until more conclusive evidence for an alternative role for DosS is forthcoming. DosS and DosT are autophosphorylating heme-dependent sensors that, together with the DNA-binding protein DosR, control the expression of a family of genes that are essential for entry into and survival in the latent, dormant state. The DosS and DosT proteins, under conditions that cause their activation, autophosphorylate a conserved histidine residue and then transfer it to DosR. Phosphorylated DosR then binds to DNA regulatory elements to alter gene expression. Two major questions remain concerning the structures and mechanisms of DosS and DosR. First, although crystal structures exist of the isolated DosS/DosT GAF-A domains in the ferrous, ferric, and oxygen bound states, no structures are available of the NO or CO ligated forms. Subtle differences that may exist in the heme domain between the OFF O


Trypanosoma brucei is a protist parasite and the causative agent of Human African Trypanosomiasis (sleeping sickness). At the base of its single flagellum is a bulb-like structure called the flagellar pocket (FP). The FP is the site of all endo-/exocytosis and thus essential for the survival of the parasite. At the neck of the FP is an electron-dense cytoskeletal structure termed the flagellar pocket collar (FPC), which currently has only one known protein component, BILBO1. Bioinformatic analysis indicates that there are four structural domains in the 67-kDa protein, including a globular N-terminal domain, two central EF-hand motifs followed by a long coiled-coil domain, and a C-terminal leucine zipper. T. brucei BILBO1 (TbBILBO1) by itself forms insoluble oligomers in vitro, which makes it intractable to any single conventional structural study method. We recently carried out structural dissection of TbBILBO1 using integrative structural biology approaches including NMR, crystallography, EM, and various biophysical methods. The high-resolution structure of its N-terminal domain reveals a variant ubiquitin-like fold with a conserved surface patch; mutagenesis of this patch causes cell death in vivo. We further found that the EF-hand motifs change their conformation upon calcium binding, the coiled-coil domain forms an antiparallel dimer, and intermolecular interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally shown to condense into fibrous bundles through lateral interactions as demonstrated by our EM studies. Based on all these experimental data, we propose a mechanism for TbBILBO1 assembly into the flagellar pocket collar.


Many economically important cattle diseases spread between herds through livestock movements. Traditionally, most transmission models have assumed that all purchased cattle carry the same risk of generating outbreaks in the destination herd. Using data on bovine viral diarrhoea virus (BVDV) in Scotland as a case example, this study provides empirical and theoretical evidence that the risk of disease transmission varies substantially based on the animal and herd demographic characteristics at the time of purchase. Multivariable logistic regression analysis revealed that purchasing pregnant heifers and open cows sold with a calf at foot were associated with an increased risk of beef herds being seropositive for BVDV. Based on the results from a dynamic within-herd simulation model, these findings may be partly explained by the age-related probability of animals being persistently infected with BVDV as well as the herd demographic structure at the time of animal introductions. There was also evidence that an epidemiologically important network statistic, “betweenness centrality” , was significantly higher for herds that supplied these particular types of replacement beef cattle. The trends for dairy herds were not as clear, although there was some evidence that open heifers and open lactating cows were associated with an increased risk of BVDV. Overall, these findings have important implications for developing simulation models that more accurately reflect the industry-level transmission dynamics of infectious cattle diseases. Infectious diseases cause significant financial losses for the cattle industry through their detrimental effects on animal health and performance . As suchFrom a practical perspective, these findings must be interpreted with some caution as most models assume that purchased cattle all carry the same risk of generating disease outbreaks in the destination herd. As numerous empirical studies have shown, the probability of any individual animal being infected or transmitting disease to susceptible cattle is strongly influenced by factors such as age, production type, and on-farm management practices -16. For BVDV is an economically important pathogen for the cattle industry due to its negative effects on herd reproduction and calf performance -21. DuriGiven the importance of BVDV, many statistical and epidemiological models have been developed to identify risk factors for BVDV transmission and opportunities for controlling disease more cost-effectively through targeted interventions ,13,30-32Our study uses three independent analytical approaches to determine the risk associated with particular types of purchased replacement breeding cattle. First, a traditional risk factor analysis is performed using serological data from beef and dairy herds that were surveyed as part of national seroprevalence studies in Scotland from 2006 to 2008 ,42. SecoThe Cattle Tracing System (CTS) database contains virtually complete records of the births, deaths, and movements of individual cattle in Great Britain since 2001. Each movement record includes basic information on the animal identification number, departure location, destination location, and movement date that may be linked with other recorded animal demographic information in the CTS database  to infer the animal’s production purpose at the time of movement. These records may also be used to generate an inventory of individual cattle present on a given farm at any given time point. This analysis focused on the movements of replacement breeding cattle since these are a well-established risk factor for BVDV transmission . Data frAn animal was classified as a purchased replacement breeding heifer (1) if the sex was registered as female, (2) if there were no recorded calvings prior to the movement, (3) if the animal was born on a different location than the destination farm, and (4) if the next recorded calving after the movement took place on the destination farm or, if there were no recorded calvings after the movement, that the animal survived beyond 30 months of age. It was assumed that animals intended for human consumption rather than breeding would be slaughtered by 30 months of age to comply with bovine spongiform encephalopathy (BSE) regulations in the United Kingdom . An animThe replacement breeding cattle groups were further subdivided by pregnancy and lactation status to generate six total production groups: open heifers, pregnant heifers, open dry cows, open lactating cows, pregnant dry cows, and pregnant lactating cows. Animals that were fewer than 280 days from calving at the time of movement were considered pregnant, while animals that were greater than or equal to 280 days from calving were considered open. Beef breed cows that were moved onto the farm at the same time as their calf were considered to be in lactation with a calf at foot, while those moved without a calf were considered to be dry cows. Dairy breed cows that were moved onto the farm within 305 days of the previous calving date were also considered to be in lactation, while those moved greater than 305 days post-calving were considered to be dry cows.A survey of 301 randomly selected beef herds was performed in Scotland between October 2006 and September 2007 to estimate the national herd-level prevalence of BVDV . During A survey of 374 dairy herds was also performed in Scotland between October 2007 and May 2008 to estimate the prevalence of antibodies to BVDV in bulk tank milk samples . The bulThe questionnaire returned by the surveyed beef and dairy farmers identified farms through the main postal address and so to link the serological results with records from the CTS database, attempts were made to match the farm address against a database of CPH codes provided by the Scottish government. Farms for which there was no available CPH code and farms for which there was an obvious discrepancy between the questionnaire estimates of herd size and CTS database estimates of herd size were excluded from the analysis. These discrepancies were most likely due to cattle being housed on a different location than the main farm address recorded in the survey and resulted in the loss of 46 beef herds  and 31 dairy herds (14% of the 220 non-vaccinating herds). The final sample therefore contained 255 beef herds (67 case herds and 188 control herds) and 189 dairy herds (122 case herds and 67 control herds).Data from the remaining 255 beef herds and 189 dairy herds were used to explore the relative risk of purchased replacement breeding cattle causing outbreaks in the destination herd. For each herd, the total number of cattle purchased in the two year period prior to serological sampling was recorded. The two year time window was selected because of the uncertainty in when BVDV may have been introduced to the herd. A series of six binary categorical variables were created representing each of replacement breeding cattle types , open cow (lactating), pregnant cow (dry) and pregnant cow (lactating)). The levels of the variables were “None purchased” and “At least one purchased”. Herds that purchased no cattle in the 2 year period prior to sampling were considered closed herds. The odds of a closed herd being seropositive for BVDV were initially calculated and the remaining analyses then focused on the subset of 233 open beef herds and 150 open dairy herds.p-value < 0.20 and were therefore retained. Thereafter, components of the final