Agrobacterium tumefaciens, and Rhodospirillum rubrum, are membrane bound and that the vacuolar proton-translocating pyrophosphatases (V-H+PPases) are present in their surrounding membranes. Volutin granules and acidocalcisomes have been found in organisms as diverse as bacteria and humans.Volutin granules appear to be universally distributed and are morphologically and chemically identical to acidocalcisomes, which are electron-dense granular organelles rich in calcium and phosphate, whose functions include storage of phosphorus and various metal ions, metabolism of polyphosphate, maintenance of intracellular pH, osmoregulation and calcium homeostasis. Prokaryotes are thought to differ from eukaryotes in that they lack membrane-bounded organelles. However, it has been demonstrated that as in acidocalcisomes, the calcium and polyphosphate-rich intracellular "volutin granules (polyphosphate bodies)" in two bacterial species, +PPase pumps, which acidify the acidocalcisome lumen and are diagnostic proteins of the organelle, also reveal the presence of this enzyme in all three superkingdoms suggesting it is ancient and universal. Since V-H+PPase sequences contained limited phylogenetic signal to fully resolve the ancestral nodes of the tree, we investigated the divergence of protein domains in the V-H+PPase molecules. Using Protein family (Pfam) database, we found a domain in the protein, PF03030. The domain is shared by 31 species in Eukarya, 231 in Bacteria, and 17 in Archaea. The universal distribution of the V-H+PPase PF03030 domain, which is associated with the V-H+PPase function, suggests the domain and the enzyme were already present in the Last Universal Common Ancestor (LUCA).Here, we show volutin granules also occur in Archaea and are, therefore, present in the three superkingdoms of life . Molecular analyses of V-H+PPase function and the evolutionary dynamics of these domains support the early origin of the acidocalcisome organelle. In particular, the universality of volutin granules and presence of a functional V-H+PPase domain in the three superkingdoms of life reveals that the acidocalcisomes may have appeared earlier than the divergence of the superkingdoms. This result is remarkable and highlights the possibility that a high degree of cellular compartmentalization could already have been present in the LUCA.The importance of the V-HThis article was reviewed by Anthony Poole, Lakshminarayan Iyer and Daniel Kahn According to the theory of serial endosymbiosis , the symProkaryotic cells have been simplistically portrayed in the general scientific literature as a plasma membrane "sack" containing DNA, filled with cytoplasm and surrounded by a cell wall. However, provocative findings have challenged the notion that the prokaryotic cell lacks a sophisticated cytoplasmic organization. For years, it was believed that the shape of bacterial cell was maintained by the organization of cellulose fibers within the cell wall. However, a protein similar to actin, which was once thought to be exclusive to the eukaryotic cytoskeleton, was discovered in bacteria -8. This Agrobacterium tumefaciens [Rhodospirillum rubrum [i), calcium, magnesium, and other elements. In addition, the organelle contains a variety of cation pumps including Ca2+/H+ and H+ pumps. In particular, the vacuolar proton translocating pyrophosphatase (V-H+PPase) proteins have been localized in the acidocalcisomes of bacteria, parasitic protozoans, algae, plants, and recently in cockroaches [+PPase arose early in the evolution of life on Earth. As mentioned before, the main components of the acidocalcisome are polyphosphates, which includes PPi. V-H+PPases use PPi as an energy source to pump protons into the acidocalcisome lumen, thereby generating an electrochemical gradient. This strongly suggests that both the V-H+PPase and the acidocalcisome share similar evolutionary histories.Even more recently, the idea that organelles similar to those present in eukaryotes are absent in bacteria was challenged by the discovery of an organelle similar to the acidocalcisome of unicellular eukaryotes within the bacterium efaciens . The exim rubrum . The acim rubrum . This mekroaches . The wid2+, pH, and osmotic homeostasis, in which polyP is a key player. However, the study of polyP has been neglected for years. An important feature of polyP is its negative charge, which enables it to interact with nucleic acids and act as a regulator of RNA polymerases [Acidocalcisomes are morphologically and chemically similar to the structures historically described as volutin or polyphosphate bodies, which have been identified in a variety of microorganisms, including bacteria, archaea, algae, and protozoans . Meyer  describeymerases  and protymerases . In addiymerases -20.Other membranous structures have also been reported in bacteria as well . However+PPases, to infer the phylogenetic history of this protein and an ancient origin of the acidocalcisome. Within protein molecules, certain parts can interact more strongly with each other than with other parts, and are usually highly conserved, making up modules, called protein domains [+PPase proteins. We then examine the distribution of the main functional V-H+PPase domain on the tree using Protein family (Pfam) database definitions.Debates about organellar evolution have been revitalized and streamlined during the post-genomic revolution. The availability of multiple complete genome sequences from the whole spectrum of life, along with corresponding protein sequence data sets and more sophisticated bioinformatics tools opens a window into the intricate mechanisms of cellular evolution. In the present study, we use sequences and domain information from a diagnostic protein of the acidocalcisome, the proton pump V-H domains . These s domains . The com domains . Using B+PPases and volutin granules as opportunity to infer the likely origin of the acidocalcisome. In addition, we discuss the implications that these findings may have on our understanding of the cellular complexity of the ancestor of cellular life.Here, we delineate the connections between the evolution of V-H+PPase is commonly present in the membrane of acidocalcisomes which couple PPi hydrolysis to active proton transport across the organellar membrane. The enzyme is found in organisms from all superkingdoms of life and should be considered ancient. Its ubiquitous nature already suggests it appeared before the last universal common ancestor (LUCA) of diversified life [+PPase is a highly conserved enzyme that acidifies the lumen of the acidocalcisome [+PPase has been detected by immunogold labeling localization in acidocalcisomes of bacteria, and several eukaryotic microorganisms [+PPases in its surrounding membrane, was found inside the protein storage vacuole of seeds [+PPases has been identified in the egg yolk of cockroaches [+PPase function exists in bacteria, microbial eukaryotes, and higher eukaryotes.V-Hied life . The V-Halcisome . V-H+PParganisms ,10,27-33of seeds . Additiokroaches . These sAgrobacterium and Rhodospirillum and eukaryotes. While it may come as a surprise that volutin electron-dense granule from different organisms bear close similarity to acidocalcisomes, now we know that acidocalcisomes are virtually identical in size, composition and morphology to volutin-polyP bodies found in a vast array of organisms, including Archaea. Although the volutin granules present in Methanosarcina have not been yet confirmed to be acidocalcisomes, they are morphologically and chemically similar to the acidocalcisomes of Agrobacterium and Rhodospirillum [Methanosarcina have the same chemical profile, morphological characteristics (sponge like-structure) and high levels of phosphorous compounds and calcium [Agrobacterium acidocalcisomes  events among Bacteria, Archaea and Eukarya, some of which may have occurred early in evolution. Alternatively, these unusual placements might simply be the result of phylogenetic error associated with using only a single gene for analysis [The tree of V-Hs Figure . It alsoanalysis .+PPase sequences group many familiar clades monophyletically, and are also ubiquitous among superkingdoms although with poor phylogenetic signal, suggests these proteins are truly ancient. To further explore their ancestral nature we focused on structure, which is highly conserved in evolution [+PPase sequences, mapped them onto the V-H+PPase tree, and determined how many species in the three superkingdoms harbored these domains.The fact that V-Hvolution . We ther+PPase proteins have not been crystallized, and there is no structural entry associated with them in the Protein Data Bank (PDB). This absence probably stems from the limitation of NMR and X-ray crystallographic techniques in their ability to acquire high-resolution structures from proteins associated with membranes [+PPase. However, domain search using Hidden Markov Models (HMMs) of fold superfamily and fold family structural recognition revealed no hits. It has already been established that V-H+PPase proteins have domains with sequence motifs that are highly conserved in evolution [+PPase are very ancient. Because Pfam attempts to classify protein domains and families using sequence alignments and profile HMM analysis of sequence motifs [+PPase proteins. A Pfam search revealed that all V-H+PPase sequences have a single domain that is annotated as inorganic H+ pyrophosphatase, PF03030. This domain in our analysis is distributed in 279 species including Eukarya, Bacteria, and Archaea  is distributed universally. For these reasons, we hypothesize that this organelle or its precursors were very early cellular innovations that occurred prior to major organismal lineage diversification events. As the lineages of the tree of life began to diverge, this organelle or its precursors were inherited in the three domains by direct descent from the LUCA. Under this scenario, the putative absence of the organelle in an organism must be explained as a loss.Several hypotheses attempt to answer the question of when and how the acidocalcisome arose. The canonical explanation that the acidocalcisome was acquired either through lateral transfer or via an endosymbiotic event is, however, unlikely. Furthermore, sequence and domain analysis of the V-He Figure . NeitherIt is outside of the scope of the present work to discuss the characteristics of the LUCA, but the above hypothesis implies that the ancestral lineage may have had a high degree of cellular sophistication, as others have suggested -46. For +PPase pumps is not yet available, a recent analysis revealed that sequences from this protein family has a very ancient and strongly conserved 57-residue block with unique properties [+PPases appeared very early in evolution of