BACKGROUND
protein domain predictions are a starting point for a range of functional analyses and can either newly predict or further refine functional predictions  <cit> .
indeed, domains form structural, evolutionary and functional units of proteins  <cit> .
the combination and order of domains in a protein is frequently considered as a fundamental level of protein functional complexity.
the majority of proteins is composed of multidomain proteins and the domain composition of multidomain proteins is critical for their specialized functions  <cit> .
furthermore, domain combinations are not random, which may indicate functional cooperation  <cit> .
plant “lesion mimic mutants”  show spontaneous necrotic lesion resembling the so-called hypersensitive response , a form of programmed cell death associated with plant defense  <cit> .
the arabidopsis thaliana vad <dig>  mutant is a lmm altered in a negative regulator of pcd and defense responses harboring a gram domain predicted to bind lipids  <cit> .
contrary to most lmm genes characterized to date, vad <dig> is expected to control the cell-to-cell propagation of pcd instead of its initiation  <cit> .
gram is a ~ <dig> amino-acids domain predicted to mediate intracellular protein binding or lipid binding during membrane-associated processes  <cit> .
this domain is related to the ph domains and is found in animal glucosyltransferases, rab-like gtpase activators, myotubularins and other membrane-associated proteins.
the gram domain of human myotubularins is able to bind phospholipids and is involved in membrane signaling  <cit> , but its exact function often remains enigmatic  <cit> .the presence of a gram domain in vad <dig> protein suggests that lipid binding could be required for vad <dig> function, but the role of vad <dig> at the subcellular level is currently unknown.
remarkably, a significant proportion of plant lmms show mutations in genes associated with lipid biosynthesis and homeostasis  <cit> .
this notably concerns sphingolipid metabolism: the acd <dig> and acd <dig> mutants carry mutations in a ceramide kinase and a putative sphingosine transfer protein, respectively  <cit> .
conversely, erh <dig> is a positive regulator of the hr encoding a functional inositolphosphoryl-ceramide  synthase which converts ceramide to ipc  <cit> .
furthermore, edr <dig> was isolated as a negative regulator of pcd and defense responses encoding a multi-domain protein featuring a duf <dig> domain, a ph domain and a start domain  <cit> .
like cert ph domain, edr <dig> ph domain preferentially binds to pi4p  <cit> .
nevertheless, the mechanisms by which lipid-binding domain containing proteins regulate pcd in plants are largely unknown.
here, we analyzed sequence conservation in the vad <dig> family and identified an uncharacterized conserved domain we designated as vast .
using sequence- and structure-based phylogenetic analyses we demonstrate that this domain is present in all major eukaryotic lineages but no molecular function has been assigned to it.
vast is related to bet v1-like, a superfamily including lipid- and hormone-binding domains.
the vast domain will be referred to with accession number pf <dig> in release  <dig>  of the pfam database  <cit> .
these findings open new perspectives for the functional analysis of vast-domain containing proteins such as a. thaliana vad <dig>  yeast ysp <dig> and human gram1a, b and c.
RESULTS
proteins in the vad <dig> family contain an uncharacterized conserved domain
to get insights into vad <dig> putative biochemical function, we analyzed protein sequence conservation among vad <dig> homologs.
first, to identify vad <dig> homologs, we used the full length sequence of vad <dig> protein in stringent phmmer searches against the uniprot database.
we identified  <dig> atvad <dig> homologs with e-value below 1e- <dig> across twelve angiosperm plant species, including monocots  and eudicots .
in this stringent approach, all species showed a single vad <dig> copy except g. max that had three copies.
the retrieved homologs showed at least 55% identity and all contained a clearly identified gram domain.
to identify conserved regions in these  <dig> sequences, we aligned them using the multiple sequence alignment  tool mafft  <cit>  and we plotted the consensus conservation score along the alignment using a ten amino-acids  sliding window .
two major conserved regions were clearly apparent.
the first one spanning positions  <dig> to  <dig> in the alignment, and the second spanning positions  <dig> to  <dig>  to characterize and precisely delimit vad <dig> conserved regions we mapped vad <dig> gene and protein annotations onto the conservation plot .
the n-terminal conserved region  overlapped largely with the predicted gram domain .
the second conserved region  corresponded to an uncharacterized domain of approximately  <dig> aa.
this domain is encoded by a region spanning from atvad <dig> exon  <dig> to exon  <dig>  we therefore set the limits of this uncharacterized domain at positions  <dig> and  <dig>  for a total length of  <dig> aa .
based on further characterization described hereafter, we designated this domain as the vast  domain.
a close up view on the msa of the vast domain revealed a high degree of conservation among plant homologs, with an average  <dig> % identity over the vast domain .
since no annotation could be mapped onto the vast domain, it represents a yet uncharacterized protein domain highly conserved in plants.
