BACKGROUND
the coiled-coil was one of the earliest protein structures described and first discovered in the two-stranded coiled-coil protein alpha-keratin  <cit> .
coiled-coils consist of two or more alpha-helices winding around each other in a supercoil, a simple yet versatile protein fold  <cit> .
mutations in coiled-coil proteins have been implicated in a large variety of human diseases such as severe skin fragility, muscular dystrophies, neurodegenerative diseases, progeria, and cancer  <cit> .
spurred by medical interest, the number of investigated long coiled-coil proteins in yeast and animals has rapidly grown in recent years.
recently, a database of all long coiled-coil proteins in the model plant arabidopsis was established to facilitate the identification and characterization of long coiled-coil proteins in plants  <cit> .
in contrast to eukaryotic organisms, only few long coiled-coil proteins have been characterized in prokaryotes.
examples include chaperonins and nucleases, secretion proteins, and cytadherence factors  <cit> .
the foremost feature of coiled-coil domains appears to be their ability to act as "cellular velcro" to hold together molecules, subcellular structures, and even tissues.
they can act as protein-protein interaction motifs, for examples as dimerization domains in transcription factors and receptor kinases  <cit> .
they function as "zippers" in membrane fusion proteins  <cit> , and as adapters between molecules and solid state cellular structures, such as in microtubule organizing centers, the nuclear pores and lamina, actin- and microtubule-associated proteins and cytoskeleton-associated e <dig> ubiquitin ligases  <cit> .
extracellular coiled-coil proteins include cell adherence factors and surface receptors, vertebrate blood components such as apolipoproteins and fibrinogen-like clotting factors, and extracellular matrix components such as laminins and cartilage matrix proteins forming tissue scaffolds in metazoa  <cit> .
besides associating with and interconnecting other molecules and macromolecular structures, long coiled-coil domains exhibit a number of structural and mechanical functions  <cit> .
typically, long coiled-coil domains form rod-like tertiary structures  <cit>  and assemble to dynamic fibers, meshworks and scaffolds.
examples are the intermediate filaments of the cytoskeleton and nuclear lamina  <cit> .
recent evidence suggests an important role for the dynamic properties of cytoplasmic intermediate filaments in neurodegenerative diseases  <cit> .
other coiled-coils act as spacers, for example in the yeast spindle pole body where the distance between the plaques is determined by the length of the coiled-coil domain in the connecting proteins  <cit> .
membrane-bound coiled-coil proteins such as the spectrins and golgins form scaffolds for membrane structures within the cell  <cit> .
in combination with other functional domains, coiled-coil domains are an integral part of molecular motors, such as the actin motor myosin and the microtubule motors kinesin and dynein  <cit> .
other coiled-coil proteins with atpase and gtpase domains often function in folding and repair, e.g.
as chaperonins in protein folding, and topoisomerases and nucleases in dna remodeling  <cit> .
on a primary structure level, amino acid sequences with the capacity to form left-handed alpha-helical coiled-coils are characterized by a heptad repeat pattern in which residues in the first and fourth position are hydrophobic, and residues in the fifth and seventh position are predominantly charged or polar  <cit> .
this pattern of hydrophobic and polar residues interferes with sequence comparison algorithms, which often lead to false predictions of homology between long coiled-coil proteins based on the low complexity and repeat nature of the underlying sequence motif.
on the other hand, this repeat pattern can also be used to predict coiled-coil domains in amino acid sequences by computational means  <cit> .
in the post-genomics era, such structure-prediction algorithms can now be applied to whole proteomes.
based on the prediction algorithm coils, roughly 10% of all proteins encoded by eukaryotic genomes contain coiled-coil domains whereas prokaryotic genomes contain only 4–5%  <cit> .
using the multicoil program, one in every  <dig> proteins in yeast was predicted to contain a coiled-coil sequence  <cit> .
however, these studies did not use a cut-off for domain length to determine coiled-coils.
a minimum length of three to four heptad repeats is required for the formation of a stable coiled-coil using synthetic peptides  <cit> .
using this minimum domain length of  <dig> amino acids ,  <dig> % of the predicted orfs in the fully sequenced arabidopsis genome were found to encode coiled-coil proteins  <cit> .
in a comparative genomics approach, we determined the coiled-coil content of  <dig> predicted whole proteomes using the prediction pipeline and processing software developed to create the arabi-coil database  <cit> .
the  <dig> genomes analyzed included four archaeal genomes, ten bacterial genomes , and eight eukaryotic genomes .
RESULTS
prediction and selection of coiled-coil proteins was performed using the multicoil algorithm  <cit>  and the extractprop processing software  <cit> .
for the purpose of this study, "long coiled-coil" proteins were defined according to the parameters used to establish the arabi-coil database and included all sequences with at least one coiled-coil domain and minimum domain length of  <dig>  two domains and minimum domain length of  <dig>  and three or more domains and minimum domain length of  <dig>  <cit> .
eukaryotic genomes contain higher percentages of long coiled-coil proteins than prokaryotic genomes
proteins predicted to form coiled-coil domains were present in all genomes analyzed  and comprised between 2% and 8% of the total proteomes.
the most pronounced difference between prokaryotic and eukaryotic genomes was in the percentage of genes per genome predicted to encode long or multiple coiled-coil domains.
with increasing coiled-coil domain length cut-off, lower percentages of proteins were identified in bacterial genomes.
with the exception of bacillus subtilis, multicoil predicted no coiled-coil proteins with domains longer than  <dig> amino acids in the bacterial genomes analyzed.
however, archaeal and eukaryotic genomes contain proteins predicted to form coiled-coils of this length.
strikingly, prediction of coiled-coil domains over  <dig> amino acids in length was completely absent in bacterial genomes, but present in eukaryotes as well as two archaea, sulfolobus solfataricus and archeoglobus fulgidus.
these numbers however do not take discontinuous coiled-coil prediction into account, as evident in the case of prokaryotic smc proteins .
proteome sequence sets were downloaded from the european bioinformatics institute   <cit>  or the institute for genome research   <cit> .
the number of protein sequence entries reflects the annotation of orfs at the time of download.
prokaryotic long coiled-coil proteins
archaea
four archaeal genomes were included in this study and tables with coiled-coil protein details are available in additional file  <dig> .
2–3% of the genes in these archaea were found to code for coiled-coil proteins.
in contrast to eubacteria, all of the coiled-coil size-classes analyzed are represented in this group, with proteins predicted to form coiled-coils longer than  <dig> residues present in methanococcus jannaschii and archeoglobus fulgidus proteomes .
eubacteria
bacterial genomes for this study were chosen from different families to represent a wide range of prokaryotic species.
three gram-positive bacterial genomes , and seven gram-negative bacterial genomes  were analyzed.
the largest prokaryotic coiled-coil domains were identified in proteins of the smc, rad <dig>  sbcc and mukb families.
these proteins contain globular head and tail domains separated by a coiled-coil rod with a hinge  <cit> .
figure  <dig> summarizes schematic diagrams of the domain structures of the prokaryotic smc and smc-like proteins identified in this study based on our coiled-coil prediction data and conserved domains as identified through conserved domain database  searches  <cit> .
figure  <dig> shows a summary of additional long coiled-coil proteins with domains of at least  <dig> amino acids in length present in prokaryotic genomes.