life. Our analysis of the distribution of the main Pfam domain along the V-H+PPase phylogenetic tree reinforces this hypothesis  [While the 3D structure of the proton V-Hoperties . This fis Figure . In addiappeared ,49. Whenappeared . Howeverphonates . Alternarushite) . Brushitrushite) .i and polyP can be used as a source of energy. However, the developments of proton gradients are a more sustainable strategy to generate potential energy available for work in a cell. Proton gradients are as universal as proteins and nucleic acids in living organisms. Therefore, the acidocalcisome, acting like an energy source, might have played a critical function for survival of the LUCA cells. Interestingly, V-H+ATPases and ATPsynthases, which are capable of creating proton gradients across the cell membranes, are among the most ancient protein structures that are known [+ATPase was found in the acidocalcisome of several eukaryotic species together with the V-H+PPase [+PPase and V-H+ATPase couple the transport of protons into cellular compartments and enhance the acidification of the lumen of this organelle [+ATPases can "run in reverse" to synthesize ATP when subjected to a sufficiently large H+ electrochemical gradient [+PPsaes can have the same ability to regenerate pyrophosphate[i can be used as an alternative to ATP, because of its similarity to the ATP bond structure. In addition, PPI acts as a biological energy donor in photosynthesis [i might have been involved as an energy carrier during the early evolution of life [+PPases, which together with the V-H+ATPase make the acidocalcisome capable of sustained energy generation. As a result, the energy demands of the LUCA in the harsh environments of early Earth might have been met by a combination of polyP hydrolysis, creation of a H+ gradient, and regeneration of ATP and PPi. Moreover, polyP may have facilitated the assembly and orientation of the key molecules such as phospholipids, nucleic acids, and proteins [PPre known . In addire known -56. Rema-H+PPase ,57. Bothrganelle . Moreovegradient , while Vphosphate,59. PPi ynthesis ,61. Ther of life . Taken tproteins . The mulproteins . Althougproteins . Therefo+PPases together with the characteristics and universal distribution of the volutin granules and its main component, polyP, offer pertinent clues about the evolutionary history of the acidocalcisome. Our findings suggest that the V-H+PPases, which are universally distributed and tightly associated with the acidocalcisome, originated prior to the divergence of the three superkingdoms. Interestingly, volutin granules-polyphosphate bodies that have been detected in Methanosarcina acetivorans and other Archaea [Agrobacterium and Rhodospirillum [The sequence and domain analysis of the V-H Archaea -37 are rpirillum ,10. ConfSequences for vacuolar pyrophosphatases were downloaded in FASTA format obtained from the Pfam database (see below), and were aligned with Clustal W . Ambiguo+PPase sequences from organisms spanning the three superkingdoms of life including the unicellular parasites Trypanosoma, Toxoplasma and Plasmodium [Chlamydomonas reinhardtii [+PPase) domain. Consequently, we retrieved all the protein sequences that harbor the domain from the Pfam database. As a result, 776 protein sequences in total were obtained and then aligned and edited, and imported into phylogenetic reconstruction using Bayesian MCMC methods. By manually examining the topology of the phylogenetic tree, we removed duplicated sequences with no genetic distance and sequences that are not identified taxonomically. Subsequently, sequences that have paralogous relationships in the tree were totally removed in this analysis because it is difficult to know which sequences of paralogs are true orthologs for the pyrophosphatase on the tree. We finally selected 279 out of 776 pyrophosphatase sequences that consist of 31 eukaryal, 231 bacterial and 17 archaeal species  at room temperature. Epoxy embedding was carried out by resuspending the sample once in 1:1 ethanol/acetonitrile, twice in 100% acetonitrile, and then 30 min in 1:1 epoxy/acetonitrile, 1.5 h in 3:1 epoxy/acetonitrile, and 4 h in 100% epoxy. Embedded samples were polymerized for 20 h at 85°C. Epoxy blocks were ultrathin-sectioned, sections were deposited on 300-mesh copper grids and grids were stained with uranyl acetate for 30 min and triple lead stain  for 1 min. For Methanosarcina acetivorans, cells were processed for electron microscopy as described in [ribed in .+PPases: Vacuolar proton-translocating pyrophosphatases; Pfam: Protein family; LUCA: Last universal common ancestor; LGT: Lateral gene transfer; PDB: Protein Data Bank; HMMs: Hidden Markov Models; SAM: Sequence alignment and modeling systemV-HThe authors declare that they have no competing interests.MJS, JW and GCA conceived and designed the experiments. MJS, JW, GCA, KMK and AV performed the experiments and/or participated in data analysis.MJS, JW, GCA, and KMK wrote the paper. All authors read, edited and approved the final manuscript.Anthony Poole The work presented here makes an interesting case for the existence of compartmentation early in evolution, based on the presence of volutin granules/acidocalcisomes in all three domains of life. The authors present data indicating that vacuolar pyrophosphatases are found in all three domains of life, and interpret this as consistent with their conjecture that acidocalcisomes predate the divergence of the three domains. It is clear that the phylogeny of vacuolar pyrophosphatases cannot be used to strongly argue for presence of this gene in the LUCA, partly because of poor resolution in the dataset, and partly because of unambiguous evidence for horizontal gene transfer within the dataset. While there are some clear cases of late transfers in their phylogeny, it is harder to establish events deeper in the tree. Mapping the domain architecture of PF03030 onto this tree does not obviously strengthen the evidence, though it is striking to note the high level of conservation of the domain itself.While it certainly seems reasonable to expect that a complex organelle may only have arisen once in evolution , such expectation is not evidence in itself. In further testing this conjecture, it will therefore be interesting to identify a broader set of common signature proteins associated with this organelle, and examine their evolutionary histories. In addition, examining whether patterns of gain and loss of both the organelles and key associated proteins can be ascertained using more detailed phylogenies and character analyses within a phylogenetic framework  358, 191-203) will provide an important means of testing the conjecture presented here.The following major claims are made on the basis of the data presented:1. That Archaea possess membrane-bound acidocalcisomes/volutin granules.2. That volutin granules evolved prior to the divergence of the three domains.These are bold claims. However, my reading of the manuscript leads me to conclude that the authors do not currently have sufficient evidence to fully support either of these claims. I address each in turn.1. That Archaea possess membrane-bound acidocalcisomes/volutin granules.Methanosarcina acetivorans cells (panels c & d) wherein intracellular structures are clearly visible as either black (electron dense) or white (putatively empty). The resolution of panels c & d make it difficult to see any evidence for a membrane; plate d in particular is pixellated. I therefore find it difficult to evaluate the presented evidence as being either supportive or dismissive of a membrane surrounding these structures. No other cell biological evidence is given to support the interpretation presented in the paper, in notable contrast to the careful and extensive experimental evidence previously presented by the lead author for the characterization of acidocalcisome structures in the bacterium Agrobacterium tumefaciens . The results in figure Figure Pyrobaculum aerophilum  -- it seems somewhat surprising that no reference to this paper is made. From what I can ascertain, no data indicate an association of these pumps with any internal membrane structure in Pyrobaculum. I am therefore curious as to whether acidocalcisome-like structures have been characterized in this archaeon. Finally, given that those authors were able to express the Pyrobaculum pyrophosphatase in yeast, have antibodies have been successfully raised against this enzyme? This is helpful information, since my reading of the literature indicates that these pumps are known to associate with acidocalcisomes, though not exclusively; with that in mind, it is rather a leap to equate presence of H+-PPases in Archaea with the presence of acidocalcisomes. Experiments of the sort presented in the above-noted JBC paper are essential for any claim for the existence of such structures in Archaea.I note that proton pyrophosphatase pumps have previously been identified in Archaea, having been experimentally characterized in +-PPases in Genbank. What we are postulating is that the volutin granules observed with electron microscopy in Methanosarcina are indeed very similar to the volutin granules of Agrobacterium and Rhodospirillum, which now are known to be acidocalcisomes. In addition, our hypothesis is based on previous publications where micrographs of the volutin granules of Methanosarcina and other Archaea such as Acidithiobacillus ferrooxidans and Sulfolobus metallicus are structurally very similar to the acidocalcisomes found in bacteria. Our intention of showing these structures is to provide a visual connection between volutin granules (which we do have direct evidence of) and acidocalcisomes (an inference that requires considerably more evidence to demonstrate)Authors' Response: In this paper we neither directly claim that the volutin granules of Archaea are acidocalcisomes nor do we explicitly extrapolate acidocalcisome presence based on identification of V-H.