the vast domain is conserved among eukaryotes
since none of atvad <dig> homologs had characterized biochemical functions, we extended our search of related protein domains using hidden markov models  to get insights into the vast domain putative function and evolution.
for this, we built a hmm using the  <dig> plant homologs of atvad <dig> vast domain.
to highlight important sequence motifs in this hmm, we examined the corresponding sequence logo .
amino acids w <dig>  r <dig>  p <dig>  f <dig> and i <dig> were strongly conserved in the model, suggesting an important contribution of these residues to the protein function.
to identify protein domains related to the vast domain, we searched the uniprot database with the vast hmm model.
after three iterations of jackhammer search, we retrieved  <dig> hits distributed exclusively in eukaryotic proteins, including yeasts and other fungi, oomycetes, mammals, and plants .
most of the identified proteins contained one copy of the vast domain , with a few containing two or more vast domains .
as little as ~ <dig> % of the protein hits contained vast as the only domain identified.
the vast domain is frequently associated with lipid binding domains such as gram , c <dig> and pex24p domains, suggesting a functional link between vast and lipid-binding domains.
among proteins related to vad <dig> retrieved by our hmm search was ysp <dig>  a major cell death regulator in yeast  <cit> .
however, there was no protein for which a biochemical function has been described.
multiple domain combinations contributed to the diversification of vast-containing proteins
to document the evolution of the vast domain, we examined the phylogenetic relations of vast domains in  <dig> fully sequenced species representing all major eukaryotic lineages , corresponding to a total of  <dig> protein sequences .
.
sequences clustered into nine groups defined by their taxonomic range and domain organization.
to highlight the phylogenetic relationship between copies of the vast domain present in a single protein , we have connected together vast copies present in the same protein .
group  <dig> gathered sequences from mammals and other metazoans.
in this group, the vast domain is found either  alone,  associated with a gram domain or  associated with another vast domain.
the two copies of vast in gram-vast-vast proteins clustered in the same clade indicating that the duplication of vast domain is recent.
this group included human gramd1a, gramd1b and gramd1c proteins.
group  <dig> exclusively consisted in sequences from fungi, with the same domain structures as found in group  <dig>  group  <dig> contained the yeast ysp <dig> protein.
group  <dig> and  <dig> contained n-terminal and c-terminal vast domains respectively of proteins with a c2-vast--gram-vast domain architecture.
group  <dig> and  <dig> are restricted to plants, including the moss physcomitrella patens, and phylogenetically distant, suggesting that duplication of the vast domain early in land plant evolution allowed the divergence of two vast copies in these proteins.
group  <dig> contained proteins from stramenopiles, ciliates and amoebozoa, with either a vast domain alone or a gram-vast architecture.
group  <dig> contained exclusively plant sequences with a gram-vast structure, include a. thaliana vad <dig>  group  <dig> contained exclusively stramenopile sequences with either a vast domain alone or a fch-gram-vast architecture.
group  <dig> contained exclusively ciliate sequences comprising either a vast domain alone, or gram domain with one to three copies of vast.
finally, group  <dig> contained sequences from amoebozoa, tracheophyta  and stramenopiles with diverse domain architectures:  vast alone,  gram-vast,  c2-gram-vast or  vast-vast-pex24p.
groups  <dig>   <dig>   <dig> and  <dig> show support value of  <dig>  suggesting that the function of vast domains from these groups could have diverged, and that this divergence could be essentially driven by the association with the c <dig> and the gram domains.
next, we attempted to reconstruct the evolutionary history of domain combinations in vast-containing proteins .
similar to gram  <cit> , the vast domain would originate from the last common eukaryote ancestor .
since gram and vast domains are associated in nearly all eukaryotic lineages the gram-vast combination probably dates back from the lcea.
sequences harboring a vast domain alone were also found in all lineages except plants and fungi, suggesting that a copy of the ancestral vast domain gene has been maintained in most phyla.
alternatively, the gram domain could have been lost from a putative gram-vast ancestor in several lineages.
adjacent vast duplications within a single protein are observed in ciliates, stramenopiles, fungi and mammals that probably arose recently, judging from high sequence similarity between adjacent copies.
consistent with  <cit> , vast association with both gram and c <dig> appeared plant-specific.
a parsimonious scenario for the emergence of the complex c2-vast-c2-gram-vast domain architecture specific to plants could be the combination of a c <dig> domain with ancestral vast alone and gram-vast proteins, followed by the fusion of a c2-vast and a c2-gram-vast module early in the evolution of plants .
the c2-vast and c2-gram-vast modules could have been maintained in vascular plants but not in mosses.
alternatively, a vast-gram-vast fusion could have emerged in an ancestral eukaryotic lineage , combined with two c <dig> domains in plants, then c2-vast and c2-gram-vast could have emerged from the split of a c2-vast-c2-gram-vast plant ancestor in vascular plants but not in mosses.
the association of vast with pex24p or fch domains seems to be innovations from the oomycete lineage.