a number of these proteins are involved in membrane events, such as chemosensing via methyl-accepting chemotaxis proteins  <cit>  and membrane fusion and vesicle formation mediated by acra, tola, and inca proteins  <cit> .
others function as adhesion proteins, for example the lambda phage side tail fiber protein  <cit>  and the hmw <dig> protein of the attachment organelle of mycoplasma pneumoniae  <cit> , or as enzymes of the cell wall such as the nlpc/p <dig> proteins  <cit> .
long coiled-coil domains cause clustering of unrelated coiled-coil sequences
sequences predicted to form long coiled-coil domains were analyzed for family relationships and conservation across species in an all-against-all approach using the smith-waterman sequence comparison algorithm followed by clustering based on an adaptation of kruskal's minimum cost spanning tree algorithm  <cit> .
in a pilot analysis to test the feasibility of the clustering approach, all prokaryotic sequences meeting the aforementioned criteria for "long coiled-coil" proteins were included in the clustering.
due to the larger number of qualified sequences in the eukaryotic species, only the longest domains  or sequences largely covered by coiled-coil  were included in the combined pilot sequence set comprising  <dig> unique sequences.
a maximum p-score of  <dig> e- <dig> was used as the critical threshold when selecting only the most prominent sequence similarities in this test group.
in all,  <dig>  pair-wise p-score values were selected, defining as many unique relationships from the  <dig>  possible pair-wise relationships.
sequences were then grouped using kruskal's minimum cost spanning tree algorithm using the p-score value as the edge weight for the selected p-score values.
<dig> independent non-overlapping sequence subsets  were defined in this manner.
the largest grouping consisted of  <dig> sequences, representing over half of the sequences in the pilot sequence set and including functionally distinct families such as for example myosins, golgins, and smc proteins.
distinct clusters of long coiled-coil proteins besides this large, heterogeneous group were formed by the animal and yeast tropomyosins , the laminins, the casp/cdp-family and the nuclear lamins.
masking of coiled-coil domains before clustering
to prevent clustering based on the inherent coiled-coil repeat similarities, amino acids predicted to form coiled-coil domains were computationally masked out before being subjected to sequence similarity comparison .
the clustering of the sequences with masked coiled-coil domains yielded a much more accurate grouping of known long coiled-coil protein families such as the myosins, golgins, and smc proteins .
the largest group of long coiled-coil proteins with  <dig> sequences comprised the myosin motor proteins.
the laminins, casp/cdp, and nuclear lamins still exhibited the prior cluster profile, however the tropomyosin clusters did not appear after masking the coiled-coil domains.
the coiled-coil coverage for many of the tropomyosins was predicted as 100% in our analysis, effectively excluding this protein family from the sequence comparison after masking.
largest clusters identified in a pilot analysis using all prokaryotic long coiled-coil proteins and eukaryotic proteins with coiled-coil domains longer than  <dig> amino acids or more than 60% coverage.
only clusters with four or more members are listed.
rock, rho-associated coiled-coil containing kinase  <cit> ; elks/erc <dig>  rab6-interacting protein  <cit> , slap, sarcolemmal-associated protein  <cit> ; doc <dig>  downregulated in ovarian cancer  <dig>  <cit> ; cenp-f, centromer protein f  <cit> .
for species name abbreviations, see table  <dig>
clustering analysis with masked coiled-coil domains
after determining the consistency of clusters formed after masking coiled-coil domains with well-known coiled-coil protein families such as the smc proteins, myosins and kinesins, we proceeded to cluster all  <dig> predicted long coiled-coil sequences from the  <dig> genomes.
the clustering algorithm was further improved to first preclude transitively similar sequences by requiring all sequences in each cluster to satisfy the p-score threshold for all pair-wise relationships within the cluster and secondly to identify "bridge" sequences meeting these criteria for multiple clusters .
a p-score threshold of 10e- <dig> was selected as the appropriate balance of sequence coverage and cluster discrimination.
table  <dig> gives an overview of the sequences from each species contributing to the clustering analysis using the  <dig> e- <dig> p-score cut-off.
the high number of species-specific sequences found in rice is caused by retrotransposon repeats in the rice genome containing predicted coiled-coil domains within a putative transposase orf.
figure  <dig> shows the distribution of clusters among the different kingdoms.
sequence annotation including species origin provided further insight into functions and relationships among sequences in each cluster.
additional information was obtained using conserved domain database searches, multiple sequence alignments, and phylogenetic tree analysis of selected clusters .
contribution of genomes to cross-species clusters .
for species name abbreviations, see table  <dig>
coiled-coil proteins conserved between prokaryotes and eukaryotes
the smc proteins were identified as the single major cluster of long coiled-coil proteins containing sequences from eukaryotic as well as prokaryotic genomes .
another group of conserved proteins with long coiled-coils comprised a number of eukaryotic ser/thr-kinases and a homolog from the cyanobacterium synechocystis .
however, proteins belonging to this cluster could not be found in any other prokaryotic genome.
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
dap, death-associated protein kinase  <cit> ; dmk, myotonic dystrophy kinase  <cit> ; gin <dig>  growth inhibitory gene  <dig>  <cit> ; rock, rho-associated coiled-coil containing kinase  <cit> .
for species name abbreviations, see table  <dig>
a number of smaller cluster were formed containing proteins with shorter coiled-coil domains close to the cut-off for our analysis.
one cluster comprised the translation initiation factor if- <dig>  containing the respective sequences from drosophila, e. coli, mouse, rice and yeast.
another cluster with sequences conserved in prokaryotes as well as eukaryotes contained the aaa+ family atpase clpb/hsp <dig> represented by plant, yeast and bacterial sequences.
this protein functions as a protease/chaperonin in eubacteria, plants and mitochondria  <cit> .
two small clusters combined sequences from prokaryotes and plant genomes.
one cluster comprised mitochondrial seryl-trna synthetases conserved in plant mitochondria as well as archaea while the second cluster comprised the pspa-like vipp <dig> protein from plastids and the cyanobacterium synechocystis.
vipp <dig> is involved in thylakoid biosynthesis in both chloroplasts as well as cyanobacteria, possibly acting in thylakoid membrane trafficking  <cit> .
prokaryotic coiled-coil protein clusters
prokaryotic clusters comprised membrane-bound proteins and signal transducers, as well as membrane-spanning transporters and secretion proteins such as the hlyd family  <cit> .
the only cluster specific to prokaryotes represented by more than ten sequences in this study comprised the methyl-accepting chemotaxis proteins .
smaller prokaryotic clusters contained the aforementioned abc-atpases rad <dig> and sbcc involved in dna repair and a highly conserved group of archaeal proteins of unknown function .
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
for species name abbreviations, see table  <dig>
eukaryotic coiled-coil protein clusters
the main clusters formed by eukaryotic sequences only  were the eukaryotic motor proteins: the actin motor myosin and the microtubule motor kinesin and the related kinesin-like calmodulin-binding protein kcbp  <cit> .
the proteins of the smc <dig> and smc <dig> families formed a eukaryotic cluster instead of clustering together with the condensin/cohesin smcs 1– <dig> and the prokaryotic smc proteins in our analysis .
eukaryotic rad <dig> proteins clustered separately from prokaryotic rad50s as well, indicating a higher convergence of the non-coiled-coil rad <dig> atpase domains as compared to the smc 1– <dig> head and tail domains.
additional larger clusters included eukaryotic ser/thr-kinases and a family comprised of the retinoblastoma-associated protein rbp <dig>  ring finger proteins  <dig> and  <dig>  and yeast bre1p  <cit>  .
formin-related proteins associated with growing actin fibers  <cit>  were found in animal/yeast and animal/plant cluster combinations.
smaller conserved eukaryotic clusters included a number of proteins involved in vesicle transport, such as a rab <dig> gtpase-activating protein involved in retrograde transport  <cit> , the golgin casp  <cit>  and the vesicular transport proteins p <dig> , autophagy protein apg <dig>  <cit> , and early endosome antigen  homologs .