+PPase in Pyrobaculum was made more than 10 years ago and we considered this information to be common knowledge . Interestingly, Drozdowicz et al. (1999) also made an early suggestion of the possibility that the V-H+PPases could have originated in the last universal common ancestor. However, and as the reviewer points out, no data is presented in the paper that indicates an association of these pumps with any internal membrane structure in Pyrobaculum. This is not surprising. At the time of the publication of this paper, the association between volutin granules and acidocalcisomes in bacteria had not been reported. Moreover, the absence of organelles (similar to eukaryotic organelles) in bacteria was at that time already considered a dogma in microbiology. Consequently, the search for a membrane bound organelle in bacteria was not undertaken. In the case of Agrobacterium for example, the presence of volutin granules and vacuolar pyrophosphatases were described a long time ago, but it was not until 2003 when the volutin granules of Agrobacterium were rediscovered as acidocalcisomes and the V-H+PPase was located in their surrounding membrane. Even more eloquent is the fact that in Rhodospirillum rubrum, the microbe where the V-H+PPase was first discovered, the volutin granules were never investigated until 2004, when they were confirmed as acidocalcisomes and the V-H+PPase also localized in their surrounding membranesWe consider that the information we present in this paper is significant because it provides concrete facts and clues about the possible presence of this organelle in Archaea. In addition, this work prompts further inquiry and exploration of this organelle in other organisms (including the full characterization of the volutin granules in Archaea as suggested by the reviewer). The full characterization of the volutin granules of Methanosarcina is not a trivial matter. Based on similar previous studies, it might well take 1-2 years of work to complete. Therefore, the present scope of this paper is not to prove that the volutin granules of Methanosarcina are acidocalcisomes, but instead illustrate our hypothesis that they are promising candidates (among others) to be acidocalcisomes. We appreciate the note about the work of Drozdowicz et al. (1999). The reference has now been included. Originally, we did not include this reference because the discovery of V-H.2. That volutin granules evolved prior to the divergence of the three domains.The authors also present genomic evidence (H+-PPase sequences) in support of acidocalcisomes in archaea. The implicit argument in this part of the paper is that one can extrapolate acidocalcisome presence based on identification of H+-PPases in Genbank.+PPases  is not evidence against an ancient pre-LUCA origin. Having said that, it is not evidence in favor of it either. The dual forces of mutation and gene transfer are having a major impact on the reconstruction of the deep evolutionary past and it may be that signal has in most cases been heavily degraded, to the point that it cannot be informatively identified.There are two interesting points here. First, it is nice to see that the authors are honest regarding the limits of phylogeny in this particular instance. The previously inflammatory acronym MUTOG  does need to be taken seriously following the recognition that most genes do not carry sufficient phylogenetic signal to trace the relationships between species across the three domains. Moreover, with HGT an indisputable factor in gene distribution, this caution is not only welcome, but necessary. Against this backdrop, that a clearly resolved three-domain tree cannot be generated for the V-H+PPases and these organelles across representatives from all three domains of life. This would suggest that multiple losses have occurred throughout evolution, from a more compartmentally complex common ancestor. What we still don't know, and are challenged to think about by this paper, is whether complex internal membrane structures such as these could arise multiple times independently. In this respect, comparative analyses do have an important role to play in establishing what the minimum number of components required to support an acidocalcisome or volutin granule may be. The variability in membrane number and architecture observed across Planctomycetes (Fuerst) does suggest a greater capacity for evolution of novel membranes than suggested by a stringently 'genetic' view  of membrane evolution. More fine-scale examination of whether the distribution of key proteins localized to acidocalcisomes/volutin granules completely overlaps with the existence of these organelles. In addition, looking at evolution within a group (e.g. relatives of Agrobacterium tumefaciens) may help elucidate whether or not horizontal gene transfer does play a role in the emergence of such complex cell structure. It might be time to ask just whether our assumptions about how 'hard' it is to evolve a compartment are in need of scrutiny.The second point lies at the heart of this paper. The triumph of endosymbiotic theory has been the recognition that some key eukaryotic organelles did not evolve de novo, but are instead the result of one cell becoming resident within another. The emerging view of complex membrane structures has therefore been one of continuity (TCS), though this may yet be challenged by the recognition that both bacteria and archaea can harbor complex cell structures. In the case of acidocalcisome/volutin granule organelles, it seems plausible to suggest a deep evolutionary origin, given the presence of both the H+PPase gene sequences, a common finding in many genes that exist in a genome, tells little about the ancestrality of the V-H+PPase gene. Lack of phylogenetic signal in the V-H+PPase sequences is probably the result of (i) high sequence conservation of segments that correspond to the Pfam 03030 domain that are present in each and every one of the sequences examined, including sites necessary to maintain fold structure and sites needed for catalytic activity, and (ii) segments of sequence that are highly variable and prone to sequence saturation that contain highly degraded phylogenetic signal and probably arise from the complex and frustrated interplay between stability and function that exists in proteins Authors' Response: We agree with the reviewer that the poor phylogenetic resolution of the tree generated from V-H[+PPase sequences and in every other V-H+PPase sequence we examined provides strong support to the suggestion that the functional and structural core of this domain was already present in LUCA and was vertically distributed without loss in all lineages of the tree of life. The size of the domain (an average of 603 amino acids covering 94% of the sequence with a model length of 682 amino acids) was calculated based on start and end positions on the HMM (Pfam) alignment of sequences obtained by searches in the Sanger database. The average identity of the complete alignment is 44%. Despite sequence variability, the domain is therefore highly conserved and universally present. More information about seed sequences and PF03030 domain distributions can be found elsewhere. (http://pfam.sanger.ac.uk/family/PF03030#tabview=tab6).. In contrast, the universal distribution of the Pfam 03030 domain in the tree of V-Hproteins . In contThe second point raised by the reviewer is important for the genesis of organelles and cellular structure. The proteome of LUCA was recently reconstructed and was shown to embody the functions of a complex organism, with pathways for the biosynthesis of membrane sn1,2 glycerol ester and ether lipids [. An ancient volutin/acidocalcisome structure present in LUCA implies gradual evolution of internal cellular structure in the universal ancestor of cellular life. By definition, LUCA implies the absence of cellular lineages. Consequently, a process of endosymbiosis between lineages similar to that used to explain the origin of mitochondria or plastids could not be responsible for the rise of volutin/acidocalcisome organelles without invoking endosymbiosis was an ancient and pervasive property of LUCA and that these polyphosphate harboring cellular structures were remnants of that cellular state. Alternatively, cellular structure other than an external membrane may be the logical consequence of energetic and architectural constraints operating on membrane organization [. In vitro experiments of membrane biogenesis show considerable diversity of cellular shape and structure, with cellular engulfment being a common outcome [r lipids . An ancinization . In vitr outcome ,85.+PPases). We do not yet know whether independent recruitment  of key functionalities, such as the H+PPases, are sufficient for the independent evolution of a new membrane-bound organelle. The authors make the reasonable assumption that loss is more likely, and bolster their argument by relating their provocative model a possible early role for pyrophosphate/polyphosphate in the origin and early evolution of life. The obvious problem with scenarios that invoke loss from a more complex ancestral state is that one also needs to explain the original gain. The present manuscript does not give us an answer, but this and other results do challenge us to keep an open mind.The argument presented here, that compartmentation is much older than we may expect based on the presumed late origin of the eukaryote nucleus and endomembrane system, has been given an additional boost in this manuscript from Seufferheld and colleagues. However, the jury is certainly still out -- on the present data, it is difficult to distinguish between either a deep evolutionary origin with numerous losses, or 'convergent' origins + PPase domain, a component of the acidocalcisome in the eukaryotes and bacteria. They argue that the acidocalcisome was present in LUCA along with the V-H+ PPase.Seufferheld and colleagues perform a phylogenetic analysis of the V-HCritique:+ PPases suggests a complicated evolutionary history with loss and possible lateral transfer playing a role in their distribution. However, reconstruction to LUCA is not strongly supported. Firstly this domain family is not widespread in the archaea. Further, the major archaeal branches (e.g. eury- and cren- archaea) also do not cluster with each other. Even within the bacteria, homologs from monophyletic groups do not group with each other. Given the presence of this family in diverse bacteria, I would find it more parsimonious to reconstruct the evolutionary history of this family as having evolved in the bacteria with subsequent lateral transfer to the archaea and eukaryotes.The phylogenetic analysis of the V-HThe paragraph on the Pfam domains does not fit into the paper since these proteins show no domain complexity. Further, in this case the Pfam domain deposited in the database only captures the key catalytic motif of the protein. If the authors wish to discuss the domain in terms of sequence, they can write about the various transmembrane helices and the context of the catalytic residues.