in addition to data presented in figure  <dig>  proteins with a ph-vast architecture were found in some fungal species.
homology modeling of the vast domain 3d structure
classical sequence-based phylogeny did not allow identifying protein domains of known function related to vast.
structure-based phylogenetic network inference may be used to improve the resolution of deep evolutionary relationships and assist in inference of protein function  <cit> .
to analyze relationships between vast and protein domains of known three-dimensional structure, we conducted a structure-based clustering of atvad <dig> vast domain and its closest analogs.
first, to obtain atomic coordinates of a 3d model for atvad <dig> vast domain, we submitted its  <dig> aa sequence to the homology and threading structure prediction server i-tasser  <cit> .
the best model  showed a two-layer sandwich alpha beta fold  containing three alpha helices , six beta-sheets  and two loops  numbered from n to c terminus .
this model had a c-score of - <dig> indicating that quality predictions can be estimated with more than 90% confidence  <cit> , and expected tm-score of  <dig>  suggesting a correct topology.
eight of the top  <dig> threading alignments had normalized z-score higher than  <dig>  thus accuracy of the model is expected to be high  <cit> .
the predicted vast model encompasses a large hydrophobic cavity delimited by sheets β <dig>   <dig> and  <dig>  loop Ω <dig> and helices α <dig> and  <dig> .
structure-based phylogeny reveals relationships between the vast domain and bet v1-like domains
to investigate structural relationships between atvad <dig> vast domain and its closest analogs, we conducted a structure-based tree inference analysis including atvad <dig> vast domain, predicted three-dimensional structure for  <dig> vast homologs, and the top structural analogs retrieved by fold recognition searches.
the best structural analogs retrieved by i-tasser and ncbi vector alignment search tool fold recognition searches were human mln <dig>  star-related lipid transport domain  and streptomyces zhui polyketide aromatase/cyclase .
close analogs also included human cert ceramide trafficking protein  and arabidopsis thaliana pyl <dig> aba receptor .
gene ontology terms associated with atvad <dig> vast domain based on the 3d model included hormone binding , isoprenoid binding  and monocarboxylic acid binding .
to build a structure-based phylogenetic tree, we modeled the three-dimensional structure of  <dig> vast domains using atvad <dig> vast as a template, and searched for atvad <dig> vast closest structural analogs in the medium redundancy subset of the molecular modeling database .
we next performed a multiple structure alignment, calculated normalized pairwise rmsd distances for aligned cα atoms , and used this distance matrix to produce a neighbor-joining tree .
structures clustered into five major groups .
group i contained plant bet v <dig> phytohormone-binding proteins and pathogenesis-related  10-like proteins.
group ii contained arabidopsis thaliana pyl <dig> abscisic acid  receptor, belonging to the pyrabactin resistance  <dig> /pyr1-like /regulatory components of aba receptors  family and uncharacterized bacterial proteins.
group iii contained mammalian start proteins binding sterols and sphingolipids, and streptomyces aromatase-cyclases.
group iv contained vast-domains.
finally group v contained mammalian phosphatidylinositol transfer protein , bacterial oxygenase and hydrolases, and other bet v1-like domains.
with the exception of the n-terminal vast domain of phytophthora infestans predicted protein pitg_ <dig> , all vast domains clustered together into structural group iv.
a separate sub-group within structural group iv was formed by vast domains from sequence-based phylogenetic group  <dig>  supporting a possible functional divergence.
this analysis revealed that vast domains are structurally related to bet v1-like domains  known to bind bulky hydrophobic ligands such as phytohormones, lipids and polyketides.
association with lipid-binding domains in large multidomain proteins is typical for start and vast domains.to further test whether atvad <dig> vast domain and the well-characterized start domain of the cert protein could be evolutionary related, we closely examined the ligand-binding pockets of these two proteins.
for this, we performed a structural alignment of atvad <dig> vast and cert start domain models.
the structural alignment of atvad <dig> vast domain and cert start domain highlighted a good conservation of secondary structure elements lining the substrate binding pocket .
although the overall sequence conservation between the two proteins is limited, residues binding to c <dig> ceramide in cert showed conservation or similar environments in atvad <dig> vast domain.
these observations are consistent with hidden homology between vast and bet v1-like domains.
alternatively, the analogy between vast and bet v1-like domains could result from convergent evolution.
testing the importance of the predicted ligand-binding pocket residues for vast function could help discriminate between these hypotheses.
discussion
we report the identification of the vast protein domain in the vad <dig> plant cell death regulator.
this domain is conserved across eukaryotes and is structurally related to bet v1-like domains, including start lipid-binding domains.
the predicted structure of vad <dig> vast domain is consistent with a function in binding large hydrophobic ligands.
our findings open new perspectives for the analysis of functions of the vast domain associated with the gram, c <dig> and ph lipid-binding domains and the characterization of novel mechanisms regulating pcd in plants.