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
mkrps, mitochondrial kinesin-related proteins  <cit> ; nack/hinkel, npk1-activating kinesin  <cit> ; met,  <cit> .
for species name abbreviations, see table  <dig>
yeast, yeast-plant, and yeast-animal coiled-coil protein clusters
eukaryotic genomes included the baker's yeast  and fission yeast  as eukaryotic, unicellular organisms.
protein clusters found to be specific for yeast were typically small  and comprised proteins involved in rna export, such as gle <dig>   <cit>  and mlp <dig>  <cit> , the spindle assembly checkpoint protein mad <dig>  <cit> , and grip-domain golgins  <cit> .
these proteins have known homologs in other eukaryotic proteomes, which did not cluster together with the yeast proteins, likely due to a high overall coverage with coiled-coil sequences .
another functional group of yeast proteins were cell polarity proteins such as spa <dig> and tea <dig>  <cit> .
tea <dig> clustered together with a number of plant sequences of unknown function containing kelch repeats  <cit>  in combination with coiled-coil domains.
proteins that were found in clusters specific to yeasts and animals  included the microtubule motor dynein as well as proteins involved in endocytosis and microtubule dynamics, such as intersectin, restin and cytoplasmic linker proteins   <cit> .
a number of myosin subclusters, for example myosin type ii, was represented only by yeast and animal but not plant sequences, consistent with previous findings  <cit>  .
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
clip, cytoplasmic linker protein  <cit> ; drfs, diaphanous-related formins  <cit> ; eps <dig>  epidermal growth factor receptor substrate  <dig>  <cit> ; png- <dig>  postmitotic neural gene- <dig>  <cit> .
for species name abbreviations, see table  <dig>
animal coiled-coil protein clusters
from the metazoan kingdom, genomes from nematodes , flies , and mammals  were analyzed.
clusters that appeared to be specific to animals  comprised a variety of proteins crosslinking cytoskeletal components with membranes, such as spectrin- and periplakin-like membrane-actin and membrane-if crosslinkers  <cit> , the plasmamembrane-scaffolding liprins  <cit> , the family of merlin and ezrin/radixin/moesin  proteins  <cit> , and a number of golgi- and vesicle-associated proteins.
other groups comprised centrosome-associated and mitotic spindle checkpoint proteins.
type x myosins grouped together in a metazoan cluster without plant or yeast sequences.
another animal-specific group contained coiled-coil proteins involved in structural integrity such as the extracellular scaffolding protein laminin  <cit>  and intermediate filament proteins including the nuclear lamins and neurofilaments  <cit> .
smaller animal-specific clusters contained protein sequences involved in cell attachment and motility, embryogenesis, spermatogenesis, and immune cell movement.
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
aspp, apoptosis stimulating of p <dig> protein  <cit> ; pp <dig>  protein phosphatase  <dig>  <cit> ; rufy, run and fyve domain containing proteins  <cit> .
for species name abbreviations, see table  <dig>
a number of the clusters containing animal sequences were limited to mammalian sequences only .
the hair fiber protein keratin was found to form the largest group of proteins specific to mammals.
other mammlian clusters comprised neurofilament proteins and crosslinkers of the actin cytoskeleton and were found to overlap with clusters containing invertebrate sequences as well.
a number of smaller mammalian clusters  contained sequences of unknown function which have so far only been characterized as autoantigens or cancer antigens.
smaller clusters included the centrosomal protein ninein, which is involved in anchoring microtubule minus ends  <cit> , and a number of other centrosomal proteins including taccs, c-nap <dig>  and centriolin  <cit> .
other clusters included mammalian reproductive organ-specific proteins, such as sperm tail-associated proteins and mammary gland-specific proteins, vertebrate-specific transcription factors and coactivators such as the sox proteins  <cit> , and regulators of endothelial cell motility and clotting factors in blood vessels.
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
see table s <dig> for protein details on clusters smaller than  <dig>  for species name abbreviations, see table  <dig>
plant coiled-coil protein clusters
as representatives for the plant kingdom, a dicot  and a monocot  plant genome were analyzed.
clusters of long coiled-coil proteins specific to arabidopsis and rice contained mostly sequences of so far unknown function .
the rice genome contains a large number of transposon-derived orfs which are predicted to contain coiled-coil domains, therefore a large number of plant-specific clusters was represented by rice sequences only.
these have been omitted from table  <dig>  plant-specific clusters represented by both plant species analyzed included kinase interacting protein  <dig>  and its relatives  <cit> , the family of filament-like plant proteins, fpps  <cit> , and a cluster of putative zinc finger transcription factors homologous to the x <dig> gene of maize  <cit> .
smaller clusters  included nuclear matrix constituent protein  <dig>  and relatives  <cit> , and the chloroplast unusual positioning  <dig>  actin-interacting protein  <cit> .
several clusters showed overlap between the plant and animal kingdoms .
these included a number of kinesin subclusters, vesicle trafficking proteins, and guanylate-binding proteins .
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
see table s <dig> for protein details on clusters smaller than  <dig>  fpps, filament-like plant proteins  <cit> .
for species name abbreviations, see table  <dig>
cluster size
numbers include "bridge" sequences qualifying for more than one cluster.
only clusters represented by at least  <dig> sequences are listed.
fra, fragile fiber  <cit> ; pakrp, phragmoplast-associated kinesin-related protein  <cit> ,  <cit> .
for species name abbreviations, see table  <dig>
discussion
the smc proteins are the most widely conserved coiled-coil proteins
the most widely conserved family of long coiled-coil proteins found in our study comprised the smc proteins.
representatives from almost all species analyzed were found in this cluster, with a few exceptions such as the gram-negative bacterium e. coli.
this is consistent with previous findings that smc proteins are present in eukaryotes as well as all gram-positive bacteria and nearly all archaea, but only less than half of the gram-negative bacteria.
it has been proposed that eukaryotic smc genes evolved from archaeal precursors by two consecutive gene duplications  <cit> .
bacteria without smc proteins often contain an smc-related long coiled-coil protein involved in chromosome segregation or dna repair, such as mukb or sbcc  <cit> .
prokaryotic coiled-coil filament proteins
while prokaryotic genomes contained less long coiled-coil proteins than eukaryotes, we found a number of so far uncharacterized long coiled-coil proteins as candidates for filament-forming prokaryotic coiled-coils.
these included heliobacter pylori proteins previously suggested as candidates for bacterial filament proteins  <cit> .
metazoan mitotic motor proteins lack homologs in plants
the presence of a nucleus in eukaryotic cells is closely linked with the presence of a motile cytoskeleton, in particular the mitotic structures necessary to orchestrate nuclear division, and the endocytic pathway.
dolan et al.