+PPase proteins include the amino acid sequence of the entire membrane-associated protein and not only of the domain regions. Furthermore, our phylogenetic tree describes the evolution of the sequence in 279 organisms spanning the three superkingdoms of life, Archaea (17 species), Bacteria (231), and Eukarya (31). These 279 sequences were selected from an analysis of a larger set of 776 sequences, following removal of paralogs or sequences with no genetic distance or that have no clear taxonomical descriptions. We find that the V-H+PPases are present in every one of the 776 organism analyzed and that the Pfam domain PF03030 is always present and captures the main sequence profile of the structure of these proteins. The claim of the reviewer that the domain is absent in Archaea and that its evolutionary history follows a complex series of losses and possible gains through LGT is therefore incorrect. The protein and associated domain is simply present everywhere. While variants could have been recruited by LGT and replaced, the protein was never lost in any lineage examined. We agree that LGT events are possible. They could have been responsible for the lack of phylogenetic signal that limits the correct grouping of organisms in the V-H+ PPase tree. However, the topology of the tree has clearly defined groups that match taxonomical classification, including the Euryarchaeota, Proteobacteria, Thermotogales, Actinobacteria and Firmicutes. Some cases show non-monophyletic groupings that could have resulted from LGT. Nevertheless, it is important to notice that in several of these non-monophyletic groupings the phylogenetic support is very poor 0.5 to 0.76. While the tree of the vacuolar pyrophosphatase is one gene tree and has a largely star-like internal topology, it is significant that the phylogenetic reconstruction of this gene maintains essentially the three superkingdoms of life.Authors' Response: Our phylogenetic analyses of the V-HAn analysis of the mean distance between protein sequences using the JTT model with gaps excluded in the analysis showed that levels of sequence identity were 64.4%, much larger the 30% expected in the presence of high conservation levels. Using JALVIEW, we extracted conservation scores  for every nucleotide site with a proportion of gaps that is less than 25%. Analysis of the 523 sites  that fulfills that criterion, shows the vast majority of sites have conservation scores above 7  and many have scores above 10  . These results support the notion that V-H+PPases do not exhibit a complex domain organization. Complex domain arrangements are the hallmark of recently evolved proteins in the protein world While a minority of sequences contain a duplicated PF03030 arrangement or are associated with another domain (PB008043), most contain a single Pfam domain. Thus, and as the reviewer correctly points out, our analyses show V-H[+PPases provides additional clues in support of their ancient origin. Remarkably, an analysis of trans-membrane helices (TMH) of the protein shows also high levels of structural conservation. The number of TMHs ranges 9-11with an average of 10.2 ± 0.75 (SD) and TMHs hold an average of 243.2 ± 20.30 amino acid residues. TMHs therefore represent on average 42.1 ± 3.46% of the protein. Figure . Consequently, the domain make up of V-H3+PPases are therefore evolutionarily conserved at different levels, including sequence, domain structure, and TMH make-up, a fact that speaks about their ancient origins.illustrates the position of the two PF03030 domain and the 10 TMHs in the sequence of R. rubrum YP426905, the model system for acidocalcisome studies. The V-Hin world . ConsequDaniel Kahn This reviewer provided no comments for publicationMultiple alignments of PF03030 domain sequences distributed in the three superkingdoms . Shown above is the strongly conserved 57-residue region of the V-H+-PPase identified by Hedlund et al [nd et al  and viewClick here for fileTaxa and accession numbers used in the phylogenetic analyses.Click here for file


N-acetyl-Ser–Asp–Lys–Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P2 position (compound 33RE) displayed potent inhibition (Ki=11.21±0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr369. This work further elucidates the molecular basis for N-domainselective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders.ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP ( The present paper reports the development of an N-domain selective ACE inhibitor and the molecular basis for the interaction of key active site residues with the ligand. In vivo both domains clear the vasodilator peptide bradykinin with approximately equal efficiencies [N-acetyl-Ser–Asp–Lys–Pro) [ACE  is a zinc dipeptidyl carboxypeptidase that plays a critical role in blood pressure regulation and electrolyte homoeostasis . ACE conciencies –9, the CLys–Pro) –10. Ac-SLys–Pro) ,12. MoreLys–Pro) –25. FurtLys–Pro)  and suggThere are a number of ACE inhibitors that have been approved for clinical use . These iN-acetyl group, P2 aspartate residue and C-terminal amide as contributors to the observed N-selectivity [2 aspartate residue and N-protecting group resulted in a 1000-fold shift towards N-selectivity [RXP407 is a phosphinic peptidomimetic ACE inhibitor that displays approximately three orders of magnitude N-selectivity . Detaileectivity . Using tectivity .369 and Arg381 (by the P2 aspartate residue) were exploited by the inhibitor [Elucidation of the ACE domain structures has allowed for the understanding of active site architecture and the relative positioning of amino acids that differ between the two domains ,6. Furthnhibitor . This obnhibitor . StructuIt was the purpose of the present study to exploit unique N-domain residue interactions with novel chemotypes using a receptor-based SHOP (scaffold hopping) GRID-based molecular modelling approach ,37. Suchhttp://www.clinsci.org/cs/126/cs1260305add.htm). Four amino acids differing in chemical nature from their C-domain counterparts were selected. Residues Tyr369 and Arg381 are located in the S2 subsite and have been shown by both mutagenesis and structural studies to be important for selective RXP407 binding [391 and Glu403, respectively, in the C-domain . Thr496 is located on the border of the S2 and S1 subsites [518), it was selected as a side-chain for further selective binding exploitation by hydrogen bonding. Thr358 is located in the S2′ subsite and differs from the corresponding C-domain residue Val380 [496, identification of functional groups that allows for specific interaction with the threonine side chain by hydrogen bonding could assist in generating N-selective inhibitors. Novel inhibitor structures that met the criteria were selected for further study. Final compound 33RE (http://www.clinsci.org/cs/126/cs1260305add.htm).SHOP methodology was employed to screen for commercially available building blocks that would possess similar or improved active site interaction properties . Assays were performed on wild-type N-domain, C-domain and N-domain S2 mutants Y369F, R381E and YR/FE as described previously [λex=320 nm and λem=420 nm using a fluorescence spectrophotometer . Initial change of fluorescence over time was converted into arbitrary rate units and inhibitor-binding affinities determined using the Dixon method [Parent molecule RXP407 and compound eviously ,35. Brie33RE (3.3 mM) at room temperature (20°C) for 2 h before crystallization. Pre-incubated sample (1 μl) was mixed with the reservoir solution consisting of 30% PEG550 MME/PEG20000, 100 mM Tris/Bicine, pH 8.5, and 0.06 M divalent cations (Molecular Dimensions), and suspended above the well. Crystals appeared within 3 days.N-domain ACE was expressed and purified to homogeneity from CHO (Chinese-hamster ovary) cells . The cry33RE complex were collected on the PX station IO4-1 at Diamond Light Source (Didcot). A total of 720 images were collected using a Quantum-4 CCD (charge-coupled-device) detector (ADSC Systems). No cryoprotectant was used and the crystal was kept at constant temperature (100 K) under the liquid nitrogen jet during data collection. Raw data images were indexed and scaled with XDS [Fo–Fc electron density peaks exceeded 3σ and potential H-bonds could be made. On the basis of the electron density interpretation, the inhibitor and glycosylated carbohydrate moieties were added in the complex structure and further refinement was carried out. The co-ordinate and parameter files for 33RE were generated using SKETCHER [X-ray diffraction data for the N-domain ACE–with XDS  and the with XDS . Initialwith XDS . The atowith XDS   in a similar range to parent molecule RXP407 (i=K21.03±0.27 nM). Characterization of the C-domain showed micromolar inhibition with compound 33RE (iK=10 395±593 nM) as well as RXP407 (iK=60 826±9 175 nM), thus both compounds displayed three orders of magnitude N-selectivity  compared with wild-type N-domain. Upon substitution of both these residues in the double mutant YR/FE an additive effect was observed (iK=2794±156 nM), which led to a drastic decrease in 33RE selectivity (only ~4-fold N-selectivity compared with 927-fold with wild-type). Given the importance of these two residues in conferring selectivity for both 33RE and parent molecule RXP407, the kinetic results suggest that the actual interaction energies for the two inhibitors with these residues is critical [Assessment of inhibitor potential was carried out using a continuous fluorogenic assay (Abz)-FRK(Dnp)P assay. Compound ectivity . The actcritical .33RE  and Thr358 (S2′ subsite), where RXP407 has minimal contact with these unique N-domain residues, fragments were identified that had high predicted interaction energies. One such scaffold was synthesized using approaches that attempted to enhance the turnaround time of inhibitor production.Using the above receptor-based SHOP methodology, eight scaffolds were identified with known fragments that displayed equal or improved predicted interaction energies towards identified residues compared with RXP407. Specifically, the approach was able to identify fragments in the P33RE to C- and N-domains, and therefore N-selectivity, is in the same range as that of the parent molecule. The data for RXP407 presented here compare well with previously published reports [33RE would be comparable to RXP407. Kinetic analysis indicated three orders of magnitude N-selectivity for both compounds and serves as a proof of concept for using the discussed SHOP methodology in ACE inhibitor design. The co-crystal structure of N-domain ACE with 33RE confirmed the interactions common to RXP407 and highlighted the importance of the P2 subsite for N-selectivity. The main difference in binding was seen in the loss of a salt bridge with Arg381 but enhanced interactions with Tyr369. Importantly, these two residues are not conserved in the C-domain (replaced by Glu403 and Phe391 respectively). Kinetic characterization revealed an 8-fold decrease in binding affinity for 33RE upon mutating Arg381 to Glu, suggesting an alternative orientation of this residue than that seen in the crystal structure. A 36-fold decrease in binding affinity was observed for Y369F confirming the structural findings of a loss of direct and