what is the physiological role of vast-containing proteins?
most of the proteins containing vast domain have no characterized function to date.
the a. thaliana vad <dig> is the only exception in plants.
in the context of pathogen attack, controlled programmed cell death  is one of the prevailing plant defense responses, allowing confinement of the pathogen locally in dead cells.
the vad <dig> mutant exhibits spontaneous pcd lesions initiated in cells surrounding vascular tissue progressively expand to the whole leaf, hence its classification as “propagation lesion mimic mutant”  <cit> .
this phenotype suggests that vad <dig> is impaired in the control of cell-to-cell propagation of pcd, involving a yet unknown mechanism.amiodarone is a ca2+ channel-targeted drug inducing apoptosis mediated by reactive oxygen species , via the same pathway as natural pheromones  <cit> .
genetic screens have revealed the function of the ysp <dig>  in enhancing survival after amiodarone treatment.
ysp <dig> is a mitochondrial membrane protein involved in mitochondrial fragmentation, probably acting downstream of ros production triggered by intracellular acidification  <cit> .
ysp <dig> harbors a gram and a vast domain but its molecular function is unknown.
in human, a polymorphism in the gramd1b gene has been associated with susceptibility to chronic lymphocytic leukemia  <cit> .
recently, a whole genome modified-sirna screen identified gramd1b as a protein associated with chemoresistance in epithelial ovarian cancer  cells.
consistent with the view that acquired chemoresistance is a major contributor to patient mortality from ovca, reducing gramd1b expression increased overall survival in ovca patients and decreased tumour burden in mouse models  <cit> .
gramd1c has been identified as part of a quantitative trait locus associated with hepatic iron overload in mice, but its function has not been validated  <cit> .
the molecular function of gramd <dig> proteins has not been investigated but their association with several disorders supports the relevance of vast-containing proteins for cell integrity.
what may be the signal associated to vad <dig> that mediate propagation of pcd?
the vast domain is related to domains from the bet v1-like superfamily  that bind large hydrophobic ligands such as lipids, hormones and antibiotics  <cit> .
in the bet v1-like superfamily, pr- <dig>  bet v <dig> and pyr/pyl/rcar domains  typically bind phytohormones such as brassinosteroids, cytokinins and abscisic acid  <cit> .
some other bet v1-like domains  bind secondary metabolites such as flavonoids, polyketides and various antibiotics  <cit> .
these ligands are diffusible molecules that could act as intercellular signals regulated by vad <dig>  domains belonging to the start subfamily of bet v1-like domains bind lipids such as sterols and sphingolipids  <cit> .
in animal cells, intercellular transport of sterols and sphingolipids is mainly mediated by non-vesicular transport via the action of dedicated lipid transport proteins  or via spontaneous lipid exchange  <cit> .
phytohormone- and secondary metabolite-binding proteins in the bet v1-like superfamily often function as single-domain proteins, or multimers of single domain-proteins, whereas start and vast-containing proteins are generally large multidomain proteins.
notably, the mammalian cert and arabidopsis vad <dig> proteins share a common domain structure involving a ph superfamily domain  and a bet v1-like superfamily domain .
cooperation between the ph and start domains in cert is critical for its function as a ceramide transport protein  <cit> .
ceramides and other sphingolipids are important regulators of cell death programs in animals and plants  <cit> .
vad <dig> may therefore sense or transport lipids to modulate cell death signals intercellularly.
what is the evolutionary history of the vast domain?
our sequence- and structure-based phylogenetic analyses suggest that the vast domain evolved from a primordial bet v1-related protein that existed in the last universal common ancestor, and emerged with the divergence of eukaryotes.
alternatively, the analogous structure of vast and bet v1-like domains could result from convergent evolution.
by contrast to the pr10-like subfamily of bet v <dig> domains, the vast domain is conserved across all major eukaryotic lineages, and therefore probably serves a function relatively conserved across all eukaryotes.
radauer et al.
proposed that the primordial bet v <dig> protein would bind lipids, and would have evolved by addition of secondary structural elements or fusion to other domains into multi-domain proteins  <cit> .
our results suggest that the vast domain has been associated with the gram domain very early in the history of eukaryotes, and was later combined with c <dig> domains in plants and with pex24p domains in oomycetes.
the 3d model we obtained for vad <dig> vast domain features a long loop connecting helix α <dig> and sheet β <dig>  instead of a beta-sheet in typical bet v <dig> domains, leading to a β-α2-β5-α instead of β-α2-β6-α secondary structure arrangement.
vast domains of plants span across four phylogenetic groups , suggesting the emergence of novel adaptations in the plant kingdom, that may either reflect the evolution of new catalytic activities or the adaptation to plant-specific ligand.