<cit>  proposed a list of motility proteins involved in mitotic processes as candidates for homology searches in prokaryotes to determine their evolutionary origin.
we found 70% of the suggested proteins  among the long coiled-coil proteins identified in our analysis, however none of them clustered together with sequences from archaea or bacteria.
interestingly, with the exception of the kinesins, we also could not find any of these proteins clustering with plant sequences.
with the exception of dynein, kinesin and mad proteins, we could not find clustering of these mitotic motility proteins with yeast sequences either.
the organization of mitotic microtubule nucleation and the composition of the nuclear envelope in plant cells differ significantly from metazoan cells  <cit> .
one hypothesis to explain these differences is the separate development of specialized mechanisms to orchestrate open mitosis in metazoan and plant lineages, leading to the evolution of different nuclear envelope compositions, targeting mechanisms, and mitotic spindle nucleation in the plant and animal kingdoms.
this model explains the absence of many metazoan mitotic motility proteins in plants as well as yeast, which undergoes closed mitosis, and suggests that this group of proteins evolved after the occurrence of open mitosis.
we could not find any plant-specific classes of coiled-coil motor proteins, but noted kinesin subclusters largely represented by plant sequences only, indicating an expansion of this group of motor proteins during plant evolution .
it has been noted before that arabidopsis contains a surprisingly large number of kinesins  <cit> , and it has been suggested that plant-specific kinesin subfamilies might be involved in stress responses or pathogen defenses  <cit> .
differences and similarities in cytoskeletal and membrane infrastructure between plants and animals
besides the motor proteins , membrane tethering and vesicle transport proteins appear to be specific for eukaryotes in our clustering analysis, indicating another major class of specialized coiled-coil proteins that evolved after the formation of eukaryotic cells.
it has been previously suggested that the higher content of long coiled-coil domains in metazoa compared to plants and protists indicates the presence of extensive coiled-coil matrices in animal cells and tissues  <cit> .
one of the groups of coiled-coil proteins apparently absent in plants and yeasts are the nuclear matrix and intermediate filament proteins.
no lamin sequences could be identified from the plant genomes.
other differences we noted between the plant and animal kingdoms are the lack of membrane-cytoskeleton crosslinkers and scaffolding proteins, such as spectrin-like proteins and many actin- and microtubule-associated proteins, in plant proteomes.
this might indicate differences in the overall organization and networking of membrane systems and the actin and microtubule cytoskeleton in plant and animal cells.
differences in coiled-coil content between genomes
earlier surveys of coiled-coil sequences in genbank had suggested that invertebrate genomes contain more coiled-coils than vertebrates, and that animal genomes contain four times more "extended" coiled-coils  than plant genomes  <cit> .
while we could not find such a difference for the overall coiled-coil content or the group of proteins defined as "long" coiled-coils in this study, we did note a significantly lower percentage of coiled-coils longer than  <dig> amino acids in yeast as well as plants compared to the animal genomes .
on average, the yeasts contained one third of the percentage of coiled-coils present in vertebrate genomes with domains longer than  <dig> and longer than  <dig> residues , whereas invertebrates contained about two thirds .
the plant genomes, however, contained on average 57% of the percentage of proteins with coiled-coil domains longer than  <dig> amino acids, but only 22% of the coiled-coils with  <dig> amino acids and longer when compared to vertebrates.
an interesting observation is that the human genome appears to contain more extended coiled-coil proteins than the mouse genome.
our data suggests that this is caused by the human proteome sequence set containing more unique long coiled-coil proteins without homologs in other species , as well as more redundant sequences in clusters .
comparison with other genome-wide coiled-coil predictions
comparable with the arabidopsis coiled-coil protein database arabi-coil, this study takes a more restrictive approach to identifying coiled-coil proteins than previous genome-wide approaches to predict coiled-coil proteins  <cit> .
in contrast to the older studies, our prediction criteria included a minimum coiled-coil domain length corresponding to about three heptad repeats to eliminate sequences with short stretches of predicted coiled-coils unlikely to form stable structures  <cit> .
using these parameters, on average about  <dig> % of all proteins in the eukaryotic proteomes and about  <dig> % in the prokaryotic proteomes  contained coiled-coil domains.
our results were consistent with the study of liu and rost  <cit>  in that most eukaryotic genomes contained more coiled-coil proteins than prokaryotic genomes, and most bacterial genomes more than archaea.
the more restrictive parameters used here resulted in predicting on average about 65–70% of the number of proteins found in those previous studies.
liu and rost  <cit>  further found an exceptionally high coiled-coil content in heliobacter pylori with a higher percentage than c. elegans, and an exceptionally low coiled-coil content in mycobacterium tuberculosis.
our analysis was consistent with these previous observations and resulted in  <dig> % coiled-coil for heliobacter pylori versus  <dig> % in c. elegans and only  <dig> % in mycobacterium tuberculosis, the lowest percentage for all  <dig> genomes analyzed here.
limitations of the prediction and clustering analysis
discontinuous coiled-coil domain predictions
multicoil provides a more stringent coiled-coil prediction than other programs such as coils, resulting in less false positive predictions.
in tests on the pdb database of solved protein structures, two-thirds of the sequences predicted by coils did not contain coiled-coils  <cit> .
by comparison, the programs paircoil and multicoil perform significantly better  <cit> .
occasionally, however, the increased stringency might lead to prediction of fragmented domains where continuous domains have been experimentally verified, as evident in the case of the smc proteins .
selection of long coiled-coil proteins only
in this study, we focused on proteins potentially involved in structural functions.
as the emphasis was placed on proteins with long or multiple coiled-coil domains, it is possible that our selection criteria resulted in the exclusion of homologs of proteins with short stretches of coiled-coil that barely qualified for the analysis.
the selection criteria applied in this study have been shown to exclude 97% of the known bzip proteins from arabidopsis  <cit> .
other examples we noted are the translation initiation factor if- <dig>  mitochondrial and prokaryotic seryl-trna synthetases, and the clpb/hsp <dig> family of heatshock proteins.
members of these protein families failed to meet the selection criteria for long coiled-coil domains, making it difficult to draw conclusions for these protein families from our clustering analysis.
we therefore focused our attention on clusters with mainly proteins containing longer coiled-coils .
effect of coiled-coil masking in the clustering analysis
when clustering sequences with long coiled-coil domain in the pilot analysis, the majority of proteins with long coiled-coil domains was grouped together in one large cluster.
many of the proteins with unknown functions in this group were annotated as "myosin-like", however only about 20% of the proteins in the cluster actually contained a myosin motor domain.
in the other cases, the only similarity to myosin was the presence of a long coiled-coil domain similar to the myosin coiled-coil tail.
this illustrates the ease with which long coiled-coil domains can lead to misannotations in databases with annotations based on sequence similarity searches.
masking the coiled-coil domains before sequence comparison and clustering significantly increased the specificity of the clustering analysis, however protein sequences with high coiled-coil coverage were lost in the subsequent clustering as the masking left little to no sequence for comparison.
examples are the animal and yeast tropomyosins, many of which were predicted to contain 100% coiled-coil coverage, paramyosin, and the plant cytoskeletal protein cip <dig> with more than 80% coiled-coil coverage  <cit> .