water-mediated hydrogen bonds to the P2 tetrazole of 33RE. Thus, Tyr369 appears to be the major contributing residue towards the N-selectivity of compound 33RE. The importance of the P2 subsite for selectivity was further emphasized by the additive effect observed in YR/FE constituting a 250-fold decrease in 33RE-binding affinity compared with wild-type, similar to that seen previously for RXP407 [388, thus potentially improving affinity to both domains with a subsequent slight decrease in N-selectivity compared with parent molecule.Assessment of inhibition was conducted using a fluorogenic (Abz)-FRK(Dnp)P assay. This analysis indicated that binding affinity of compound  reports ,35. Sinc2 candidate (compound 33RE) was synthesized and displayed potent and N-selective inhibition. This novel phosphinic ACE inhibitor provides a basis for incorporation of non-amino acid and non-carboxylate P2 functionalities into clinically relevant inhibitor backbones. It is noteworthy that a tetrazole moiety represents a bioisosteric replacement for a carboxylic acid group. N-domain selective inhibitors with drug-like characteristics could be useful in the treatment of tissue injury and fibrosis, which currently have limited treatment options.In conclusion, a modified SHOP methodology was employed to identify novel chemotypes with improved interactions with certain unique N-domain amino acid residues. A shortlist of scaffolds was identified and predicted to have equal or improved N-domain interactions compared with parent molecule RXP407. The most promising non-carboxylate P•N-acetyl-Ser-Asp-Lys-Pro. Thus a fragment-based approach was used to design a novel inhibitor that was specific for the N-domain.The N-domain catalytic site of ACE is highly selective for the antifibrotic and anti-inflammatory tetrapeptide •2 position displayed potent inhibition of the N-domain and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of the inhibitor in complex with the N-domain revealed its mode of binding through aromatic stacking with His388 and direct hydrogen bonding with the N-domain-specific Tyr369.A molecule incorporating a tetrazole moiety in the P•This work further elucidates the molecular basis for N-domain-selective inhibition and assists in the design of novel N-selective ACE inhibitors that have potential for the treatment of fibrosis disorders


Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley ( Saccharomyces cerevisiae, causing defects in kinetochore assembly, and also as a protein that interacts with the SCF ubiquitin ligase complex SGT1 increases the susceptibility of plants to pathogen attack and growth. Plant SGT1 is also involved in the auxin and jasmonate response, which is mediated by SCF-ubiquitin ligase complexes SGT1, a suppressor of the G2 allele of skp1, is a highly conserved  and esseArabidopsis thalianaArabidopsis thaliana, the expression of two SGT1 isoforms, AtSGT1a and AtSGT1b, depends on the TPR domain sequence The SGT1 protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2), which lie between the conserved domains  [8]. So SGT1 by testing the susceptibility of wild-type Nicotiana benthamiana plants for to PVX virus infection and discovered a dominant-negative mutation in AtSGT1b (Glu119Gly) It has recently been observed that plant and yeast SGT1 proteins can dimerize in a low ionic strength environment and at high protein concentrations Small-angle X-ray scattering (SAXS) is one of the most effective experimental methods for analysis of protein structure in solution. This method is especially useful for proteins that are difficult to crystallize  (for review see: Hordeum vulgare). In solution, the plant SGT1 protein exists in monomer-dimer equilibrium; with increasing ionic strength, this equilibrium is shifted towards the monomeric state. Using a novel SAXS data analysis method ab-initio methods.There is limited structural information on full-length SGT1 proteins. In this study, we used SAXS and synchrotron radiation to determine, for the first time, the structure and properties of the full-length SGT1 protein from barley (Escherichia coli BL21 DE3 Codon Plus. The cells were cultured in Luria Broth (LB) liquid medium supplemented with ampicillin (50 µg/ml) and chloramphenicol (30 µg/ml) , and a total of 1.6 L of LB medium was inoculated with 40 ml of overnight culture. The culture was incubated at 37°C with shaking (200 rpm) until the OD600 nm reached 0.6–0.8; IPTG was then added at a final concentration of 1 mM. Cells were grown for 5 hours and harvested by centrifugation at 10000 g at 4°C for 30 minutes and were subsequently frozen at −20°C until further use. The frozen cells were than resuspended at 4°C in lysis buffer , disrupted by sonication (5 times 60 sec. on ice) and centrifuged at 10000 g. In the next step, the supernatant was applied to a HisTrap 1 ml FF Ni-NTA column  using Akta Explorer X10. The column was washed with a binding buffer , and the barley SGT1 protein with a His-TEV site was eluted using a gradient of elution buffer . The fractions that contained the barley SGT1 were concentrated using the Amicon 10 kDa MWCO (Millipore) centrifugal filter device and purified using Superdex 200 pg HiLoad 16/60 . The first fraction of the SGT1 peak was cleaved using His-Tev  The full length barley SGT1 cDNA cloned into the pET151-D TOPO  vector was used for the transformation of Hordeum vulgare SGT1 (6–124) was amplified from the pET151-HvSgt1 plasmid by standard PCR reaction and cloned into the pETM11 vector E.coli BL21(DE3) Codon Plus cells. Transformed cells were grown at 37°C (to OD600 nm = 0.6–0.8) and induced with 1 mM IPTG. Induced cells were grown overnight at 20°C. After expression, bacterial cells were centrifuged at 4°C at 5500 g for 30 minutes and frozen at −20°C. For purification, the cells were thawed on ice and resuspended in buffer A  supplemented with 0.1% Triton X and protease inhibitor cocktail. The cells were disrupted by five cycles of sonication (30 sec. on/1 minute on ice) and centrifuged for 1 hour at 10000 g at 4°C. The cleared lysate was loaded onto the HisTrap FF 1 ml column washed with 10CV of buffer A and eluted with gradient 0–100% of buffer B (50 mM sodium phosphate pH 7.5 500 mM NaCl 500 mM imidazole and 10 mM 2-mercaptoethanol) over 20CV. The fractions containing TPR domain protein were collected, supplemented with TEV protease for his-tag cleavage and dialyzed against buffer A overnight at 4°C. The TEV protease and unclaved TPR domain were removed by the HisTrap FF 1 ml column. The TPR protein was concentrated and purified by size exclusion chromatography using HiLoad 16/60 Superdex 200 pg column equilibrated in buffer C . The purified protein was concentrated to 10 mg/ml, snap frozen in liquid nitrogen and stored at −80°C for further use.The cDNA sequence of TPR domain from 2PO4 pH 7.5, 2 mM 2-mercaptoethanol and dialysed overnight for 12 hours against buffer A.The variants of barley SGT1 protein: TPR-CS (1–261) and CS-SGS (174–373) obtained after the expression in vector pnEA-vH Arabidopsis thalina SGT1a protein were cloned into vectors pETM41 2PO4 pH 7.5, 5 mM 2-mercaptoethanol.CS domains from barley SGT1 protein and from Arabidopsis thalina (46.7 µM) CS domain form were studied from 205 nm to 280 nm. All CD data were collected at a 50 nm/sec scanning speed and 1 nm band-width. Buffer subtraction and spectra deconvolution procedures were conducted using the Jasco Spectra Manager II software and using CDSSTR module on Dichroweb server The protein samples for CD measurements were dialyzed overnight against a 10-mM sodium phosphate buffer (pH 7.5) at 4°C. Following dialysis, the sample was centrifuged at 14000 rpm for 30 minutes at 4°C and filtered through a 0.1-µm filter. CD measurements were conducted at room temperature using diluted protein samples (c = 10–47 µM) and a Jasco J-815 spectropolarimeter  with a 0.02 cm cell length path. For full-length SGT1 (c = 12 µM) a total of six scans, ranging from 176 nm to 280 nm, were collected. CD data for TPR-CS (c = 10.4 µM), CS-SGS (12.8 µM) were collected from 190 nm to 280 nm. CS domain from barley SGT1 protein (c = 47 µM) and CS domain of SGT1a from ab initio protein folding and structure prediction, QUARK (TPR domain) and iterative structure assembly and refinement approach implemented in I-TASSER (CS and SGS domains) ab initio modeling server QUARK (http://zhanglab.ccmb.med.umich.edu/QUARK/) and using first template search in PDB data base and then replica-exchange Monte Carlo simulations with additional loop modeling with structure refinement in the third step (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).The three-dimensional structures of individual SGT1 domains  were generated using a computer algorithm for 2), with a sample-to-detector distance of 2.7 m, which corresponds to an s-axis range of 0.06 to 6 nm−1, using synchrotron radiation (wavelength λ = 0.15 nm). All measurements were performed using a flow cell (70 µl volume) with polycarbonate windows −1 cm−1. The solution scattering data were corrected for detector response and normalized to the incident beam intensity, and the scattering of the buffer was subtracted using the program package PRIMUS Streptomyces rubiginosus were used as references for the molecular weight calibration SAXS measurements were performed on the X33 Beamline of EMBL Hamburg Outstation on the DORIS storage ring at DESY 2) and with a sample-to-detector distance of 2.867 m, (s-axis range of 0.06 to 6 nm−1). For small angle X-ray scattering experiment the TPR domain was dialyzed against low salt buffer . The samples were prepared by mixing dialyzed protein with high salt buffer  for final KCl concentration: 0, 100, 200, 400, 600 oraz 800 mM and protein concentration 2 mg/ml. All experiments were done at 15°C using continuous flow cell (sample volume = 30 µl). SAXS data were processed using the same procedures and software as for full length barley SGT1.Solution scattering data for TPR domain were collected on the BM29 BioSAXS Beamline T to reconstruct the data set A. To minimize errors in the SVD analysis, we used experimental data to 2 nm−1. Using PRIMUS, we established that only two eigenvectors were statistically important. The PRIMUS result was also confirmed by analyzing the autocorrelation of the calculated eigenvectors from matrix U, which showed that only two eigenvectors had non-zero autocorrelation functions. Pure scattering function reconstruction of the components was conducted using the Multivariate Curve Resolution Alternating Least-Squares (MCR-ALS) algorithm implemented in MATLAB ab-initio SAXS modeling.To estimate the number of species in solutions of the barley SGT1 protein and TPR domain, we employed singular value decomposition (SVD) analysis using the PRIMUS Two