multivariate models were determined by a backwards stepwise elimination process in which variables with the highest p-values were sequentially dropped from the model until all the remaining variables had a p-value < 0.05. Forwards stepwise selection was then performed adding in each of the eliminated variables in turn and checking for p-values of < 0.05 to ensure that no variables were excluded based on the order of elimination.Preliminary univariate screens were performed to select variables for inclusion in the final multivariate logistic regression models. All six movement variables were associated with BVDV seropositivity at a A stochastic individual-based within-herd simulation model was then developed to determine whether the general trends in the empirical risk associated with different types of replacement breeding cattle movements could be explained by the combination of (1) the theoretical risk of the animal being infected with BVDV or carrying an infected calf at the time of purchase, which is a function of the animal’s age and pregnancy status as well as the expected disease prevalence and (2) the theoretical risk of disease subsequently spreading to at least one susceptible dam during the gestational risk period for generating PI calves, which is a function of the herd demographic structure at the time of purchase as well as the ability for disease to spread within and between management subgroups.This analysis focused on the subset of 2895 beef herds and 546 dairy herds in Scotland with exclusively beef or dairy calvings (1) that housed cattle continuously over the period from July 2004 through June 2007 to ensure that sufficient post-hoc data was available to classify animals into production groups, (2) that purchased at least one replacement breeding animal in the period from July 2004 through June 2005 since the focus of the analysis was on BVDV introductions through replacement breeding cattle movements, and (3) that had at least 20 recorded calvings per year to rule out hobby farms and farms that ceased production during the study period. The within-herd model included a demographic component to capture the typical management subgroups of Scottish beef and dairy herds, a seeding component to describe the introduction of BVDV through one of the purchased replacement breeding cattle, and a disease component to describe the subsequent transmission of BVDV within and between management subgroups after the animal was introduced. The distribution of animals across the different management subgroups within each herd and the movements on and off the farm were modelled directly from CTS records to account for real world heterogeneity in herd demographic structure. The simulation model was implemented in the C programming language.Records for all cattle present in the study herds from 01 July 2004 through 01 July 2007 were extracted from the CTS database. Each animal was initialized as a virtual object that carried information on its age, production subgroup, pregnancy status, and disease status at any given time point. Based on expert opinion from farmers and veterinarians on the typical management structure of Scottish beef and dairy herds, animals were assigned into one of the production subgroups shown in Figure In beef herds, all calves born in the herd remained with their dams until a fixed weaning age of 213 days (7 months). At weaning, male calves were transferred into the “other” group and female calves were transferred into the “young heifer” group. At the minimum age at first breeding for the farm, heifers that subsequently delivered a calf or survived beyond 30 months of age were transferred into the “breeding heifer” group. All other heifers were assumed to be intended for fattening and transferred into the “other” group. The minimum age at first breeding was calculated by taking the minimum age at first calving for heifers over the three year study period and subtracting the average gestation length of 280 days. Heifers remained in the “breeding heifer” group until calving or until reaching 48 months of age. Given limitations in the CTS data, it was not possible to further separate animals into spring and fall calving units on farms with year-round calving patterns or to identify exposure to male cattle kept or purchased as breeding bulls.In dairy herds, calves were removed from their dams immediately at birth. All male calves and crossbreed calves  were transferred into the “other” group. Female calves remained in the “calf” group until a fixed age of 183 days (6 months) and were then transferred into the ‘young heifer’ group until the minimum age at first breeding for the farm. Heifers remained in the “breeding heifer” group until calving or until reaching 48 months of age. After calving, the dams were transferred to the “lactating cow” subgroup until 42 days prior to the next calving, reflecting the average dry period for dairy cattle, or until more than 365 days into lactation if the animal failed to conceive. Animals remained in the “dry cow” subgroup until the next recorded calving date or movement off the farm. An average gestation length of 280 days was again assumed.On each day of the simulation, the herd demographic structure was updated in four steps: (1) animals within the herd were transitioned between production subgroups as appropriate, (2) animals were removed from the herd based on an event list of deaths and off-movements, (3) the pregnancy status of animals was updated based on an event list of breeding dates derived by subtracting 280 days from the next recorded calving date, and (4) animals were added to the herd based on an event list of births and on-movements.Each disease simulation-run for an individual herd began on 01 July 2004. All animals in the herd were initially assumed to be susceptible to BVDV to represent a true outbreak scenario. In each simulation run, only one of the replacement breeding cattle movements occurring from 01 July 2004 through 30 June 2005 was selected as the potential disease “seeding event” and all other cattle moved onto the farm before or after this movement were assumed to be susceptible to BVDV . Since it was not possible to determine the true BVDV status of the source herds at the time of movement, it was assumed that all source herds were potentially infected with BVDV. The objective was not to make inferences about the true within-herd and between-herd dynamics of BVDV outbreaks, but rather to provide a simple framework for comparing the relative risk associated with the different types of replacement breeding cattle movements and the demographic structure of the herds at the time when replacement breeding cattle were introduced. Over the one year period from 01 July 2004 through 30 June 2005, the beef study herds collectively purchased 44 485 replacement breeding cattle and the dairy study herds collectively purchased 10 023 replacement breeding cattle.pi) based on its age (Equation 0) in infected herds was 3% and the probability of being PI decayed as an exponential function of age with a half-life (t1/2) of 365 days to reflect the decreased survival rate of PI calves [The infection status of each replacement breeding animal was determined stochastically at the start of each simulation according to the decision tree process presented in Figure Equation . It was I calves -48.Figur0 is equal to the average prevalence of PI cattle at birth, Age is equal to the animal’s age at the time of purchase in days, and t1/2 is equal to the half-life of PI cattle.Where ρAgesero) sampled from an exponential distribution with a half-life of 1095 days . The outcome measure for each individual simulation run was a binary response variable of whether or not the purchased animal caused at least one additional dam in the herd to generate a PI calf over the two year period as this is an important prerequisite for disease persisting within the cattle herd . The resinf) on any given day of the simulation.The model for the within-herd transmission dynamics of BVDV following disease introduction was adapted directly from work by Ezanno et al. . DiseaseX denotes one of the two the disease states PI, TI, then Xg(t) is the number of animals in infectious state X within the same production subgroup, g, at time (t), Xa(t) is the number of animals in infectious state X in all other production subgroups at time (t), β1 is the transmission rate from PI animals within the same production subgroup, β2the transmission rate from TI animals within the same production subgroup, β3the transmission rate from PI animals in all other production subgroups, Ng is equal to the total number of animals in the same production subgroup, and Na is equal to the total number of animals in all other production subgroups.Where, if Within each production subgroup, contact with both persistently infected (PI) and transiently infected (TI) animals was assumed to lead to possible virus transmission, while between production subgroups, only PI cattle were assumed to be able to transmit virus due to their much higher viral excretion rates. The probability of an individual animal acquiring BVDV Equation  was updaγ) before seroconverting and moving to the recovered (R) state. Immunity to BVD was assumed to be lifelong. For S dams infected during early gestation (days 0 to 42), there was a probability αe of embryonic loss or abortion. For S dams infected during mid-gestation (days 43 to 150), there was a probability αm of abortion and if the calf survived, a probability θPI of being PI, a probability θR of being R, and a probability θM being born protected by maternal antibodies (M). Temporary immunity to BVD through maternal antibodies was assumed to last for 183 days (ω) after which the animal joined the S group. All dams infected during early gestation (days 1 to 42) and late gestation (days 151 to 280) gave birth to M calves, all PI dams gave birth to PI calves, and all S dams gave birth to S calves. Given the short duration of the model simulation and the simple outcome measure, we assumed no increase in mortality amongst PI calves.All TI cattle remained infectious for a period of 10 days  with a binomial distribution. Data from the beef herds were analysed separately from the dairy herds due to inherent differences in management practices. The response variable was the counts of successes and failures for each seeding event and the predictor variable was the seeding event type . Herd was included as a random effect to account for covariance between observations on the same farm. The results from the regression model were reported as odds ratios (ORs) with the corresponding 95% confidence intervals (CIs).Another possible explanation for the differential risk associated with the different types of replacement breeding cattle is that the source herds for these movements are also not epidemiologically equal. As there was no information on the disease status of the source herds at the time of our study, we used a simple measure from network analysis, between centrality, as an approximation of the likelihood of the source herds being infected with BVDV. Betweenness centrality measures the number of times the shortest paths between any two farms in the network pass through a particular farm . In prevIn our study, the individual movements of cattle in Great Britain from July 2005 through June 2006 were selected as a representative year and used to reconstruct the static movement network. For the purpose of this analysis, a farm was defined as any location classified as an agricultural holding or landless keeper (farmers housing cattle on rented land) with a Scottish county designation that housed cattle for at least one day during the study time period. For each movement, the departure location, destination location, and date were recorded. Movements that occurred through livestock markets were treated as direct movement between farms by pairing the movement onto the market location with the corresponding movement off the market location. The movements of replacement breeding cattle were identified and classified into the six production groups as previously described. The betweenness centrality scores for each source herd that supplied replacement breeding cattle to a Scottish beef or dairy herd were calculated using the igraph package for the C programming language . For eacp = 0.041 for beef herds and OR: 0.38, 95% CI: 0.18 – 0.78, p = 0.008 for dairy herds). Amongst the open herds, beef herds that purchased replacement breeding cattle had a 2.09 times greater odds of being seropositive for BVD  compared with herds that purchased store cattle only. Similarly, open dairy herds that purchased replacement breeding cattle were 2.67 times more to be seropositive for BVDV  than open herds that purchased store cattle only.Only 22 of the 255 surveyed beef herds (9%) and 39 of the 189 surveyed dairy herds (21%) remained completely closed to cattle movements in the two year period prior to serological testing. This practice was associated with a significantly decreased odds of being seropositive for BVDV  and 2.09 times greater with the purchase of open cows with a calf at foot . For open dairy herds, the odds of being seropositive for BVDV increased with the purchase of open heifers, open dry cows, and open lactating cows in the univariable analysis (Table p = 0.004).Results from the empirical risk factor analysis predicted that the risk of a herd being seropositive for BVDV varied based on the types of replacement breeding cattle purchased during the two year period prior to serological testing. Open beef herds that purchased pregnant heifers, open cows with a calf at foot and pregnant dry cows were at significantly increased risk of being seropositive for BVDV in the univariable analyses Table . In the p < 0.001) of generating additional PI calves and the movements of open beef cows with a calf at foot had 1.23 times greater odds  despite both groups having a lower probability of being PI cattle based on their age at the time of movement. For dairy herds, the movements of open heifers had the greatest odds of generating additional PI calves compared with all other animal production types. The movements of open dry cows had the lowest risk compared to the movements of open heifers for both beef and dairy herds, although these movements were relatively infrequent.Results from the within-herd simulation models are presented in Table There were a total of 4934 Scottish beef herds and 804 Scottish dairy herds that sold replacement breeding cattle from 01 July 2005 through 30 June 2006. The median source herd betweenness centrality scores for herds that supplied each type of replacement breeding cattle is shown in Table The predicted risks associated with the different types of replacement breeding cattle movements across the three study analyses are summarized in Table Similar to other empirical risk factor analyses ,32,58, tThe within-herd simulation model used published estimates from the literature to determine the age-related probability of purchased cattle being persistently infected, transiently infected, or immune from previous exposure to BVDV. By using movement records from the CTS database to characterize herd demographic structures, the models also explicitly accounted for the probability of purchased cattle being introduced to the herd when susceptible dams were in the gestational risk period for generating additional PI calves. To our knowledge, only one other published study to date has used CTS records in this manner  and thisPregnant replacement breeding cattle are a known epidemiological control challenge for beef herds since there are few reliable prenatal diagnostic tests to identify animals carrying a PI calf -40 and oIn contrast, the purchase of open heifers and open breeding cattle in general was a significant risk factor for seropositivity in dairy herds, but not for beef herds. One possible explanation is that beef heifers were purchased at a much younger age than dairy heifers and under certain circumstances, it has been shown that exposing susceptible heifers to PI cattle can be beneficial in inducing protective immunity to prevent future transient BVDV infections during early gestation . Most opIn this interpretation, we also assumed that replacement breeding cattle movements were a cause of BVDV infections rather than a consequence. Herds that are actively infected with BVDV may need to purchase additional replacement breeding animals to compensate for the negative effects on fertility, reproductive performance, and mortality  -64. As hper se that matters, but could be the connectedness of herds to others that tend to transfer that particular type of animal. A recent study from Sweden found that herds with large ingoing infection chains (another measure which accounts for both the number and connectivity of source herds for purchased cattle) were significantly more likely to be seropositive for bovine coronavirus and bovine respiratory syncytial virus than more isolated herds [There was also evidence that our findings may be related to the differing epidemiological risk associated with herds that sell particular types of replacement breeding cattle. Herds that sold pregnant replacement beef heifers and open beef cows with a calf at foot had higher median betweenness centrality scores than herds that sold other types of cattle, which may indicate an increased likelihood that they were infected with BVDV. In short it is possible that it is not the type of replacement animal ed herds . With thed herds . We alsoAny test which seeks to detect an antibody that arises from historic infection, present infection, vaccination and introduction of an antibody positive animal is likely to be an imperfect test. This applies to the testing for BVDV antibodies in bulk milk and interpretation of the test is made more confused by the fact that currently most dairy herds in Scotland buy in replacement heifers. Such factors probably explain our observation that the trends in movement and network risks associated with purchased replacement breeding cattle were less clear for dairy herds than for beef herds ,69. AlthThere were also limitations in how we used the CTS data to define animal production types and epidemiologically relevant cattle movements. For example, it was not possible to distinguish heifers that were purchased as store cattle for fattening from those that were purchased as replacement breeding animals, but culled prior to calving. This may have underestimated the number of herds that purchased replacement breeding cattle as well as the potential risk associated with purchasing open heifers. Additional bias may have been introduced to the empirical risk factor analysis through the exclusion of farms that could be linked to a valid CPH code through their reported address. However, we believe this is unlikely to have impacted the main study findings since there is no obvious reason why the unmatched farms would have a higher prevalence of BVDV. This highlights the importance of recording accurate farm identification information in future epidemiological studies.The primary motivation behind this study was to emphasize that not all cattle movements and not all cattle herds are epidemiologically equal. Although BVDV was used as a case example, the basic principles and methodologies are equally relevant to the many other infectious cattle diseases that spread through cattle movement networks, such as bovine tuberculosis, bovine paratuberculosis, bovine herpesvirus 1, bovine leukaemia virus, and contagious mastitis. Most importantly, this analysis demonstrates how the basic records of individual births, deaths, and movements available through national cattle movement databases can be used in future research studies to better quantify disease specific risk factors for infectious diseases and to determine the relative importance of high risk movements to disease transmission dynamics at the population level.


To identify the thermal transitions on the DSC profiles , we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.Myosin head  consists of two major structural domains, the motor  domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF The molecular mechanism of muscle contraction and many other events of biological motility is based on cyclic interaction of myosin heads with actin filaments coupled to myosin–catalyzed ATP hydrolysis. The myosin head which is referred to as myosin subfragment 1 (S1) consists of two major structural domains, the N-terminal globular motor  domain and the C-terminal regulatory domain. The motor domain is responsible for ATP hydrolysis and actin binding. The regulatory domain is a long α-helix stabilized by non-covalent interactions with the essential and the regulatory light chains  .The present concept of the myosin motor function includes rotation of the regulatory domain relative to the motor domain. During this rotation the regulatory domain acts as a semi-rigid “lever arm”, which amplifies and transmits conformational changes occurring in the motor domain during ATP hydrolysis. Local conformational changes in the myosin ATPase site spread to the entire motor domain, resulting in global structural changes in the motor domain and leading to rotation of the converter by ~60° , 4. The in vitro motility assay strongly depends on the length of the lever arm . Two parameters obtained from this fitting were T0.5, the transition midpoint , and d which reflects the curve slope at a transition midpoint and the width of the temperature range in which the changes in the fluorescence parameters occur.Fluorescence studies were performed on a Cary Eclipse spectrofluorimeter (Varian) equipped with a Peltier-controlled cell holder and thermoprobes. All measurements were performed in a 30 mM Hepes-KOH buffer (pH 7.3) containing 2 mM MgClctively) , 35. Upovely) [A . This inx and S1-ADP-AlF4 complexes was heated with a constant rate of 1°C/min in the medium containing 30 mM Hepes (pH 7.3), 2 mM MgCl2, and 100 mM NaCl., and the S1 aggregation was recorded by an increase in light scattering intensity at 350 nm on a Cary Eclipse spectrofluorimeter (Varian). During the heating, 100 μL aliquots of the sample were taken, cooled down, and subjected to ultracentrifugation at 140,000 g for 20 min on a Beckman airfuge . The protein composition of supernatants obtained was analyzed by SDS–PAGE [This approach was based on the fact that irreversible thermal denaturation of S1 is accompanied by its aggregation which can be detected by a significant increase in either turbidity or light scattering , 28, 36.SDS–PAGE . Quantifi.e., to define their correspondence to structural domains of the S1 molecule. For this purpose the temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation were recorded at the same heating rate (1°C/min) as for DSC experiments and were compared with the DSC data . Using this approach, we have identified the more thermostable calorimetric domain as the S1 motor domain, because changes in fluorescence and ATPase activity were only observed within the same temperature range as this domain on the DSC profile. On the other hand, thermal denaturation of the least thermostable domain was not accompanied by any changes in fluorescence and activity, and therefore we proposed that this calorimetric domain (which melts at ~40°C) can be assigned to the S1 regulatory domain which does not contain tryptophan residues [4- and S1-ADP-BeFx (with the exception of ATPase measurements as S1 has no ATPase activity in these complexes).In the previous work we applied DSC for a detailed comparative analysis of thermal denaturation and domain structure of two S1 isoforms containing different ELC  in the nucleotide-free state. We revealed two calorimetric domains in the S1 molecule, where the more thermostable domain in turn denatured in two steps. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the least thermostable domain, within the temperature range of 35–45°C , suggestresidues . Here wex , as well as T0.5 values for the parameter A, are summarized in x and S1-ADP-AlF4- complexes each thermal transition corresponds to separate calorimetric domain.).x  and S1-Ax  mutants which were fluorescently labeled at a single cysteine residue at different positions in the C-terminal half of the LC1 molecule and then exchanged into S1. The temperature induced changes in the fluorescence parameters of these LC1 mutants associated with the S1 regulatory domain, which provided information on the thermal unfolding of this domain, were then compared with the corresponding DSC data and with the changes in tryptophan fluorescence, which reflected thermal unfolding of the S1 motor domain.L for fluorescence of labeled LC1 in S1 were almost identical for both these LC1 species, both for nucleotide-free S1 (T0.5 = 46 ± 0.5°C) (x complex (T0.5 = 52 ± 0.5°C)  near the C-terminus and were labeled by AEDANS under similar conditions and then exchanged into S1. The temperature dependences of parameter ± 0.5°C)  and for ± 0.5°C) . Thus, wx and S1-ADP-AlF4 complexes, we studied the temperature dependences of fluorescence of these LC1 mutants both free in solution and bound to the regulatory domain of the nucleotide-free S1. Isolated LC1 species did not demonstrate any changes in the fluorescent parameter L upon heating (data not shown). Temperature-induced changes in parameter L for fluorescence of these LC1 mutants associated with the regulatory domain of nucleotide-free S1 are presented on A for the tryptophan fluorescence, which reflect thermal unfolding of the S1 motor domain. The values of T0.5 and d extracted from the analysis of the temperature-induced changes in parameters A and L are summarized in We prepared several recombinant LC1 mutants, each containing a single cysteine residue at different positions in the C-terminal half of the LC1 molecule , which were fluorescently labeled at these cysteines with 1,5-IAEDANS and then exchanged into S1. Prior to the experiments on the S1-ADP-BeFL occur at somewhat lower temperature than the changes in parameter A. In most cases, with the only exception of LC1 Cys-99 mutant, the T0.5 value for parameter L is lower, by 1–2°C, than that for parameter A  than the parameter A (~45°C)  . At the redicted . These ricted [L  might beL registered either in the absence or in the presence of HspB5. The results showed that HspB5 effectively prevents the thermally-induced aggregation of S1 by shifting the aggregation curve by ~20°C towards higher temperature (L (L for the fluorescent label (IAEDANS) specifically attached to the reactive SH1 group (Cys-707 in the S1 heavy chain) located in the S1 motor domain. These dependences obtained in the absence or presence of HspB5  and very similar to those obtained for parameter A of the S1 tryptophan fluorescence  effectively prevents the heat-induced aggregation of S1 with almost no effect on the S1 thermal unfolding measured by DSC . Here weperature . As a reature   . The widameter A , and thiT0.5 values for parameter L presented in T0.5 values for calorimetric transition 1 obtained from the DSC experiments on the S1-ADP-BeFx and S1-ADP-AlF4 complexes  in the course of its thermal denaturation, we can conclude that calorimetric transition 1 observed on the DSC profiles of the S1-ADP-BeFx and S1-ADP-AlF4 complexes corresponds to the thermal denaturation of the S1 regulatory domain containing associated LC1.Analyzing the omplexes . Moreoveeriments , with thsition 1 , probablsition 1 . Neverthmidpoint . Taking x and S1-ADP-AlF4 complexes, were similar for all LC1 mutants, independently of the position of a labeled cysteine residue in the C-terminal half of LC1   , Table 3 complex , should x and S1-ADP-AlF4 complexes. The samples were heated with a constant rate of 1°C/min and aliquots were withdrawn at appropriate temperature, cooled down, subjected to ultracentrifugation to separate LC1 from denatured and precipitated S1 heavy chains, and then protein composition of the