CONCLUSIONS
our genome-wide identification of coiled-coil proteins and subsequent clustering provides data suggesting evolutionary conservation or uniqueness of coiled-coil proteins among  <dig> fully sequenced genomes.
we found smc, mukb, sbcc and rad <dig> proteins to be the proteins with the longest coiled-coil domains occurring in prokaryotes, whereas eukaryotic proteomes also contained proteins with stretches of coiled-coil longer than the smc rod domains.
the high conservation of the smc proteins and their structural relatives involved in chromosome maintenance and repair demonstrates the universal importance and conservation of dna housekeeping mechanisms.
long coiled-coil proteins specific to eukaryotes are predominantly involved in subcellular infrastructure maintenance and trafficking control.
table  <dig> gives an overview of the functional classes of long coiled-coil proteins found in our analysis and their representation in different kingdoms.
the genomes of higher plants lack sequences coding for intermediate filament proteins.
many of the known mitotic spindle associated coiled-coil motor proteins in animals lack homologs in plants, consistent with the absence of a centrosomal microtubule organization center in plant cells.
however, the kinesin family of microtubule motor proteins appears to have expanded during the evolution of higher plants.
the repeat nature of the coiled-coil motif makes it difficult to clearly determine sequence homology relationships between long coiled-coil proteins.
functional studies will have to reveal whether so far uncharacterized prokaryotic and plant coiled-coil proteins fulfill similar functions to metazoan counterparts.

 the coiled-coil was one of the earliest protein structures described and first discovered in the two-stranded coiled-coil protein alpha-keratin. coiled-coils consist of two or more alpha-helices winding around each other in a supercoil, a simple yet versatile protein fold. mutations in coiled-coil proteins have been implicated in a large variety of human diseases such as severe skin fragility, muscular dystrophies, neurodegenerative diseases, progeria, and cancer. spurred by medical interest, the number of investigated long coiled-coil proteins in yeast and animals has rapidly grown in recent years. recently, a database of all long coiled-coil proteins in the model plant arabidopsis was established to facilitate the identification and characterization of long coiled-coil proteins in plants. in contrast to eukaryotic organisms, only few long coiled-coil proteins have been characterized in prokaryotes. examples include chaperonins and nucleases, secretion proteins, and cytadherence factors. the foremost feature of coiled-coil domains appears to be their ability to act as "cellular velcro" to hold together molecules, subcellular structures, and even tissues. they can act as protein-protein interaction motifs, for examples as dimerization domains in transcription factors and receptor kinases. they function as "zippers" in membrane fusion proteins, and as adapters between molecules and solid state cellular structures, such as in microtubule organizing centers, the nuclear pores and lamina, actin- and microtubule-associated proteins and cytoskeleton-associated e ubiquitin ligases. extracellular coiled-coil proteins include cell adherence factors and surface receptors, vertebrate blood components such as apolipoproteins and fibrinogen-like clotting factors, and extracellular matrix components such as laminins and cartilage matrix proteins forming tissue scaffolds in metazoa. besides associating with and interconnecting other molecules and macromolecular structures, long coiled-coil domains exhibit a number of structural and mechanical functions. typically, long coiled-coil domains form rod-like tertiary structures and assemble to dynamic fibers, meshworks and scaffolds. examples are the intermediate filaments of the cytoskeleton and nuclear lamina. recent evidence suggests an important role for the dynamic properties of cytoplasmic intermediate filaments in neurodegenerative diseases. other coiled-coils act as spacers, for example in the yeast spindle pole body where the distance between the plaques is determined by the length of the coiled-coil domain in the connecting proteins. membrane-bound coiled-coil proteins such as the spectrins and golgins form scaffolds for membrane structures within the cell. in combination with other functional domains, coiled-coil domains are an integral part of molecular motors, such as the actin motor myosin and the microtubule motors kinesin and dynein. other coiled-coil proteins with atpase and gtpase domains often function in folding and repair, e.g. as chaperonins in protein folding, and topoisomerases and nucleases in dna remodeling. on a primary structure level, amino acid sequences with the capacity to form left-handed alpha-helical coiled-coils are characterized by a heptad repeat pattern in which residues in the first and fourth position are hydrophobic, and residues in the fifth and seventh position are predominantly charged or polar. this pattern of hydrophobic and polar residues interferes with sequence comparison algorithms, which often lead to false predictions of homology between long coiled-coil proteins based on the low complexity and repeat nature of the underlying sequence motif. on the other hand, this repeat pattern can also be used to predict coiled-coil domains in amino acid sequences by computational means. in the post-genomics era, such structure-prediction algorithms can now be applied to whole proteomes. based on the prediction algorithm coils, roughly 10% of all proteins encoded by eukaryotic genomes contain coiled-coil domains whereas prokaryotic genomes contain only 4–5%. using the multicoil program, one in every proteins in yeast was predicted to contain a coiled-coil sequence. however, these studies did not use a cut-off for domain length to determine coiled-coils. a minimum length of three to four heptad repeats is required for the formation of a stable coiled-coil using synthetic peptides. using this minimum domain length of amino acids, % of the predicted orfs in the fully sequenced arabidopsis genome were found to encode coiled-coil proteins. in a comparative genomics approach, we determined the coiled-coil content of predicted whole proteomes using the prediction pipeline and processing software developed to create the arabi-coil database. the genomes analyzed included four archaeal genomes, ten bacterial genomes, and eight eukaryotic genomes.  prediction and selection of coiled-coil proteins was performed using the multicoil algorithm and the extractprop processing software. for the purpose of this study, "long coiled-coil" proteins were defined according to the parameters used to establish the arabi-coil database and included all sequences with at least one coiled-coil domain and minimum domain length of two domains and minimum domain length of and three or more domains and minimum domain length of. eukaryotic genomes contain higher percentages of long coiled-coil proteins than prokaryotic genomes proteins predicted to form coiled-coil domains were present in all genomes analyzed and comprised between 2% and 8% of the total proteomes. the most pronounced difference between prokaryotic and eukaryotic genomes was in the percentage of genes per genome predicted to encode long or multiple coiled-coil domains. with increasing coiled-coil domain length cut-off, lower percentages of proteins were identified in bacterial genomes. with the exception of bacillus subtilis, multicoil predicted no coiled-coil proteins with domains longer than amino acids in the bacterial genomes analyzed. however, archaeal and eukaryotic genomes contain proteins predicted to form coiled-coils of this length. strikingly, prediction of coiled-coil domains over amino acids in length was completely absent in bacterial genomes, but present in eukaryotes as well as two archaea, sulfolobus solfataricus and archeoglobus fulgidus. these numbers however do not take discontinuous coiled-coil prediction into account, as evident in the case of prokaryotic smc proteins. proteome sequence sets were downloaded from the european bioinformatics institute or the institute for genome research. the number of protein sequence entries reflects the annotation of orfs at the time of download. prokaryotic long coiled-coil proteins archaea four archaeal genomes were included in this study and tables with coiled-coil protein details are available in additional file. 2–3% of the genes in these archaea were found to code for coiled-coil proteins. in contrast to eubacteria, all of the coiled-coil size-classes analyzed are represented in this group, with proteins predicted to form coiled-coils longer than residues present in methanococcus jannaschii and archeoglobus fulgidus proteomes. eubacteria bacterial genomes for this study were chosen from different families to represent a wide range of prokaryotic species. three gram-positive bacterial genomes, and seven gram-negative bacterial genomes were analyzed. the largest prokaryotic coiled-coil domains were identified in proteins of the smc, rad sbcc and mukb families. these proteins contain globular head and tail domains separated by a coiled-coil rod with a hinge. figure summarizes schematic diagrams of the