CONCLUSIONS
local variations in membrane protein and lipid composition create subcellular compartments with diverse physico-chemical properties.
such local variations in lipid and protein content may be critical for defining the specific structure membrane compartments and flagging them for the addressing of proteins and other signals  <cit> .
the trafficking routes between membrane compartments, and the proteins implicated, are just starting to be uncovered.
our analyses revealed the vast domain as a member of the bet v1-like superfamily predominantly associated with lipid binding domains such as gram, c <dig> and ph domains.
this finding opens new perspectives for molecular and genetics studies of the function and regulation of vast domain containing proteins.

 protein domain predictions are a starting point for a range of functional analyses and can either newly predict or further refine functional predictions. indeed, domains form structural, evolutionary and functional units of proteins. the combination and order of domains in a protein is frequently considered as a fundamental level of protein functional complexity. the majority of proteins is composed of multidomain proteins and the domain composition of multidomain proteins is critical for their specialized functions. furthermore, domain combinations are not random, which may indicate functional cooperation. plant “lesion mimic mutants” show spontaneous necrotic lesion resembling the so-called hypersensitive response, a form of programmed cell death associated with plant defense. the arabidopsis thaliana vad mutant is a lmm altered in a negative regulator of pcd and defense responses harboring a gram domain predicted to bind lipids. contrary to most lmm genes characterized to date, vad is expected to control the cell-to-cell propagation of pcd instead of its initiation. gram is a ~ amino-acids domain predicted to mediate intracellular protein binding or lipid binding during membrane-associated processes. this domain is related to the ph domains and is found in animal glucosyltransferases, rab-like gtpase activators, myotubularins and other membrane-associated proteins. the gram domain of human myotubularins is able to bind phospholipids and is involved in membrane signaling, but its exact function often remains enigmatic.the presence of a gram domain in vad protein suggests that lipid binding could be required for vad function, but the role of vad at the subcellular level is currently unknown. remarkably, a significant proportion of plant lmms show mutations in genes associated with lipid biosynthesis and homeostasis. this notably concerns sphingolipid metabolism: the acd and acd mutants carry mutations in a ceramide kinase and a putative sphingosine transfer protein, respectively. conversely, erh is a positive regulator of the hr encoding a functional inositolphosphoryl-ceramide synthase which converts ceramide to ipc. furthermore, edr was isolated as a negative regulator of pcd and defense responses encoding a multi-domain protein featuring a duf domain, a ph domain and a start domain. like cert ph domain, edr ph domain preferentially binds to pi4p. nevertheless, the mechanisms by which lipid-binding domain containing proteins regulate pcd in plants are largely unknown. here, we analyzed sequence conservation in the vad family and identified an uncharacterized conserved domain we designated as vast. using sequence- and structure-based phylogenetic analyses we demonstrate that this domain is present in all major eukaryotic lineages but no molecular function has been assigned to it. vast is related to bet v1-like, a superfamily including lipid- and hormone-binding domains. the vast domain will be referred to with accession number pf in release of the pfam database. these findings open new perspectives for the functional analysis of vast-domain containing proteins such as a. thaliana vad yeast ysp and human gram1a, b and c.  proteins in the vad family contain an uncharacterized conserved domain to get insights into vad putative biochemical function, we analyzed protein sequence conservation among vad homologs. first, to identify vad homologs, we used the full length sequence of vad protein in stringent phmmer searches against the uniprot database. we identified atvad homologs with e-value below 1e- across twelve angiosperm plant species, including monocots and eudicots. in this stringent approach, all species showed a single vad copy except g. max that had three copies. the retrieved homologs showed at least 55% identity and all contained a clearly identified gram domain. to identify conserved regions in these sequences, we aligned them using the multiple sequence alignment tool mafft and we plotted the consensus conservation score along the alignment using a ten amino-acids sliding window. two major conserved regions were clearly apparent. the first one spanning positions to in the alignment, and the second spanning positions to to characterize and precisely delimit vad conserved regions we mapped vad gene and protein annotations onto the conservation plot. the n-terminal conserved region overlapped largely with the predicted gram domain. the second conserved region corresponded to an uncharacterized domain of approximately aa. this domain is encoded by a region spanning from atvad exon to exon we therefore set the limits of this uncharacterized domain at positions and for a total length of aa. based on further characterization described hereafter, we designated this domain as the vast domain. a close up view on the msa of the vast domain revealed a high degree of conservation among plant homologs, with an average % identity over the vast domain. since no annotation could be mapped onto the vast domain, it represents a yet uncharacterized protein domain highly conserved in plants. the vast domain is conserved among eukaryotes since none of atvad homologs had characterized biochemical functions, we extended our search of related protein domains using hidden markov models to get insights into the vast domain putative function and evolution. for this, we built a hmm using the plant homologs of atvad vast domain. to highlight important sequence motifs in this