independent programs, DAMMINv51 ab initio modeling, we also applied rigid body modeling, using the program BUNCH To confirm the barley SGT1 models obtained from Modelling of the TPR dimer against SAXS data was performed in SASREF 6.0 ab-initio modeling from BUNCH, to model multiple chain and domain proteins Modeling of the barley SGT1 dimer was performed using the CORAL program from the ATSAS package, and the scattering curve was obtained from the MCR-ALS analysis. CORAL combines two algorithms, rigid body modeling from SASREF and multidomain protein modeling using rigid body and ab-initio modeling was necessary because there is no structural model of the barley SGT1 protein. The structure of the three domains of the barley SGT1 protein was modeled using the ab-initio modeling platform QUARK and meta-threading approach approach implemented in I-TASSER, which were chosen because are ranked as the best platform in the free-modeling and protein prediction competition in CASP9 Arabidopsis SGT1 homologs and has a typical beta sandwich structure (see The use of ture see . The strhttp://zhanglab.ccmb.med.umich.edu/QUARK/). The model has a canonical TPR structure with three tetratricopeptide repeats, each repeat being formed by two anti-parallel helices. A solvating helix is located at the C terminus of the domain. This helix is typical for all structures of TPR domains solved so far, and it is hypothesized to increase the solubility and stability of the TPR fold G) characterizing the TPR domain in solution were 1.66 nm and 2.04 nm for TPR monomer and dimer, respectively. In max) characterizing the TPR dimer and monomer were estimated from the p(r) functions  was obtained using the online modeling platform QUARK , as well as CD spectrum for CS-SGS protein (obtained in the presence of 30% Ficoll 70) are presented in The CD spectra (from 190 nm to 280 nm) collected for TPR-CS and CS-SGS proteins together with their deconvolution are presented also in Circular dichroism spectroscopy clearly shows the modular architecture of the barley SGT1 protein, with the α-helical TPR domain, the β-sheet sandwich CS domain, and the disordered SGS domain separated by the VR1 and VR2 regions. The α-helix content of the barley SGT1 protein is 33.9%, which corresponds to the TPR domain. The CS domain of the studied SGT1 is responsible for 21.9% of the β-sheet secondary structure. The SGT1 also has 11.8% of the β-turns and 33.5% of the random coil structure. VR1 and VR2 represent 67 of the total 373 amino acids (18%). Assuming that both VR1 and VR2 are in a random coil conformation, almost 12% of the random coil content must originate from the rest of the protein.The β-turn content (12%) can be ascribed to some unstructured regions outside the VR1 and VR2 regions. The SGS domain consists of 84 amino acids. Because the isolated SGS domain shows NMR spectra characteristic of unfolded proteins The content of α-helices determined for TPR-CS protein from CD data was 36% (found mainly in TPR domain), while the content of β-strands was 17% (mainly in CS domain)  that characterize the SGT1 protein were found to increase with decreasing ionic strength, suggesting that in solution, SGT1 exists in equilibrium between the monomeric and dimeric states. It indicates that the dimerization process takes place at low ionic strength conditions. The protein sample at the high salt concentration (1 M NaCl) was characterized by the radius of gyration RG = 5.02 nm, and the value of RG increased to 5.52 nm in a low ionic strength environment (0 M NaCl).We assumed that the barley SGT1 protein underwent dimerization but not aggregation because the scattering data fitted into the linear range of the Guinier plot, as shown in Arabidopsis thaliana and Homo sapiens have been solved, using NMR spectroscopy and X-ray diffraction methods −1 . This range of SAXS data can be used for shape determination methods and rigid body modeling. In the first stage, using the SVD method max) characterizing the barley SGT1 dimer and monomer were estimated from the p(r) functions and were found to be 17 nm and 22 nm for the monomer and the dimer, respectively. The pair distance distribution function of the monomeric barley SGT1 has two maxima that are characteristic of a multidomain particle. For the barley SGT1 dimer, the first maximum is slightly shifted towards greater distances, and the second maximum is much broader; both peaks are less resolved, a consequence of the dimerization process. The calculated p(r) functions for the monomer and dimer have their maxima in the same positions as the maxima of the functions calculated using raw experimental data at various salt concentrations (data not shown), confirming that the MCR-ALS analysis is accurate.As noted previously, a three-dimensional structure of the full-length SGT1 protein has not yet been proposed. Only the structures of the CS domains of the SGT1 protein of ab initio shape determination method. The pure scattering function for the monomeric SGT1 protein, taken from the MCR-ALS analysis, was used to obtain the low-resolution structure in solution using the ab-initio approach implemented in the DAMMIN program The first insights into the full-length structure of the barley SGT1 protein were performed using the ab initio methods As previously shown, SAXS can help to predict the solution structure of a particle on the basis of ab initio modeling of low-resolution structures, we also applied rigid body modeling, using the program BUNCH To confirm the barley SGT1 models obtained from G = 3.67 nm, and a pool of models with the SGS domain represented as dummy residues (unfolded domain) is characterized by a RG value of 4.10 nm  was chosen  4.10 nm . After mG value of 5.08 nm. The model with the unfolded SGS domain is characterized by RG value of 5.39 nm which is slightly higher than the RG value calculated directly from the experimental data using 1 M NaCl. In both the cases, bell-shaped RG histograms were observed, with an additional maximum just above 4 nm for the model with the folded SGS domain. On the basis of analysis of the RG histogram . This difference suggests that the variable regions (VR1 and VR2) of barley SGT1 provide some flexibility but also ensure the rigidity and extended conformation of the protein. The fit to the extracted scattering data for the monomeric SGT1 model is slightly better for the EOM procedure than for the structure with an unfolded SGS domain (χ2 = 1.267), in comparison to the folded SGS domain model (χ2 = 1.289). The observed difference is most likely caused by the quality of the SGS domain model generated by I-TASSER.The model with the folded SGS domain is characterized by an average Ristogram , we concArabidopsis thaliana and Saccharomyces cerevisiae form dimers Nicotiana benthamianaIt is known that the human SGT1 protein does not dimerize under the conditions in which the SGT1 proteins from In the current study, the structure of the full-length barley SGT1 protein in solution has been determined. As can be expected from a flexible multidomain protein, all three structural domains behave independently. Similar observations were made when the results of the NMR structural studies of the isolated domains and the full-length human SGT1 protein were compared It was possible to extract the pure scattering function of monomeric and dimeric species from the monomer-dimer mixture using SVD and MCR-ALS analysis. This allowed the modeling of a low-resolution structure. The barley SGT1 monomer, in solution, has an elongated shape with a slight bend in the middle, with the domains behaving similar to rigid beads on a string. The bend occurs at the site at which the CS domain is located. The dimeric form of the barley SGT1 has a similar elongated shape. It was possible to fit all three models of SGT1's domains into our low-resolution model of the dimer. The TPR domains form a hub from which the rest of the protein protrudes in opposite directions. Both ends of the SGT1 protein can help bring molecular complexes together to perform special tasks inside the cell. As shown by rigid body modeling, the regions between TPR, CS and SGS domains are natively unfolded and have a stretched conformation that confers flexibility and dynamics upon the barley SGT1 protein. Additionally, on the basis of our CD experiments, we can assume that all 84 amino acids of SGS are rather unfolded; 22% of secondary structure content should be in the random coil conformation in comparison to the estimated 12%. Therefore, the SGS domain studied may not be fully unfolded. SGS domain is the most conserved part of SGT1 proteins. The second class of proteins with SGS domain are Calcyclin-binding proteins (CacyBP) EOM studies revealed that the best fit to the rigid body model of the barley SGT1 protein was obtained using an ensemble of models with the average radius of gyration equal to 5.10 nm, larger than that for the monomeric form. Dimerization also does not have any impact on the conformation of the whole protein, because only TPR domains participate in this process. It is also possible that the VR regions may play some functional role. The long unfolded region with clustered hydrophobic residues may bind unfolded or hydrophobic regions of other proteins. SGT1, as a protein interacting with HSP90, can also bind other unfolded proteins quite well. Thus, the SGT1 protein can function to load proteins onto HSP90. A similar condition was observed for the p23 protein, which has a 50 aa C-terminal tail that is required for its intrinsic chaperone activity Figure S1http://ekhidna.biocenter.helsinki.fi/dali_lite/start) Structural alignment of various TPR domains to the barley SGT1 TPR domain model. Hop TRP1A – PDB ID: 1ELW (TIF)Click here for additional data file.Figure S2Arabidopsis thaliana – PDB ID: 2JKI chain S Homo sapiens – PDB ID: 1RL1 chain A Hordeum vulgare (pink) used in SAXS modeling of full length SGT1 dimer and monomer. Human and barley SGT1 CS domains possesses 40% sequence identity and 1.0 Å RMSD between aligned structures. Arabidopsis thaliana SGT1a CS domain possesses 60% sequence identity and 1.6 Å RMSD between aligned structures. Alignment was prepared using Dali structure alignment server  Experimentally determined structures of SGT1 CS domain from (TIF)Click here for additional data file.Figure S3Arabidopsis thalina (reference protein) (blue).The experimental CD data (A) for barley CS-SGS protein (red) and CD spectrum obtained for CS-SGS protein in the presence of 30% Ficoll 70 (blue). CD spectra (B) for CS domain from barley SGT1 protein (red) and CS domain of SGT1a from (TIF)Click here for additional data file.Table S1Structural alignment of various TPR domains to the barley SGT1 TPR domain model.(DOCX)Click here for additional data file.