domain structures of the prokaryotic smc and smc-like proteins identified in this study based on our coiled-coil prediction data and conserved domains as identified through conserved domain database searches. figure shows a summary of additional long coiled-coil proteins with domains of at least amino acids in length present in prokaryotic genomes. a number of these proteins are involved in membrane events, such as chemosensing via methyl-accepting chemotaxis proteins and membrane fusion and vesicle formation mediated by acra, tola, and inca proteins. others function as adhesion proteins, for example the lambda phage side tail fiber protein and the hmw protein of the attachment organelle of mycoplasma pneumoniae, or as enzymes of the cell wall such as the nlpc/p proteins. long coiled-coil domains cause clustering of unrelated coiled-coil sequences sequences predicted to form long coiled-coil domains were analyzed for family relationships and conservation across species in an all-against-all approach using the smith-waterman sequence comparison algorithm followed by clustering based on an adaptation of kruskal's minimum cost spanning tree algorithm. in a pilot analysis to test the feasibility of the clustering approach, all prokaryotic sequences meeting the aforementioned criteria for "long coiled-coil" proteins were included in the clustering. due to the larger number of qualified sequences in the eukaryotic species, only the longest domains or sequences largely covered by coiled-coil were included in the combined pilot sequence set comprising unique sequences. a maximum p-score of e- was used as the critical threshold when selecting only the most prominent sequence similarities in this test group. in all, pair-wise p-score values were selected, defining as many unique relationships from the possible pair-wise relationships. sequences were then grouped using kruskal's minimum cost spanning tree algorithm using the p-score value as the edge weight for the selected p-score values. independent non-overlapping sequence subsets were defined in this manner. the largest grouping consisted of sequences, representing over half of the sequences in the pilot sequence set and including functionally distinct families such as for example myosins, golgins, and smc proteins. distinct clusters of long coiled-coil proteins besides this large, heterogeneous group were formed by the animal and yeast tropomyosins, the laminins, the casp/cdp-family and the nuclear lamins. masking of coiled-coil domains before clustering to prevent clustering based on the inherent coiled-coil repeat similarities, amino acids predicted to form coiled-coil domains were computationally masked out before being subjected to sequence similarity comparison. the clustering of the sequences with masked coiled-coil domains yielded a much more accurate grouping of known long coiled-coil protein families such as the myosins, golgins, and smc proteins. the largest group of long coiled-coil proteins with sequences comprised the myosin motor proteins. the laminins, casp/cdp, and nuclear lamins still exhibited the prior cluster profile, however the tropomyosin clusters did not appear after masking the coiled-coil domains. the coiled-coil coverage for many of the tropomyosins was predicted as 100% in our analysis, effectively excluding this protein family from the sequence comparison after masking. largest clusters identified in a pilot analysis using all prokaryotic long coiled-coil proteins and eukaryotic proteins with coiled-coil domains longer than amino acids or more than 60% coverage. only clusters with four or more members are listed. rock, rho-associated coiled-coil containing kinase; elks/erc rab6-interacting protein, slap, sarcolemmal-associated protein; doc downregulated in ovarian cancer; cenp-f, centromer protein f. for species name abbreviations, see table clustering analysis with masked coiled-coil domains after determining the consistency of clusters formed after masking coiled-coil domains with well-known coiled-coil protein families such as the smc proteins, myosins and kinesins, we proceeded to cluster all predicted long coiled-coil sequences from the genomes. the clustering algorithm was further improved to first preclude transitively similar sequences by requiring all sequences in each cluster to satisfy the p-score threshold for all pair-wise relationships within the cluster and secondly to identify "bridge" sequences meeting these criteria for multiple clusters. a p-score threshold of 10e- was selected as the appropriate balance of sequence coverage and cluster discrimination. table gives an overview of the sequences from each species contributing to the clustering analysis using the e- p-score cut-off. the high number of species-specific sequences found in rice is caused by retrotransposon repeats in the rice genome containing predicted coiled-coil domains within a putative transposase orf. figure shows the distribution of clusters among the different kingdoms. sequence annotation including species origin provided further insight into functions and relationships among sequences in each cluster. additional information was obtained using conserved domain database searches, multiple sequence alignments, and phylogenetic tree analysis of selected clusters. contribution of genomes to cross-species clusters. for species name abbreviations, see table coiled-coil proteins conserved between prokaryotes and eukaryotes the smc proteins were identified as the single major cluster of long coiled-coil proteins containing sequences from eukaryotic as well as prokaryotic genomes. another group of conserved proteins with long coiled-coils comprised a number of eukaryotic ser/thr-kinases and a homolog from the cyanobacterium synechocystis. however, proteins belonging to this cluster could not be found in any other prokaryotic genome. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. dap, death-associated protein kinase; dmk, myotonic dystrophy kinase; gin growth inhibitory gene; rock, rho-associated coiled-coil containing kinase. for species name abbreviations, see table a number of smaller cluster were formed containing proteins with shorter coiled-coil domains close to the cut-off for our analysis. one cluster comprised the translation initiation factor if- containing the respective sequences from drosophila, e. coli, mouse, rice and yeast. another cluster with sequences conserved in prokaryotes as well as eukaryotes contained the aaa+ family atpase clpb/hsp represented by plant, yeast and bacterial sequences. this protein functions as a protease/chaperonin in eubacteria, plants and mitochondria. two small clusters combined sequences from prokaryotes and plant genomes. one cluster comprised mitochondrial seryl-trna synthetases conserved in plant mitochondria as well as archaea while the second cluster comprised the pspa-like vipp protein from plastids and the cyanobacterium synechocystis. vipp is involved in thylakoid biosynthesis in both chloroplasts as well as cyanobacteria, possibly acting in thylakoid membrane trafficking. prokaryotic coiled-coil protein clusters prokaryotic clusters comprised membrane-bound proteins and signal transducers, as well as membrane-spanning transporters and secretion proteins such as the hlyd family. the only cluster specific to prokaryotes represented by more than ten sequences in this study comprised the methyl-accepting chemotaxis proteins. smaller prokaryotic clusters contained the aforementioned abc-atpases rad and sbcc involved in dna repair and a highly conserved group of archaeal proteins of unknown function. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. for species name abbreviations, see table eukaryotic coiled-coil protein clusters the main clusters formed by eukaryotic sequences only were the eukaryotic motor proteins: the actin motor myosin and the microtubule motor kinesin and the related kinesin-like calmodulin-binding protein kcbp. the proteins of the smc and smc families formed a eukaryotic cluster instead of clustering together with the condensin/cohesin smcs 1– and the prokaryotic smc proteins in our analysis. eukaryotic rad proteins clustered separately from prokaryotic rad50s as well, indicating a higher convergence of the non-coiled-coil rad atpase domains as compared to the smc 1– head and tail domains. additional larger clusters included eukaryotic ser/thr-kinases and a family comprised of the retinoblastoma-associated protein rbp ring finger proteins and and yeast bre1p. formin-related proteins associated with growing actin fibers were found in animal/yeast and animal/plant cluster combinations. smaller conserved eukaryotic clusters included a number of proteins involved in vesicle transport, such as a rab gtpase-activating protein involved in retrograde transport, the golgin casp and the vesicular transport proteins p, autophagy protein apg, and early endosome antigen homologs. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. mkrps, mitochondrial kinesin-related proteins; nack/hinkel, npk1-activating kinesin; met,. for species name abbreviations, see table yeast, yeast-plant, and yeast-animal coiled-coil protein clusters eukaryotic genomes included the baker's yeast and fission yeast as eukaryotic, unicellular organisms. protein clusters found to be specific for yeast were typically small and comprised proteins involved in rna export, such as gle and mlp, the spindle assembly checkpoint protein mad, and grip-domain golgins. these proteins have known homologs in other eukaryotic proteomes, which did not cluster together with the yeast proteins, likely due to a high overall coverage with coiled-coil sequences. another functional group of yeast proteins were cell polarity proteins such as spa and tea. tea clustered together with a number of plant sequences of unknown function containing kelch repeats in combination with coiled-coil domains. proteins that were found in clusters specific to yeasts and animals included the microtubule motor dynein as well as proteins involved in endocytosis and microtubule dynamics, such as intersectin, restin and cytoplasmic linker proteins. a number of myosin subclusters, for example myosin type ii, was represented only by yeast and animal but not plant sequences, consistent with previous findings. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. clip, cytoplasmic linker protein; drfs, diaphanous-related formins; eps epidermal growth factor receptor substrate; png- postmitotic neural gene-. for species name abbreviations, see table animal coiled-coil protein clusters from the metazoan kingdom, genomes from nematodes, flies, and mammals were analyzed. clusters that appeared to be specific to animals comprised a variety of proteins crosslinking cytoskeletal components with membranes, such as spectrin- and periplakin-like membrane-actin and membrane-if crosslinkers, the plasmamembrane-scaffolding liprins, the family of merlin and ezrin/radixin/moesin proteins, and a number of golgi- and vesicle-associated proteins. other groups comprised centrosome-associated and mitotic spindle checkpoint proteins. type x myosins grouped together in a metazoan cluster without plant or yeast sequences. another animal-specific group contained coiled-coil proteins involved in structural integrity such as the extracellular scaffolding protein laminin and intermediate filament proteins including the nuclear lamins and neurofilaments. smaller animal-specific clusters contained protein sequences involved in cell attachment and motility, embryogenesis, spermatogenesis, and immune cell movement. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. aspp, apoptosis stimulating of p protein; pp protein phosphatase; rufy, run and fyve domain containing proteins. for species name abbreviations, see table a number of the clusters containing animal sequences were limited to mammalian sequences only. the hair fiber protein keratin was found to form the largest group of proteins specific to mammals. other mammlian clusters comprised neurofilament proteins and crosslinkers of the actin cytoskeleton and were found to overlap with clusters containing invertebrate sequences as well. a number of smaller mammalian clusters contained sequences of unknown function which have so far only been characterized as autoantigens or cancer antigens. smaller clusters included the centrosomal protein ninein, which is involved in anchoring microtubule minus ends, and a number of other centrosomal proteins including taccs, c-nap and centriolin. other clusters included mammalian reproductive organ-specific proteins, such as sperm tail-associated proteins and mammary gland-specific proteins, vertebrate-specific transcription factors and coactivators such as the sox proteins, and regulators of endothelial cell motility and clotting factors in blood vessels. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. see table s for protein details on clusters smaller than for species name abbreviations, see table plant coiled-coil protein clusters as representatives for the plant kingdom, a dicot and a monocot plant genome were analyzed. clusters of long coiled-coil proteins specific to arabidopsis and rice contained mostly sequences of so far unknown function. the rice genome contains a large number of transposon-derived orfs which are predicted to contain coiled-coil domains, therefore a large number of plant-specific clusters was represented by rice sequences only. these have been omitted from table plant-specific clusters represented by both plant species analyzed included kinase interacting protein and its relatives, the family of filament-like plant proteins, fpps, and a cluster of putative zinc finger transcription factors homologous to the x gene of maize. smaller clusters included nuclear matrix constituent protein and relatives, and the chloroplast unusual positioning actin-interacting protein. several clusters showed overlap between the plant and animal kingdoms. these included a number of kinesin subclusters, vesicle trafficking proteins, and guanylate-binding proteins. cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. see table s for protein details on clusters smaller than fpps, filament-like plant proteins. for species name abbreviations, see table cluster size numbers include "bridge" sequences qualifying for more than one cluster. only clusters represented by at least sequences are listed. fra, fragile fiber; pakrp, phragmoplast-associated kinesin-related protein,. for species name abbreviations, see table discussion the smc proteins are the most widely conserved coiled-coil proteins the most widely conserved family of long coiled-coil proteins found in our study comprised the smc proteins. representatives from almost all species analyzed were found in this cluster, with a few exceptions such as the gram-negative bacterium e. coli. this is consistent with previous findings that smc proteins are present in eukaryotes as well as all gram-positive bacteria and nearly all archaea, but only less than half of the gram-negative bacteria. it has been proposed that eukaryotic smc genes evolved from archaeal precursors by two consecutive gene duplications. bacteria without smc proteins often contain an smc-related long coiled-coil protein involved in chromosome segregation or dna repair, such as mukb or sbcc. prokaryotic coiled-coil filament proteins while prokaryotic genomes contained less long coiled-coil proteins than eukaryotes, we found a number of so far uncharacterized long coiled-coil proteins as candidates for filament-forming prokaryotic coiled-coils. these included heliobacter pylori proteins previously suggested as candidates for bacterial filament proteins. metazoan mitotic motor proteins lack homologs in plants the presence of a nucleus in eukaryotic cells is closely linked with the presence of a motile cytoskeleton, in particular the mitotic structures necessary to orchestrate nuclear division, and the endocytic pathway. dolan et al. proposed a list of motility proteins involved in mitotic processes as candidates for homology searches in prokaryotes to determine their evolutionary origin. we found 70% of the suggested proteins among the long coiled-coil proteins identified in our analysis, however none of them clustered together with sequences from archaea or bacteria. interestingly, with the exception of the kinesins, we also could not find any of these proteins clustering with plant sequences. with the exception of dynein, kinesin and mad proteins, we could not find clustering of these mitotic motility proteins with yeast sequences either. the organization of mitotic microtubule nucleation and the composition of the nuclear envelope in plant cells differ significantly from metazoan cells. one hypothesis to explain these differences is the separate development of specialized mechanisms to orchestrate open mitosis in metazoan and plant lineages, leading to the evolution of different nuclear envelope compositions, targeting mechanisms, and mitotic spindle nucleation in the plant and animal kingdoms. this model explains the absence of many metazoan mitotic motility proteins in plants as well as yeast, which undergoes closed mitosis, and suggests that this group of proteins evolved after the occurrence of open mitosis. we could not find any plant-specific classes of coiled-coil motor proteins, but noted kinesin subclusters largely represented by plant sequences only, indicating an expansion of this group of motor proteins during plant evolution. it has been noted before that arabidopsis contains a surprisingly large number of kinesins, and it has been suggested that plant-specific kinesin subfamilies might be involved in stress responses or pathogen defenses. differences and similarities in cytoskeletal and membrane infrastructure between plants and animals besides the motor proteins, membrane tethering and vesicle transport proteins appear to be specific for eukaryotes in our clustering analysis, indicating another major class of specialized coiled-coil proteins that evolved after the formation of eukaryotic cells. it has been previously suggested that the higher content of long coiled-coil domains in metazoa compared to plants and protists indicates the presence of extensive coiled-coil matrices in animal cells and tissues. one of the groups of coiled-coil proteins apparently absent in plants and yeasts are the nuclear matrix and intermediate filament proteins. no lamin sequences could be identified from the plant genomes. other differences we noted between the plant and animal kingdoms are the lack of membrane-cytoskeleton crosslinkers and scaffolding proteins, such as spectrin-like proteins and many actin- and