hmm, we examined the corresponding sequence logo. amino acids w r p f and i were strongly conserved in the model, suggesting an important contribution of these residues to the protein function. to identify protein domains related to the vast domain, we searched the uniprot database with the vast hmm model. after three iterations of jackhammer search, we retrieved hits distributed exclusively in eukaryotic proteins, including yeasts and other fungi, oomycetes, mammals, and plants. most of the identified proteins contained one copy of the vast domain, with a few containing two or more vast domains. as little as ~ % of the protein hits contained vast as the only domain identified. the vast domain is frequently associated with lipid binding domains such as gram, c and pex24p domains, suggesting a functional link between vast and lipid-binding domains. among proteins related to vad retrieved by our hmm search was ysp a major cell death regulator in yeast. however, there was no protein for which a biochemical function has been described. multiple domain combinations contributed to the diversification of vast-containing proteins to document the evolution of the vast domain, we examined the phylogenetic relations of vast domains in fully sequenced species representing all major eukaryotic lineages, corresponding to a total of protein sequences.. sequences clustered into nine groups defined by their taxonomic range and domain organization. to highlight the phylogenetic relationship between copies of the vast domain present in a single protein, we have connected together vast copies present in the same protein. group gathered sequences from mammals and other metazoans. in this group, the vast domain is found either alone, associated with a gram domain or associated with another vast domain. the two copies of vast in gram-vast-vast proteins clustered in the same clade indicating that the duplication of vast domain is recent. this group included human gramd1a, gramd1b and gramd1c proteins. group exclusively consisted in sequences from fungi, with the same domain structures as found in group group contained the yeast ysp protein. group and contained n-terminal and c-terminal vast domains respectively of proteins with a c2-vast--gram-vast domain architecture. group and are restricted to plants, including the moss physcomitrella patens, and phylogenetically distant, suggesting that duplication of the vast domain early in land plant evolution allowed the divergence of two vast copies in these proteins. group contained proteins from stramenopiles, ciliates and amoebozoa, with either a vast domain alone or a gram-vast architecture. group contained exclusively plant sequences with a gram-vast structure, include a. thaliana vad group contained exclusively stramenopile sequences with either a vast domain alone or a fch-gram-vast architecture. group contained exclusively ciliate sequences comprising either a vast domain alone, or gram domain with one to three copies of vast. finally, group contained sequences from amoebozoa, tracheophyta and stramenopiles with diverse domain architectures: vast alone, gram-vast, c2-gram-vast or vast-vast-pex24p. groups and show support value of suggesting that the function of vast domains from these groups could have diverged, and that this divergence could be essentially driven by the association with the c and the gram domains. next, we attempted to reconstruct the evolutionary history of domain combinations in vast-containing proteins. similar to gram, the vast domain would originate from the last common eukaryote ancestor. since gram and vast domains are associated in nearly all eukaryotic lineages the gram-vast combination probably dates back from the lcea. sequences harboring a vast domain alone were also found in all lineages except plants and fungi, suggesting that a copy of the ancestral vast domain gene has been maintained in most phyla. alternatively, the gram domain could have been lost from a putative gram-vast ancestor in several lineages. adjacent vast duplications within a single protein are observed in ciliates, stramenopiles, fungi and mammals that probably arose recently, judging from high sequence similarity between adjacent copies. consistent with, vast association with both gram and c appeared plant-specific. a parsimonious scenario for the emergence of the complex c2-vast-c2-gram-vast domain architecture specific to plants could be the combination of a c domain with ancestral vast alone and gram-vast proteins, followed by the fusion of a c2-vast and a c2-gram-vast module early in the evolution of plants. the c2-vast and c2-gram-vast modules could have been maintained in vascular plants but not in mosses. alternatively, a vast-gram-vast fusion could have emerged in an ancestral eukaryotic lineage, combined with two c domains in plants, then c2-vast and c2-gram-vast could have emerged from the split of a c2-vast-c2-gram-vast plant ancestor in vascular plants but not in mosses. the association of vast with pex24p or fch domains seems to be innovations from the oomycete lineage. in addition to data presented in figure  proteins with a ph-vast architecture were found in some fungal species. homology modeling of the vast domain 3d structure classical sequence-based phylogeny did not allow identifying protein domains of known function related to vast. structure-based phylogenetic network inference may be used to improve the resolution of deep evolutionary relationships and assist in inference of protein function. to analyze relationships between vast and protein domains of known three-dimensional structure, we conducted a structure-based clustering of atvad vast domain and its closest analogs. first, to obtain atomic coordinates of a 3d model for atvad vast domain, we submitted its aa sequence to the homology and threading structure prediction server i-tasser. the best model showed a two-layer sandwich alpha beta fold containing three alpha helices, six beta-sheets and two loops numbered from n to c terminus. this model had a c-score of - indicating that quality predictions can be estimated with more than 90% confidence, and expected tm-score of suggesting a correct topology. eight of