supernatants was determined by SDS–PAGE. The results showed that the S1 heavy chain disappeared from the supernatants at temperatures corresponding to the thermal denaturation and aggregation of the S1 motor domain, i.e. above 40°C for the nucleotide-free S1 . Under these conditions, about 40% of LC1 remained in the supernatant for the nucleotide-free S1 , whereas its content in the supernatant remains constant after complete denaturation of all S1 molecules, when all denatured S1 heavy chains precipitate and disappear from the supernatants  strongly increases upon formation of its ternary complexes with ADP and Vi, BeFx or AlF4 [4- or S1-ADP-BeFx  relative to the motor domain, i.e. in the angle between these domains, with a hinge point located in the pliant region between the converter in the motor domain and the ELC in the regulatory domain . We cann4- and S1-ADP-BeFx complexes as the regulatory domain of the S1 molecule and also have shown that formation of these complexes  strongly increases the thermal stability of the S1 regulatory domain. We propose that this stabilization can be explained not only by global nucleotide-induced conformational changes of the S1 molecule leading to rotation of the converter and the lever arm, but also by a rather tight interaction of LC1 on the regulatory domain with the motor domain, which is caused by this rotation. The interaction of the C-terminal part of LC1 with the S1 motor domain can also exist in the absence of nucleotides [4- and S1-ADP-BeFx complexes that mimic the main intermediate states of the S1 ATPase cycle, in which the C-terminal part of LC1 interacts with the other sites on the motor domain [In any case, we conclude that the results obtained from our experiments on the thermal unfolding of S1 testify in favor of the earlier proposed concept of the nucleotide-induced interaction between the S1 motor domain and LC1 associated with the S1 regulatory domain. Using fluorescently labeled LC1s, we have identified the least thermostable thermal transition on the DSC profiles of the S1-ADP-AlFleotides  Fig 1A)4- and Sr domain  Fig 1B)4- and Sr domain .4- and S1-ADP-BeFx complexes leads to a significant movement of the regulatory domain (lever arm) relative to the motor domain, and as a result, in these complexes the lever arm is located rather close to the surface of the motor domain [According to crystallographic data, formation of the S1-ADP-AlFr domain  Fig 1B)4- and SS1 Figmax was equal to 491 nm for LC1 bound to the S1 heavy chain and 486.4 nm for free LC1.The value of λ(TIF)Click here for additional data file.


Molecular dynamics was used to simulate large molecules of the immune system  To characterize the relative orientation and movements of domains local coordinate systems  were generated and directional cosines and Euler angles were computed. As a most interesting result, we found that the presence of the coreceptor seems to influence the dynamics within the protein complex, in particular the relative movements of the two  C), but is also found on natural killer cells, cortical thymocytes, and dendritic cells kV of three orthonormal eigenvectors v1, v2, v3 of the covariance matrix of the coordinates of Cα atoms in domain V at frame kV. These vectors define a reference frame  for domain V.The(3)W, yielding the respective central frame kW with an eigenvector matrix TW(kW) = [w1  w2  w3]kW and a local coordinate system for W.Steps (1) and (2) are performed also for domain In order to avoid such artifacts we refrained from computing principal components repeatedly for each MD time step and adopted the following procedure, see f of a trajectory, the relative orientation of domains V and W is computed as follows:(a)f we compute the transformation of all coordinates xV(kV) of domain V from its position within the central frame into its position at frame f according to minimum RMSD using Kabsch's method [For each frame s method .(b)RV(f) of the above transformation is applied to the eigenvectors of domain V at frame kV (the reference frame) to obtain the position of the eigenvectors of V at frame f: TV(f) = [v1  v2  v3]f.The rotational part (c)W, yielding RW(f) and transformed eigenvectors TW(f) = [w1  w2  w3]f for each frame f.Steps (a) and (b) are performed also for domain (d)f we note that the directional relation between the two sets of eigenvectors [v1  v2  v3]f and [w1  w2  w3]f can be represented via a rotation matrix RVW(f) asFor each frame  which also characterizes the relative orientation of both domains. From  we obtai RVW(f) in terms of its column vectors since the inverse of an orthogonal matrix equals its transpose. Rewriting the matrix  x-convention [v1  v2  v3]f and [w1  w2  w3]f can be read asthe Euler angles in nvention  between   f (dependencies suppressed in the notation).with all quantities depending on frameAs a result, d(f), α(f), β(f), and γ(f) define the relative spatial relation between domains V and W for MD frame f.Given all MD-frames α1, CD8 α2) as domain V and domain α3 of the MHC  frames, Relative positions of both domains were then computed for each frame  as outlined above leading to the following results; see A first and direct measure is provided by the orientation cosines between corresponding eigenvectors; see To further characterize the evolution of the geometry of the complex with time we computed the autocorrelation functions (ACFs) for cosines between eigenvectors and for Euler angles; see v1 and w1, has only a short memory (the ACF passes through zero around 20 ns), a massively prolonged memory is seen for both smaller eigenvectors v2 versus w2 and v3 versus w3: they exhibit a long negative tail and do not become stochastic throughout the whole simulation time. This reflects the fact already seen in the time course itself  frames (kV = 279 ≡ 138.4 ns and kW = 345 ≡ 171.2 ns) of each, and computed corresponding eigenvectors; see v1(kV) and w1(kV) have similar directions and orientations, when computed for the central frames. As opposed to this, they show almost opposite directions when computed from the first frame of the trajectory; see v1(1) and w1(1) we actually use for frame 1  have orientations similar to v1(kV) and w1(kV), except for the actual, relative movements of both domains.The two α1 and Gα2 are characterized by cosines (see Relative motions of Gines see  and Euleines see  between ρ = 0.79 (with p < 0.01 and Nframes = 503); see The above finding is nicely quantified via the correlation coefficient α1 and Gα2 from our MD simulation of TCR/pMHC/CD8 with CD8 attached to the MHC and compared it with the above results without CD8. Interestingly, the relative motions do not differ significantly (figures not shown); however, the correlation of “rolling” oscillations is almost lost in the presence of CD8: correlation coefficient ρ = 0.6 (with p < 0.01 and Nframes = 406); see ρ = 0.79 (Nframes = 503) and  for ρ = 0.6 (Nframes = 406). These intervals do not overlap and the difference in correlation coefficients may thus be considered statistically significant.We have also analyzed the relative motions of GThe methodological parts of this work describe a new computational technique to obtain relative orientations of intramolecular domains. In the application parts this method is used to analyze the molecular dynamics of two systems, TCR/pMHC and TDC/pMHC/CD8, respectively.α3; see There is a plethora of ways to characterize relative movements of intramolecular domains e.g., , 24–27).–27.24–27 orientation is not well defined and may suddenly switch into almost opposite directions. Up to now, this was in most cases mended by some logical condition in the code selecting the appropriate orientation with reference to some atoms that have to be individually specified. These drawbacks are even more severe when eigenvectors are computed from internally deformable sets of data points, such as MD-frames.In this work we present a more sophisticated approach by attaching local coordinate systems (of eigenvectors) to each domain and calculating the rotational relations between them. It is a well-known drawback of eigenvector-based techniques that the eigenvectorWe have solved this problem by computing eigenvectors only once (for each domain) from a very specific frame , see Note that although the method seems computationally demanding, it is in fact fast. The reason is that the Kabsch algorithm is a direct matrix operation, not an iterative procedure, as one might expect. It requires no larger computational effort than singular value decomposition.Once mutual orientations have been computed (rotation matrix), some attention was given to select suitable quantities to characterize relative orientations. We presented cosines between corresponding eigenvectors, since they can easily be interpreted. In addition, we used Euler angles as a well-known concept for relative orientations of rigid bodies.v1 may also result as v1∗ = −v1, merely as a consequence of minute numerical noise in data substantially equal. The same is true for v2. The third eigenvector always has to satisfyv1 and v2. When comparing the relative orientation of two domains via their eigenvectors, orientation ambiguity has to be coped with, which could be done as follows:(i)kV of domain V, results are manually inspected and v1 given a well-defined orientation within domain V. This can be accomplished by selecting two Cα atoms and setting the orientation of v1 such that a positive cosine of the angle between v1 and the vector joining the two Cα atoms is obtained.After performing a PCA for the reference frame (ii)v2, with a second pair of appropriate Cα atoms selected. v3 is computed via the cross product of v1 and v2; see above.The same is done for In order to arrive at standardized eigenvectors allowing for comparison between different MD-runs the resulting eigenvectors should be reoriented (if necessary) after performing PCA for the reference frame kW of domain W: a criterion for reorientation could be positive cosines with the eigenvectors of domain V:PCA as such yields eigenvectors unique in directions but ambiguous in orientation. For example, the first eigenvector α3 over time  fail to indicate the relaxation but  and  clearly do. This indicates that the relaxation is made up of some rotation around the major axes, v1 and w1, respectively.The existence of an equilibration phase raises the importance of a comparison of the two presentation methods employed: cosines between eigenvectors and Euler angles see . Clearlyv2, w2) and ) show excessively long autocorrelations, pertaining throughout the whole simulation time. This finding, even more than the 50 ns equilibration of distance, indicates the necessity of additional MD simulations to arrive at a more adequate sampling.This surprising finding is supported by comparing the corresponding autocorrelation functions; see α-helices, Gα1 and Gα2. In this case, Euler angles proved the more sensitive tool. Negative correlation between cosines of  and  was in both cases beyond ρ < −0.9, and presence or absence of CD8 did not make much difference. For the Euler angles, however, we obtained the nice reduction of movement-correlation induced by the presence of CD8; see As a most interesting result, we found that the presence of CD8 seems to influence the dynamics within the MHC, in particular the relative movements of the two All in all, both sets of orientation parameters presented (cosines and Euler angles) seem to have their merits and weaknesses that have to be explored in many more situations to arrive at a comprehensive judgment.


With the continuous discovery of novel RNA molecules with key cellular functions and of novel pathways and interaction networks, the need for structural information of RNA is still increasing. In order to predict structure of long RNA and understand its natural folding mechanism, exploring the characteristic of RNA structure is an important issue.The real RNA secondary structures of all 480 sequences from the database of RNA strand, validated by nuclear magnetic resonance or x-ray are selected. For one sequence with multiple domains, the length ratios of these domains to the sequence are computed. For one sequence with one domain and multiple sub-domains, the length ratios of these sub-domains to the domain are computed. Then the ratios are compared and analyzed to seek the partition characteristic of domains and subdomains.For most RNAs, the length ratios of multiple domains to its sequence are close to equal, and those of sub-domains to its domain are also nearly identical. Most RNAs with multiple domains have two domains, so the length ratios of the domains to its sequence are close to 0.5. For sequence with one domain and no sub-domain or one sub-domain, the centre of domain and sub-domain is close to that of the sequence.A novel finding is given that RNA folding accords with the characteristic of equipartition based on statistical analysis. The characteristic reflects the folding rules of RNA from a new angle, which maybe more close to natural folding. RNAs are versatile molecules. To understand fully the various functions of RNAs, we need to first understand their structures . ExperimRNA is folded as the process of transcription into RNA from DNA. In order to predict RNA structure, a case may be made that the natural folding process of RNA and the simulated folding of RNA using an evolutionary algorithm, which includes intermediate folds, have much in common . So explWe compare the structures of the test set of all 480 sequences from RNA STRAND [s=s1s2n ...sbe a single-stranded RNA molecule, where each base i ≤ n. The subsequence i, j s= si si+1. . . j sis a segment of s, 1 ≤ i ≤ j ≤ n.Let sequence i sand j sare complementary bases , then i sand j smay constitute a base pair . A secondary structure S on s is a set of base pairs S={}, where If No sharp turns.) The ends of each pair in S are separated by at least four intervening bases; that is, if i < j-3. in For any pair (S is a matching: no base appears in more than one pair.The non-crossing condition.) If  and  are two pairs in S, then they are compatible, that is, they are juxtaposed (e.g. i <j < k <l) or nested (e.g. i <k <l <j). and i, j) and  constitute stack , and m(≥1) consecutive stacks form the helix  with the length of m+1.If  and  are incompatible, they form a pseudoknot (e.g. i < k < j < l). More complex pseudoknots may occur if three or more base pairs cross each other.If base pairs  consists of all  that satisfy,  ∈ D then i < i' < j' < j. Each base pair and each helix is placed uniquely in one domain [A domain e domain .A domain is closed by a helix or pseudoknot, as Figure i,j sbegins to transcribe from the 5'-end is. It terminates transcription at the 3'-end js, as Figure i, i+m: j-m, j) is totally folded after transcription of js.By convention, single strands of RNA sequences are written in 5'-to-3' direction. RNA is folded as the process of transcription into RNA from DNA. The subsequence For purpose of understanding the natural folding mechanism and pathway of RNA, we selected the real structures of all 480 sequences from RNA STRAND with secondary and pseudoknotted structures, validated by NMR or X-Ray, non-fragment and non-redundant sequences, and analyzed their domains and sub-domains.R as the ratios of 3'-end of domains to the length of sequence. Let L is the length of sequence. The ratio of the 3'-end of the helix  and the domain D to the length of sequence sL 1,is the ratio of j to L, that is R=j/L. We compute and analyze the value of R, and seek the partition characteristic of domains.If the structure of RNA has multiple domains, we computed SR as the length ratios of its sub-domains to the domain. If the domain D is closed by a helix , then its internal length is j-i-2m-1, and SR is equal to (q-p+1)/(j-i-2m-1) for the sub-domain D with j-m-i>q>p>m. We compute and analyze the value of SR, and seek the partition characteristic of sub-domains.If the structure of RNA has only one domain, we computed We compare the structures of all 248 sequences of synthetic RNA. The results of statistical analysis on these structures are shown in Figure Table L, 0.33L-0.66L and 0.66L-L as Table L-0.5L, 0.5L-0.75L and 0.75L-L, but we can divide the sequence into four domains, 0-0.25L, 0.25L-0.5L, 0.5L-0.75L and 0.75 L-L, then it also conforms to the characteristic of equipartition.There are 12 sequences with more than two domains as Table L, 0.25L-0.5L, 0.5L-0.75L and 0.75L- L as Table Sequences PDB_00175, PDB_00873, PDB_00447 and PDB_00340, have four domains. Their domains are 0-0.25L, 0.2L-0.4L, 0.4L-0.6L, 0.6 L-0.8L and 0.8L-L as Table Sequences PDB_01061 and PDB_00370 have five domains. Their domains are 0-0.2L, 0.17L-0.33L, 0.33L-0.5L, 0.5L-0.67L, 0.67L-0.83L and 0.83L-L, which is completely fits the characteristic of equipartition, as Table Only sequence PDB_01249 has six domains 0-0.17L and 0.5L-L, and those of 13 sequences are close to 0-0.5L and 0.5L-L, which fits the characteristic of equipartition. The domain is formed by parallel helixes or pseudoknots as Figure L and 0.33L-L, those of sequence PDB_00868 are 0-0.39L and 0.39L-L, those of sequence PDB_00709 and PDB_00710 are 0-0.4L and 0.4L-L, those of sequence PDB_00971 are 0-0.57L and 0.57L-L, and those of sequence PDB_01138 are 0-0.58L and 0.58L-L. It can be thought as the combination of some domains, and they close to 0.33L, 0.4L and 0.6L. For example, we can regard sequence PDB_00196 as three domains 0-0.33L, 0.33L- 0.66L and 0.66L-L, then 0.33L-0.66L and 0.66L-L combines into domain 0.33L-L.Table The rest of 188 sequences have one domain or one pseudokont, and the centre of domain is basically same as that of its sequence. In common, their sub-domains can be divided into three classes, no sub-domain in 157 sequences as Figure For Sequence with one domain and no sub-domain, the centre of domain is close to that of the sequence. There are 11 sequences with one pseudoknotted domain. Sequences PDB_01040, PDB_00020, PDB_01165 and PDB_01194 have one pseudoknot with three helixes, and the helix  and  in PDB_01040,  and  in PDB_00020,  and  in PDB_01165,  and  in PDB_01194, meet the characteristic of equipartition. PDB_00053, PDB_00209, PDB_00134, PDB_00842, PDB_01059 and PDB_00124 have one pseudoknot with two helixes, and the centre of the pseudoknot is same as that of its sequence. PDB_00759 has two pseudoknots with three helixes, and the centre of helix  is basically same as that of its sequence.As Table We compare the structures of all 46 sequences of tRNA. The results of statistical analysis on these secondary structures are shown in Table L and 0.5L-L, and those of 7 sequences are close to 0-0.5L and 0.5L-L, which fits the characteristic of equipartition. But there are some exceptions, the domains of sequence PDB_00681 are 0-0.33L and 0.33L-L, those of sequence PDB_01162 are 0-0.61L and 0.61L-L. It can also be thought as the combination of two domains, and they close to 0.33L and 0.66L.Table L, 0.33L -0.66L and 0.66L-L, which fits the characteristic of equipartition.There are 3 sequences have three domains. Their domains are close to 0-0.33L, 0.18L-0.34L, 0.34L-0.5L and 0.5L-L. They can be thought as two groups, one is 0-0.5L, and the other is 0.5L-L, which fits the characteristic of equipartition. For the group 0-0.5L, it is divided into three domains, 0-0.18L, 0.18L-0.34L and 0.34L-0.5L, it also fits the characteristic.Sequence PDB_00998 has four domains 0-0.18L, 0.2L-0.4L, 0.4L-0.6L, 0.6L-0.8L and 0.8L-L, which completely fits the characteristic of equipartition.Sequence PDB_00398 has five domains 0-0.2L, 0.18 L-0.34L, 0.34L -0.5L, 0.5L-0.68 L, 0.68L-0.83L and 0.83L-L, which is also fits the characteristic of equipartition.Sequence PDB_01000 has six domains 0-0.18 L, 0.33L-0.66L and 0.66L-L, which conforms to the characteristic of equipartition.The rest of 23 sequences have one domain or one pseudokont, and the centre of domain is basically same as that of its sequence. There are 12 sequences with one domain and multiple sub-domains as Table We compare the structures of all 49 sequences of Other RNA, 6 sequences of Ham Ribozyme and 9 sequences of Viral & Phag.L and 0.5L-L, which completely fits the characteristic of equipartition. There are 3 sequences have three domains. The domains of PDB_00626 and PDB_00739 are just 0-0.33L, 0.33L-0.67L and 0.67L-L, which fits the characteristic of equipartition. Sequence PDB_01261 has three domains. They can be divided into two groups, one is 0-0.5L, and the other is 0.5L-L. Then the domain 0.5L-L is divided into 0.5L-0.75L and 0.75L-L. Sequence PDB_001261 and PDB_00985 has four domains. Their domains are close to 0-0.25L, 0.25L-0.5L, 0.5-0.75L and 0.75L-L, which fits the characteristic of equipartition. Sequences PDB_01061 and PDB_00370 have five domains. Their domains are 0-0.33L, 0.33L-0.66L and 0.66L-L, which completely fits the characteristic of equipartition.The results of statistical analysis on these secondary structures are shown in Table L, 0.52LL, which fits the characteristic of equipartition, as Table For Viral & Phage, only one sequence PDB_00743 has two domains 0-0.52L, 0.5L-L, which completely fits the characteristic of equipartition, as Table For Ham Ribozyme, only one sequence PDB_00157 has two domains 0-0.5We compare the structures of all 18 sequences of Other Ribozyme. The results of statistical analysis on these secondary structures are shown in Table L, 0.25L-0.5L, 0.5-0.75L and 0.75L-L, which conforms to the characteristic of equipartition. Sequence PDB_00176 has four domains, and they meet the character of equipartition. Sequence PDB_01187 has four domains, and they can be divided into two groups 0-0.5L and 0.5L-L, which conforms to the characteristic of equipartition. There are 11 sequences with two domains. They fit the character of equipartition with three exceptions.Sequence PDB_00805 have 8 domains, and they can be divided into four groups 0-0.25L, 0.36L-0.48L, 0.48L-0.86L and 0.86L-L. They can be divided into two groups, one is 0-0.48L, and the other is 0.48L-L, which nearly fit the characteristic of equipartition. PDB_01185 has one pseudokontted domain with three sub-domains, and sub-domain 0-0.52L and 0.52L-L nearly fit the characteristic of equipartition.There are 4 sequences have only one domain. PDB_00088 has one domain with no sub-domain, PDB_00142 has one domain with one sub-domain, and they also fit the characteristic of equipartition. Sequence PDB_00078 has one pseudokontted domain with four sub-domains 0-0.36For 16S rRNA and 32S rRNA, they conform to other characteristics besides the characteristic of equipartition, it is a matter for further discussion.In this paper, we give a novel finding that RNA folding accords with the characteristic equipartition based on statistical analysis on real RNA secondary structures of all 480 sequences from RNA STRAND, validated by NMR or X-Ray. For most RNA sequences, the length of multiple domains is close to equal. For the sub-domains of one domain, the length of them is also nearly identical. Most of multiple domains are two domains, so the length ratio of the first domain to its sequence is close to 0.5. The characteristic of equipartition reflects the folding rules of RNA from a new angle, which is more close to natural folding. Applying this characteristic, algorithm can be designed to dynamically predict long RNA structure, and the dynamic folding mechanism and the relation of function, mutation and RNA structure can be deeply understood from a new view.The authors declare that they have no competing interests.This work was supported by NSFC under grant N0.61070019, 61272431, Shan Dong Province Natural Science Foundation of China under grant N0.ZR2011FL029, ZR2013FM016, the Open Project Program of the Shandong Provincial Key Lab of Software Engineering under grant No.2011SE004, and Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province.HL and DM initiated the project and carried out data analysis. KAC conducted statistical modelling and comparison. CZ and HH performed data processing. All authors read and approved the final manuscript.


Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves the way for future studies into MAPL function in alternative model organisms like Capsaspora owczarzaki and Chlamydomonas reinhardtii that will help to answer the question of MAPL’s ancestral function in ways that cannot be answered by studying animal cells alone.MAPL  is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists  Mitochondria are ubiquitous eukaryotic organelles derived from an ancient endosymbiotic α-proteobacterium. Tracing the evolutionary history of mitochondrial proteins is important in understanding how a free-living α-proteobacterium became an integrated eukaryotic organelle. Several mitochondrial proteins are closely related to α-proteobacterial proteins; however, it has been shown that many mitochondrial proteins are derived from bacteria not related to the endosymbiont ,2. The mMAPL has been implicated in several processes specific to metazoan  cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis –6. This,Human MAPL has a BAM (beside a membrane)/GIDE (growth inhibition death E3 ligase) domain located between two transmembrane domains topologically oriented such that both N- and C-termini reside in the cytosol while the BAM domain resides in the mitochondrial intermembrane space (IMS) ,11,14. BCompared to the RING domain, very little is known about the function of MAPL’s BAM domain. Previous work has shown that only the BAM domain is required for proper packaging of MAPL into MDVs trafficked to peroxisomes –10. Due The potential homology of MDVs and bacterial OMVs (outer membrane vesicles) has not gone unnoticed ,16,19. Bhttp://www.broadinstitute.org/), the National Center for Biotechnology Information (NCBI), and some private websites. A list of genomes used in this study is included in Publicly available genomes were obtained from various online resources including the Joint Genome Institute, the Broad institute (http://hmmer.janelia.org/) searching strategies of both the NCBI non-redundant database as well as individual genomes. Each putative MAPL homologue was assessed for domain structure using the Pfam database (http://pfam.xfam.org/). Only sequences that contained recognizable BAM/GIDE domains were retained for phylogenetic analysis. Some BAM/GIDE proteins were not present in predicted proteomes were reconstructed from genomic assembly sequences or EST sequence reads available from NCBI. Sequences retrieved in this study are listed in BAM domain containing proteins were retrieved using a combination of BLAST  and HMMehttp://mesquiteproject.org). Model testing was performed using ProtTestv1.3 [BAM domain-containing proteins were aligned using MUSCLE  and manuTestv1.3  with a GTestv1.3  for BayeTestv1.3  and RaxMTestv1.3  with 100Testv1.3  model. SSalpingoeca rosetta and the ichthyosporean Capsaspora owczarzaki, close single-celled relatives of animals. The presence of MAPL in organisms outside metazoa suggests an ancient origin of MAPL. In order to determine if MAPL was present in the ancestor of Opisthokonta we searched for homologues in fungi and identified MAPL-related proteins in only the chytridiomycete Spizellomyces punctatus and the neocallmastigomycete Oprinomyces sp.  and one species of choanoflagellate (S. rosetta) MAPL could not be identified in sequences of genomes from the other sequenced holozoan protists Sphaeroforma arctica or Monosiga brevicolis. These absences suggest that although MAPL has been retained in diverse lineages, it has been repeatedly lost over the evolutionary history of the Opisthokonta.Although MAPL is present in numerous opisthokonts it is noticeably absent from several lineages. We could find no evidence for MAPL in any sequenced nematode species, or any fungal lineage outside those already mentioned. Although MAPL was present in one species of filasteria   found BAnhardtii . We alsoAndrade-Navarro et al.  suggesteA. thaliana and C. rubella proteins that lack the C-terminal RING domain (MAPL-R) group weakly with other archaeplastid MAPL proteins but strongly group within the larger eukaryotic clade  was subsequently lost in the mammalian lineage . The lineage . These dstigated , 29.Arabidopsis thaliana and the closely related species Capsella rubella lacked the C-terminal domain. Our phylogenetic analysis of Archaplastid MAPL  clade are more closely related to other eukaryote sequences or prokaryote sequences, BAM protein sequences from Presence of conserved BAM-RING domain architecture in diverse eukaryotes and the relative rarity of loss of the RING domain suggests that both domains are important for the overall function of MAPL. If the domains functioned independently it would be expected that the domains would be found separated from one another more frequently over the course of eukaryote evolution. This is interesting because studies to date have not found a link between the two domains and the different functions discovered in animals seem to be distinct.Duplication and divergence in some lineages, like vertebrates and plants, suggests that functional specialization has occurred. The duplication at the base of vertebrates is of particular interest because the divergent paralogue is lost in mammals marking a relatively rare example of secondary loss in the mammalian lineage. Investigation of MAPL-like proteins in model fish, reptile, and amphibian species will be of great interest for understanding the conserved function of MAPL in mammalian cells.Although eukaryotic MAPL appears to derive from a single eukaryotic protein with similar domain architecture to extant MAPL, the ancestry of MAPL remains difficult to resolve. Previous work  suggesteMAPL is involved in organelle dynamics, mitophagy, mitochondria to nucleus signaling, and is packaged as cargo in MDVs destined for peroxisomes. The presence of MAPL in diverse eukaryotes allows us to infer its ancient origins, but several questions still remain. Specifically, although MAPL is ancient we currently have no way of knowing if it was present in the LECA or if it arose later in evolution. It is also still unclear how many HGT events may have occurred between prokaryotes.S. rosetta, the filasterian C. owczarzaki, and the green alga C. reinhardtii is critical to understanding MAPL’s ancestral function. We hope that our study provides the impetus for future functional work on MAPL in organisms outside metazoa.Its taxonomic distribution suggests that MAPL functions in several single-celled eukaryotes. As multicellular animals are cluttered with proteins and pathways required for obligate multicellular life, investigation into MAPL function in single-celled organisms like the choanoflagellate S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.