microtubule-associated proteins, in plant proteomes. this might indicate differences in the overall organization and networking of membrane systems and the actin and microtubule cytoskeleton in plant and animal cells. differences in coiled-coil content between genomes earlier surveys of coiled-coil sequences in genbank had suggested that invertebrate genomes contain more coiled-coils than vertebrates, and that animal genomes contain four times more "extended" coiled-coils than plant genomes. while we could not find such a difference for the overall coiled-coil content or the group of proteins defined as "long" coiled-coils in this study, we did note a significantly lower percentage of coiled-coils longer than amino acids in yeast as well as plants compared to the animal genomes. on average, the yeasts contained one third of the percentage of coiled-coils present in vertebrate genomes with domains longer than and longer than residues, whereas invertebrates contained about two thirds. the plant genomes, however, contained on average 57% of the percentage of proteins with coiled-coil domains longer than amino acids, but only 22% of the coiled-coils with amino acids and longer when compared to vertebrates. an interesting observation is that the human genome appears to contain more extended coiled-coil proteins than the mouse genome. our data suggests that this is caused by the human proteome sequence set containing more unique long coiled-coil proteins without homologs in other species, as well as more redundant sequences in clusters. comparison with other genome-wide coiled-coil predictions comparable with the arabidopsis coiled-coil protein database arabi-coil, this study takes a more restrictive approach to identifying coiled-coil proteins than previous genome-wide approaches to predict coiled-coil proteins. in contrast to the older studies, our prediction criteria included a minimum coiled-coil domain length corresponding to about three heptad repeats to eliminate sequences with short stretches of predicted coiled-coils unlikely to form stable structures. using these parameters, on average about % of all proteins in the eukaryotic proteomes and about % in the prokaryotic proteomes contained coiled-coil domains. our results were consistent with the study of liu and rost in that most eukaryotic genomes contained more coiled-coil proteins than prokaryotic genomes, and most bacterial genomes more than archaea. the more restrictive parameters used here resulted in predicting on average about 65–70% of the number of proteins found in those previous studies. liu and rost further found an exceptionally high coiled-coil content in heliobacter pylori with a higher percentage than c. elegans, and an exceptionally low coiled-coil content in mycobacterium tuberculosis. our analysis was consistent with these previous observations and resulted in % coiled-coil for heliobacter pylori versus % in c. elegans and only % in mycobacterium tuberculosis, the lowest percentage for all genomes analyzed here. limitations of the prediction and clustering analysis discontinuous coiled-coil domain predictions multicoil provides a more stringent coiled-coil prediction than other programs such as coils, resulting in less false positive predictions. in tests on the pdb database of solved protein structures, two-thirds of the sequences predicted by coils did not contain coiled-coils. by comparison, the programs paircoil and multicoil perform significantly better. occasionally, however, the increased stringency might lead to prediction of fragmented domains where continuous domains have been experimentally verified, as evident in the case of the smc proteins. selection of long coiled-coil proteins only in this study, we focused on proteins potentially involved in structural functions. as the emphasis was placed on proteins with long or multiple coiled-coil domains, it is possible that our selection criteria resulted in the exclusion of homologs of proteins with short stretches of coiled-coil that barely qualified for the analysis. the selection criteria applied in this study have been shown to exclude 97% of the known bzip proteins from arabidopsis. other examples we noted are the translation initiation factor if- mitochondrial and prokaryotic seryl-trna synthetases, and the clpb/hsp family of heatshock proteins. members of these protein families failed to meet the selection criteria for long coiled-coil domains, making it difficult to draw conclusions for these protein families from our clustering analysis. we therefore focused our attention on clusters with mainly proteins containing longer coiled-coils. effect of coiled-coil masking in the clustering analysis when clustering sequences with long coiled-coil domain in the pilot analysis, the majority of proteins with long coiled-coil domains was grouped together in one large cluster. many of the proteins with unknown functions in this group were annotated as "myosin-like", however only about 20% of the proteins in the cluster actually contained a myosin motor domain. in the other cases, the only similarity to myosin was the presence of a long coiled-coil domain similar to the myosin coiled-coil tail. this illustrates the ease with which long coiled-coil domains can lead to misannotations in databases with annotations based on sequence similarity searches. masking the coiled-coil domains before sequence comparison and clustering significantly increased the specificity of the clustering analysis, however protein sequences with high coiled-coil coverage were lost in the subsequent clustering as the masking left little to no sequence for comparison. examples are the animal and yeast tropomyosins, many of which were predicted to contain 100% coiled-coil coverage, paramyosin, and the plant cytoskeletal protein cip with more than 80% coiled-coil coverage.  our genome-wide identification of coiled-coil proteins and subsequent clustering provides data suggesting evolutionary conservation or uniqueness of coiled-coil proteins among fully sequenced genomes. we found smc, mukb, sbcc and rad proteins to be the proteins with the longest coiled-coil domains occurring in prokaryotes, whereas eukaryotic proteomes also contained proteins with stretches of coiled-coil longer than the smc rod domains. the high conservation of the smc proteins and their structural relatives involved in chromosome maintenance and repair demonstrates the universal importance and conservation of dna housekeeping mechanisms. long coiled-coil proteins specific to eukaryotes are predominantly involved in subcellular infrastructure maintenance and trafficking control. table gives an overview of the functional classes of long coiled-coil proteins found in our analysis and their representation in different kingdoms. the genomes of higher plants lack sequences coding for intermediate filament proteins. many of the known mitotic spindle associated coiled-coil motor proteins in animals lack homologs in plants, consistent with the absence of a centrosomal microtubule organization center in plant cells. however, the kinesin family of microtubule motor proteins appears to have expanded during the evolution of higher plants. the repeat nature of the coiled-coil motif makes it difficult to clearly determine sequence homology relationships between long coiled-coil proteins. functional studies will have to reveal whether so far uncharacterized prokaryotic and plant coiled-coil proteins fulfill similar functions to metazoan counterparts. 
long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. using the coiled-coil prediction program multicoil, we have previously identified all long coiled-coil proteins from the model plant arabidopsis thaliana and have established a searchable arabidopsis coiled-coil protein database. comparing and grouping all long coiled-coil proteins from genomes, the kingdom-specificity of coiled-coil protein families was determined. motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. at the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. multicoil predicts proteins with extended coiled-coil domains to be largely absent from bacterial genomes, but present in archaea and eukaryotes. because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. here, we have identified all proteins with long coiled-coil domains from additional fully sequenced genomes. they provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs.
 long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. they provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. using the coiled-coil prediction program multicoil, we have previously identified all long coiled-coil proteins from the model plant arabidopsis thaliana and have established a searchable arabidopsis coiled-coil protein database.  here, we have identified all proteins with long coiled-coil domains from additional fully sequenced genomes. because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. comparing and grouping all long coiled-coil proteins from genomes, the kingdom-specificity of coiled-coil protein families was determined. at the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms.  multicoil predicts proteins with extended coiled-coil domains to be largely absent from bacterial genomes, but present in archaea and eukaryotes. the structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell. 