the top threading alignments had normalized z-score higher than thus accuracy of the model is expected to be high. the predicted vast model encompasses a large hydrophobic cavity delimited by sheets β and loop Ω and helices α and. structure-based phylogeny reveals relationships between the vast domain and bet v1-like domains to investigate structural relationships between atvad vast domain and its closest analogs, we conducted a structure-based tree inference analysis including atvad vast domain, predicted three-dimensional structure for vast homologs, and the top structural analogs retrieved by fold recognition searches. the best structural analogs retrieved by i-tasser and ncbi vector alignment search tool fold recognition searches were human mln star-related lipid transport domain and streptomyces zhui polyketide aromatase/cyclase. close analogs also included human cert ceramide trafficking protein and arabidopsis thaliana pyl aba receptor. gene ontology terms associated with atvad vast domain based on the 3d model included hormone binding, isoprenoid binding and monocarboxylic acid binding. to build a structure-based phylogenetic tree, we modeled the three-dimensional structure of vast domains using atvad vast as a template, and searched for atvad vast closest structural analogs in the medium redundancy subset of the molecular modeling database. we next performed a multiple structure alignment, calculated normalized pairwise rmsd distances for aligned cα atoms, and used this distance matrix to produce a neighbor-joining tree. structures clustered into five major groups. group i contained plant bet v phytohormone-binding proteins and pathogenesis-related 10-like proteins. group ii contained arabidopsis thaliana pyl abscisic acid receptor, belonging to the pyrabactin resistance /pyr1-like /regulatory components of aba receptors family and uncharacterized bacterial proteins. group iii contained mammalian start proteins binding sterols and sphingolipids, and streptomyces aromatase-cyclases. group iv contained vast-domains. finally group v contained mammalian phosphatidylinositol transfer protein, bacterial oxygenase and hydrolases, and other bet v1-like domains. with the exception of the n-terminal vast domain of phytophthora infestans predicted protein pitg_, all vast domains clustered together into structural group iv. a separate sub-group within structural group iv was formed by vast domains from sequence-based phylogenetic group supporting a possible functional divergence. this analysis revealed that vast domains are structurally related to bet v1-like domains known to bind bulky hydrophobic ligands such as phytohormones, lipids and polyketides. association with lipid-binding domains in large multidomain proteins is typical for start and vast domains.to further test whether atvad vast domain and the well-characterized start domain of the cert protein could be evolutionary related, we closely examined the ligand-binding pockets of these two proteins. for this, we performed a structural alignment of atvad vast and cert start domain models. the structural alignment of atvad vast domain and cert start domain highlighted a good conservation of secondary structure elements lining the substrate binding pocket. although the overall sequence conservation between the two proteins is limited, residues binding to c ceramide in cert showed conservation or similar environments in atvad vast domain. these observations are consistent with hidden homology between vast and bet v1-like domains. alternatively, the analogy between vast and bet v1-like domains could result from convergent evolution. testing the importance of the predicted ligand-binding pocket residues for vast function could help discriminate between these hypotheses. discussion we report the identification of the vast protein domain in the vad plant cell death regulator. this domain is conserved across eukaryotes and is structurally related to bet v1-like domains, including start lipid-binding domains. the predicted structure of vad vast domain is consistent with a function in binding large hydrophobic ligands. our findings open new perspectives for the analysis of functions of the vast domain associated with the gram, c and ph lipid-binding domains and the characterization of novel mechanisms regulating pcd in plants. what is the physiological role of vast-containing proteins? most of the proteins containing vast domain have no characterized function to date. the a. thaliana vad is the only exception in plants. in the context of pathogen attack, controlled programmed cell death is one of the prevailing plant defense responses, allowing confinement of the pathogen locally in dead cells. the vad mutant exhibits spontaneous pcd lesions initiated in cells surrounding vascular tissue progressively expand to the whole leaf, hence its classification as “propagation lesion mimic mutant”. this phenotype suggests that vad is impaired in the control of cell-to-cell propagation of pcd, involving a yet unknown mechanism.amiodarone is a ca2+ channel-targeted drug inducing apoptosis mediated by reactive oxygen species, via the same pathway as natural pheromones. genetic screens have revealed the function of the ysp in enhancing survival after amiodarone treatment. ysp is a mitochondrial membrane protein involved in mitochondrial fragmentation, probably acting downstream of ros production triggered by intracellular acidification. ysp harbors a gram and a vast domain but its molecular function is unknown. in human, a polymorphism in the gramd1b gene has been associated with susceptibility to chronic lymphocytic leukemia. recently, a whole genome modified-sirna screen identified gramd1b as a protein associated with chemoresistance in epithelial ovarian cancer cells. consistent with the view that acquired chemoresistance is a major contributor to patient mortality from ovca, reducing gramd1b expression increased overall survival in ovca patients and decreased tumour burden in mouse models. gramd1c has been identified as part of a quantitative trait locus associated with hepatic iron overload in mice, but its function has not been validated. the molecular function of gramd proteins has not been investigated but their association with several disorders supports the relevance of vast-containing proteins for cell integrity. what may be the signal associated to vad that mediate propagation of pcd? the vast domain is related to domains from the bet v1-like superfamily that bind large hydrophobic ligands such as lipids, hormones and antibiotics. in the bet v1-like superfamily, pr- bet v and pyr/pyl/rcar domains typically bind phytohormones such as brassinosteroids, cytokinins and abscisic acid. some other bet v1-like domains bind secondary metabolites such as flavonoids, polyketides and various antibiotics. these ligands are diffusible molecules that could act as intercellular signals regulated by vad domains belonging to the start subfamily of bet v1-like domains bind lipids such as sterols and sphingolipids. in animal cells, intercellular transport of sterols and sphingolipids is mainly mediated by non-vesicular transport via the action of dedicated lipid transport proteins or via spontaneous lipid exchange. phytohormone- and secondary metabolite-binding proteins in the bet v1-like superfamily often function as single-domain proteins, or multimers of single domain-proteins, whereas start and vast-containing proteins are generally large multidomain proteins. notably, the mammalian cert and arabidopsis vad proteins share a common domain structure involving a ph superfamily domain and a bet v1-like superfamily domain. cooperation between the ph and start domains in cert is critical for its function as a ceramide transport protein. ceramides and other sphingolipids are important regulators of cell death programs in animals and plants. vad may therefore sense or transport lipids to modulate cell death signals intercellularly. what is the evolutionary history of the vast domain? our sequence- and structure-based phylogenetic analyses suggest that the vast domain evolved from a primordial bet v1-related protein that existed in the last universal common ancestor, and emerged with the divergence of eukaryotes. alternatively, the analogous structure of vast and bet v1-like domains could result from convergent evolution. by contrast to the pr10-like subfamily of bet v domains, the vast domain is conserved across all major eukaryotic lineages, and therefore probably serves a function relatively conserved across all eukaryotes. radauer et al. proposed that the primordial bet v protein would bind lipids, and would have evolved by addition of secondary structural elements or fusion to other domains into multi-domain proteins. our results suggest that the vast domain has been associated with the gram domain very early in the history of eukaryotes, and was later combined with c domains in plants and with pex24p domains in oomycetes. the 3d model we obtained for vad vast domain features a long loop connecting helix α and sheet β instead of a beta-sheet in typical bet v domains, leading to a β-α2-β5-α instead of β-α2-β6-α secondary structure arrangement. vast domains of plants span across four phylogenetic groups, suggesting the emergence of novel adaptations in the plant kingdom, that may either reflect the evolution of new catalytic activities or the adaptation to plant-specific ligand.  local variations in membrane protein and lipid composition create subcellular compartments with diverse physico-chemical properties. such local variations in lipid and protein content may be critical for defining the specific structure membrane compartments and flagging them for the addressing of proteins and other signals. the trafficking routes between membrane compartments, and the proteins implicated, are just starting to be uncovered. our analyses revealed the vast domain as a member of the bet v1-like superfamily predominantly associated with lipid binding domains such as gram, c and ph domains. this finding opens new perspectives for molecular and genetics studies of the function and regulation of vast domain containing proteins. 
proteins containing both a gram and a vast domain include notably the yeast ysp cell death regulator and numerous uncharacterized proteins. using structure-based phylogeny, we found that the vast domain is structurally related to bet v1-like domains. our findings open perspectives for the functional analysis of vast-containing proteins and the characterization of novel mechanisms regulating pcd. several regulators of programmed cell death in plants encode proteins with putative lipid-binding domains. vastvad1protein domainprogrammed cell deathgram domainbet v1-like using profile hidden markov models and phylogenetic analysis we found that this domain is conserved among eukaryotes and generally associates with various lipid-binding domains. we analyzed sequence conservation across the plant kingdom in the vad protein and identified an uncharacterized vast domain. however the function of vad at the subcellular level is unknown and the domain architecture of vad has not been analyzed in details.
 several regulators of programmed cell death in plants encode proteins with putative lipid-binding domains. among them, vad is a regulator of pcd propagation harboring a gram putative lipid-binding domain. however the function of vad at the subcellular level is unknown and the domain architecture of vad has not been analyzed in details.  we analyzed sequence conservation across the plant kingdom in the vad protein and identified an uncharacterized vast domain. using profile hidden markov models and phylogenetic analysis we found that this domain is conserved among eukaryotes and generally associates with various lipid-binding domains. proteins containing both a gram and a vast domain include notably the yeast ysp cell death regulator and numerous uncharacterized proteins. using structure-based phylogeny, we found that the vast domain is structurally related to bet v1-like domains.  we identified a novel protein domain ubiquitous in eukaryotic genomes and belonging to the bet v1-like superfamily. our findings open perspectives for the functional analysis of vast-containing proteins and the characterization of novel mechanisms regulating pcd. vastvad1protein domainprogrammed cell deathgram domainbet v1-like 