Proteins are composed of a combination of discrete, well-defined, sequence domains, associated with specific functions that have arisen at different times during evolutionary history. The emergence of novel domains is related to protein functional diversification and adaptation. But currently little is known about how novel domains arise and how they subsequently evolve.To gain insights into the impact of recently emerged domains in protein evolution we have identified all human young protein domains that have emerged in approximately the past 550 million years. We have classified them into vertebrate-specific and mammalian-specific groups, and compared them to older domains. We have found 426 different annotated young domains, totalling 995 domain occurrences, which represent about 12.3% of all human domains. We have observed that 61.3% of them arose in newly formed genes, while the remaining 38.7% are found combined with older domains, and have very likely emerged in the context of a previously existing protein. Young domains are preferentially located at the N-terminus of the protein, indicating that, at least in vertebrates, novel functional sequences often emerge there. Furthermore, young domains show significantly higher non-synonymous to synonymous substitution rates than older domains using human and mouse orthologous sequence comparisons. This is also true when we compare young and old domains located in the same protein, suggesting that recently arisen domains tend to evolve in a less constrained manner than older domains.We conclude that proteins tend to gain domains over time, becoming progressively longer. We show that many proteins are made of domains of different age, and that the fastest evolving parts correspond to the domains that have been acquired more recently. Proteins are organized in discrete functional modules called domains -3. DomaiDomains that originated in particular lineages are of special interest in helping understand the molecular basis of lineage-specific adaptations ,5,7,11. One well-characterized example of lineage-specific domain is the Kruppel-associated box (KRAB), found in several vertebrate species but greatly expanded in mammals. This domain combines with the Zn-finger motif, which is an older domain, and confers strong transcriptional repressor activity to the protein . There iDrosophila.In spite of the fact that recently emerged domains are likely to play key roles in the evolution of new protein functions, we still know very little about them. For example it is not known which fraction of young domains is located in newly evolved genes, or which fraction is formed in the context of older - previously existing - genes. Recently originated genes have special characteristics: they are poorly annotated ,18, theyWe obtained from Ensembl a set of 14,599 human genes with 1:1 orthologs in mouse . We usedAbout 12.3% of the domain types only had homologues in species separated from human in the approximately past 550 million years , suggesting that they had originated relatively recently . However, some domains were present in paralogues. One example is the Transcription elongation factor A domain (TF_A/BEX domain), present in a family of transcription factor genes located on chromosome X and which include TCEAL7, a putative tumour suppressor gene, which negatively regulates NF-kappaB mediated pathways .Recently evolved domains may be parts of new proteins or, alternatively, arise in proteins with an older origin. In order to know which proportion of young domains fall into each of these two categories we dated the age of all proteins in the dataset. We defined the age of each protein by the oldest domain it contained or, if it did not have any known domain (21.7% of proteins), by BLASTP sequence similarity searches against the 15 species proteomes. The majority of young proteins contained no annotated domains , in contrast to older proteins (only 14% did not contain known domains). We found that the number of domains per protein, as well as the complete protein length, increased with the age of the protein  and 234 of the 363 Vertebrate domain types (64.4%). In the remaining cases the younger domain could be found combined with one or more older domains , reflecting either domain fusion events or the emergence of novel domains in existing older proteins.de novo domain emergence we examined all possible domain arrangements involving Vertebrate and Old domains present in the 330 human proteins that contained both classes of domain. For each different Old-Vertebrate domain type pair we examined the individual domain occurrences. The largest group was composed of pairs in which the Vertebrate domain only combined with a particular Old domain, whereas the Old domain was found in other domain configurations as well. These cases were compatible with the gain of a novel domain in an existing protein .In order to investigate the possible importance of domain fusion versus n Figure c, probab-5, Figure We next investigated if there was any bias in the localization of Vertebrate domains in proteins also containing older domains. We found a strong bias favouring the incorporation of the Vertebrate domain at the N-terminus both in Old proteins with two domains and in Old proteins with more than two domains   for all domain regions in human and mouse orthologous proteins. We found that younger domains had significantly higher dN/dS values than older ones   Figure . Out of One example of a protein containing domains of different age is the human progesterone receptor. This protein contains three Pfam domains, an intracellular domain named ‘progesterone receptor’, which is activated by the steroid hormone progesterone, a Zn finger domain, and an extracellular ligand-binding domain. The first domain is vertebrate-specific, and has a dN/dS of 0.2, whereas the other two domains, which are older, have dN/dS values of 0.001 and 0.023, respectively. This illustrates the patterns observed in many proteins, in which the younger parts are evolving much more rapidly. This and other examples are shown in Additional file Domains typically cover the majority of a protein sequence and play a crucial role in protein evolution. The way different domains combine, and the mechanisms of domain gain in proteins, have been thoroughly studied ,9,11,31.versus 363). This is not surprising given that the number of Mammalian proteins is about one third the number of Vertebrate proteins. In addition, the percentage of Mammalian proteins with annotated domains is less than half the corresponding percentage of Vertebrate proteins. As the length of the two periods considered is not very different, this very likely reflects strong under-annotation of mammalian-specific sequences in the databases, both in relation to the number of expressed genes and to the number of functional domains in the encoded proteins. In line with this, Capra and colleagues found that younger proteins in yeast were less well covered by Pfam domains than average [Among young domains, we have been able to identify many less mammalian-specific  domains than vertebrate-specific (Vertebrate) domains (63  average .-5). In addition, the underrepresentation of domains in younger proteins is consistent with the previous finding that younger proteins are enriched in LCRs [Proteins lacking annotated domains show a slight tendency to be longer and evolve more rapidly than proteins containing annotated domains Table . The sam in LCRs . In concWe identify 330 proteins in which Vertebrate and Old domains combine. The fraction of Vertebrate domains that belong to Pfam clans (groups of evolutionary related domains) is much smaller for Vertebrate domains than for Old domains 14% vs 59%), emphasizing the recent origin of most Vertebrate domains. In general, novel domains in proteins can be gained by several mechanisms, such as gene fusion, exon extension, recombination and retro-transposition % vs 59%,,34. We hNewly evolved domains are predominantly gained at the N-terminus and, to a lesser extent, at the C-terminus. Diverse authors have found that both domain gain and loss are more frequent at the protein termini than at the protein central region ,33,36,37Drosophila[Drosophila domains of different age  library, which contains 10,340 hidden markov models derived from Pfam domains [We obtained 15,630 one-to-one orthologous human and mouse genes using version 56 of Ensembl . We took domains . We usedMus musculus, Rattus norvegicus, Bos Taurus), non-mammalian vertebrates , other metazoans  and other eukaryotes . We assigned an age group to each domain following the rank of species in which a domain was found, allowing for secondary losses. For example if a human domain was found in at least one mammalian species but in none of the other vertebrate, metazoan or eukaryotic species it was classified as Mammalian. We classified 2,294 different human domains as Eukarya, 745 as Metazoan, 369 as Vertebrate and 65 as Mammalian. The Eukarya and Metazoan groups were both considered Old (older than 550 million years) and merged into a single class  [We defined the phylogenetic age of a protein as equal to the oldest domain it contained. We obtained 11,039 proteins classified as Old, 473 as Vertebrate and 62 as Mammalian , the number of synonymous substitutions per synonymous site (dS), and the dN/dS ratio. We used the maximum likelihood approach implemented in the codeml program of the PAML software package .Domains shorter than 60 amino acids or with a dN > 0.5 or dS > 2 were discarded to ensure robustness in the evolutionary rate estimation. After the filtering process we obtained 12,647 different human domains with dN and dS data. We observed that Eukarya and Metazoan showed a very similar dN/dS distribution and for this reason we considered them as a single group (Old) in all analyses presented here.We compared the non-synonymous to synonymous substitution rates (dN/dS) of pairs of Old and Vertebrate domains located in the same protein (330 proteins). We computed the difference in dN/dS of the Vertebrate domain minus the dN/dS of the Old domain and divided it by the higher dN/dS of the two. To determine if the difference in the estimated number of non-synonymous substitutions to synonymous substitutions was statistically different between Old and Vertebrate domains we applied a binomial test comparing the total number of non-synonymous substitutions and synonymous substitutions between the two age groups.We assigned each pair of domain types Vertebrate-Old in the 330 proteins (see section above) to one of the following classes: 1. Vertebrate domain dependence on a given Old domain: when the Old domain, but not the Vertebrate domain, could be found in a different domain configuration ; 2. Vertebrate and Old domain dependence on each other: when neither the Old domain nor the Vertebrate domain could be found in a different configuration ; 3. Domain fusion: when both the Old and the Vertebrate domain could be found in a different domain configuration ; 4. Complex: when there were more than two domains in a protein and the different Old-Vertebrate pairs showed a different behaviour (31 proteins). Cases in 1 and 2 were considered to be compatible with the gain of a Vertebrate domain into an existing older protein (259 proteins). No cases where found of “Old domain dependence on a given Vertebrate domain”.The R statistical software package  was usedThe authors declare that they have no competing interests.MT-R and MMA designed the study. MT-R performed the computational analyses. MT-R and MMA wrote the manuscript. Both authors read and approved the final manuscript.Table S2. Non-synonymous to synonymous (dN/dS) substitution rates for domains classified in different age classes defined using different Hmmpfam E-value cut-offs. Table S3. List of the most abundant domains in each age group by total number of domain occurrences. Table S4. Relationship between evolutionary rates and protein domain age. Figure S5. Distribution of the non-synonymous to synonymous (dN/dS) substitution rates for each domain type. Figure S6. Examples of human proteins containing Vertebrate and Old domains. Figure S7. Distribution of non-synonymous to synonymous (dN/dS) values for D. melanogaster protein domains classified in different age groups. Table S8. Relationship between evolutionary rate and protein domain age in D. melanogaster proteins.Length distribution of domains of different age. Click here for fileAdditional Pfam annotations for proteins containing Pfam domains of different age, including Clan Accession number, Clan Identifier and Pfam Description.Click here for fileComplete list of proteins and Pfam domains employed in this study, including Ensembl Protein identifier, Pfam Accession number, age class, sequence length and non-synonymous (dN) and synonymous (dS) substitution rates.Click here for file


Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations.ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2.Transmembrane (TM) domain predictions, homology modeling, Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation  angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems. The myeloproliferative leukemia virus oncogene (MPL) encodes the thrombopoietin receptor, the major regulator of megakaryocytopoiesis and platelet formation Although JAK2 mutations at amino acid 617 and nearby positions account for the majority of patients with various forms of myeloproliferative neoplasms (MPNs), a substantial proportion of MPN patients lack a JAK2 mutation. Some JAK2 mutation-negative patients with essential thrombocytopenia and myelofibrosis have been reported to have MPL mutations, primarily at codons 515 and, to a lesser extent, 505 We have demonstrated that modern computational approaches can be used to explore the structures and dynamics of biological molecules, even without experimental structures ab inito structure prediction, to obtain possible structural models of MPL. The structural models then served as starting points for large-scale long-time molecular dynamics simulations. Possible origins of the effects of MPL mutations at positions 505 and 515 were consolidated. This work represents the first published attempt to understand pathological effects of mutations of cytokine receptors at the atomic level.In this paper we applied computational biology approaches including, transmembrane domain prediction, homology modeling, and The following sections provide detailed descriptions of the computational approaches employed to identify the possible transmembrane region of MPL, build the homology model of the transmembrane region, predict the intracellular region structure, and merge individual domains to build the final structure; the setups for molecular dynamics simulations are also described. The wild-type and four clinically observed mutants were studied: W515L, W515K, S505N, and S505A.Two transmembrane prediction servers were utilized to predict the MPL transmembrane domain: HMMTOP ab initio protein prediction technique, Rosetta algorithm Despite the numerous homologues of MPL in the human genome, there is no known 3D structure of any homologue sequence for the MPL intracellular domain. To build the initial structure of the MPL intracellular domain from residues 517 to 635, we used a well-known ab initio method were used as the template structure for the merged structure. A distance constraint between L506 and F510 was set to ensure their experimentally observed hydrogen bond MODELLER was again used to build the merged structure. The homology model of the transmembrane domain and the intracellular domain predicted by the Rosetta The merged structure then was inserted into the membrane, a bilayer of palmitoyloleoyl phosphatidylcholine (POPC) constructed by the VMD package, version 1.8.7 +: 17; Cl−: 17). All preparation steps were done using the VMD package.The MPL/membrane complex then was put in a 75 Å by 75 Å by 110 Å TIP3P water box All MD simulations were performed using the NAMD package (version 2.6) 2. Membrane, water, and ion molecules were first energy-optimized and then underwent the following simulated annealing: the temperature was first increased from 0 K to 600 K at constant volume (1 K per ps) and held there for 500 ps, and then decreased from 600 K to 300 K at 1 K per ps and held at 300 K for 500 ps. The system then was kept at 300 K for 4 ns with constant pressure at 1 atm. The procedure was then repeated, resulting in an equilibration simulation of 10 ns for water, membrane molecules, and ions.The following procedure was used prior to the data collection (production) MD simulations. All heavy atoms of MPL were first restrained at their initial structure positions with a force constant of 50 kcal/mol/Å2 during a 500 ps period, followed by a 2,000-step energy minimization for all atoms and a 300-ps heating period with the temperature increased from 0 to 300 K at a rate of 1 K per ps.This membrane/water/ion-equilibrated system served as the starting point for the production simulations. Mutant systems were created with the VMD package on the basis of the equilibrated wild-type system; hence, all mutants have the same initial structure. One or two nearby cations/anions were removed to reach electric neutrality for mutants involving charge changes, with an additional 1 ns equilibration of membrane/water/ion. The restraint force constant then was gradually reduced to 3 kcal/mole/ÅIn the production runs, a 140-ns MD simulation was performed for each mutant without any restraining potential applied. The last 100 ns of data were used for data analysis.Since all mutations simulated in this work are located in the transmembrane region, we first analyzed the impact of these mutations on the position of the transmembrane domain. The snapshot of the simulation trajectory of the wild-type MPL at 140 ns is shown in β atoms of residues 491 (upper panel), 505 (middle panel), and 515 (lower panel). The RDF is the distribution of the distance between two groups of atoms. The RDF between water molecules and a protein residue can be treated as a measure of how deep the residue is buried from the solvent. Here it is also the measure of how deep a certain residue buried in the membrane. For residue 491, located near the extracellular region  and it is difficult to draw firm conclusions based solely on tilt angle statistics, S505A and S505N do appear to exhibit significant differences in tilt angles . This finding can be explained by the fact that A505 tends to be buried in the membrane and causes the transmembrane domain to be more aligned with the membrane normal direction, while N505 will have an opposite effect. Similar observations have been reported in other systems The tilt angle alone is not enough to describe the relative position of the transmembrane region, which could also rotate relative to the intracellular domain. The averages of this rotation  and its distribution derived from simulations are shown in ab initio protein structure prediction was used to build the initial structure of the intracellular domain. Although ab initio protein structure prediction techniques are maturing, we cannot yet reliably assess the quality of our model structure of the intracellular domain. Hence, the intracellular domain structures derived from our simulations  studies have provided experimental evidence on the hydrogen bonding patterns near the intracellular domain of MPL ulations  may not Snapshots representing the conformational changes of the intracellular domain due to different mutations are shown in With the limitations of currently available computational approaches, the intracellular domain structure reported here is putative and requires further experimental verification. Nevertheless, we examined the possible effects of MPL mutations on the Box1 loop of the intracellular domain. The Box1 loop is believed to be the JAK2 binding site Traditionally the activation of membrane receptors and their partner kinases is explained by assuming that the individual domains are more-or-less rigid. Upon binding to the ligand, the receptor forms a dimer and the relative orientation between domains of a receptor dimer causes the activation processes Taken together, the above studies indicate that signal activation may involve the following events: ligand binding to the receptor causes rotation of the transmembrane domain and conformational changes of the juxtamembrane domain, which results in significant conformational changes of the intracellular domain. The anchor points of the transmembrane domains are thus critical to holding the transmembrane domain in the right tilt and azimuthal angles. Understanding of the detailed conformational changes may require dynamic descriptions rather than traditional rigid-domain models.In contrast to most explanations for the activation mechanism, where the transmembrane and intracellular domains are usually treated as being rigid and the activation of JAK2 is explained by the relative translational movement between those rigid domains An important aspect of this study is that it highlights possible limitations of crystal structures for studying membrane proteins. All analyses reported here are based on long-time (140 ns for each mutant) molecular dynamics simulations that sample over different local and global conformations of different domains of MPL. The results cannot be obtained by static models. Although crystallography techniques are critically important in structural biology, crystal structures are averaged structures and cannot provide dynamic insights. For example, the rotation angle distribution analysis in the last section gives deep insights into how the intracellular domain may be influenced by the location of the transmembrane domain. This type of analysis is not possible with static crystal structures alone. To deeply understand the activation process in signaling transduction, other approaches such as NMR techniques and molecular dynamics simulations are necessary.We present the first full-scale molecular dynamic simulations on the wild-type and clinically observed mutants of the MPL transmembrane protein, a critical element of the related JAK2 signal pathway. Simulation results suggest that S505 and W515 are important in keeping the transmembrane domain in its correct position. Mutations at these two positions cause movement of the transmembrane domain, alter the conformation of the nearby intracellular domain in an unexpected way, and may cause constitutive activation of MPL's kinase partner, JAK2. We predict the order of impact of the mutational effects to be in the following sequence: W515K > S505A > W515L > S505N.Our results suggest that mutations within the transmembrane region could cause conformational changes that are seldom considered, such as the tilt angle change and azimuthal rotational angle  change. Such changes may significantly alter the conformation of the adjacent intracellular domain. Hence, extreme caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems.