BACKGROUND
integral membrane proteins play essential roles in living cells by transporting ions and small molecules across the membrane, participating in signal transduction and light harvesting.
although they account for about 20-30% of the open reading frames of various sequenced genomes  <cit> , they represent only less than 2% of the protein data bank , due to the difficulty to obtain high-resolution structures  <cit> .
many disease-linked point mutations, which can lead to misfolding and misfunction  <cit> , occur in membrane proteins  <cit> .
there has been considerable research in the area of membrane protein structure and function, particularly with respect to sequences, topology, and the effect of mutations  <cit> .
even though the number of experimentally known membrane protein structures has increased in the last few years, an exhaustive analysis of structural features is still widely needed for enhancing the understanding of many basic phenomena underlying functions, for annotation of large scale genome sequencing data, modeling, and drug design.
proteins in general are known to be rich in small 3d structural motifs important for protein folding and stability as well as for function  <cit> .
structural motifs are commonly occurring small sections in proteins that can characterise active sites, play a structural role in protein folding, and are involved in enzyme biological functions.
sequence-structure correlation studies of small structural motifs have been carried out and several motif databases have been developed in the past few years  <cit> , including the i-sites library, developed by baker and co-workers  <cit>  and the msdmotif database at the ebi, developed from thornton and co-workers  <cit> .
most of the documented 3d motifs show unique patterns of hydrogen bonds, patterns of highly conserved residues, and particular distributions of backbone torsion angles.
when it is possible to associate sequence patterns with structural motifs, they can be used to predict the occurrence of the motifs in new sequences to improve structure prediction methods or help functional annotation such as prosite  <cit>  or profunc  <cit> .
although the structural roles of several small 3d motifs have been widely recognized, their functional roles are not always known.
numerous experiments demonstrate the important role played by helix caps in stabilizing helical termini  <cit> , and linking secondary/supersecondary structure elements.
in some cases, structural motifs have been found to be functionally very important: beta-hairpins in specific protein-protein interactions  <cit>  and nest motifs, as part of small hydrogen-bonded motifs, are prominent in p-loops  <cit> .
integral α-helical membrane proteins are composed of a bundle of α-helices that completely span the membrane.
besides motifs that are also common to globular proteins, α-helical transmembrane proteins are rich in reentrant regions  <cit> , interfacial helices  <cit> , irregular structures at the water-membrane interface, and structured extracellular or cytoplasmic loops  <cit> .
furthermore, helix-helix interaction motifs have been defined by walters and co-workers  <cit>  by few clusters of 3d helical pairs in transmembrane proteins.
among these motifs, the most important are the gasleft and gasright motifs, characterized by high propensities of the small residues gly, ala and ser to occur at periodic positions in the helix-helix interfaces.
although these transmembrane protein structure-sequence motifs are very important from a functional point of view  <cit> , very few motif databases focus on transmembrane proteins.
there are no comprehensive studies, to our knowledge, that focus on functional/structural motifs in transmembrane proteins.
among recent studies on transmembrane protein motifs, the topdom database  <cit>  collects domains and transmembrane protein sequence motifs from different motif databases and organizes them by their location in the protein with respect to the lipid layer.
in the present work we describe three novel motifs in transmembrane proteins and a novel computational approach, structural fragment clustering, which learns sequential motifs from 3d structural fragments.
the motifs are a dimer interface motif in voltage-gated chloride channels, a proton transfer motif in heme-copper oxidases and a convergently evolved interface helix motif in aspartate symporter, serine protease and cytochrome b. these motifs were chosen from among a list of  <dig> novel motifs specific to transmembrane proteins, because, besides being statistically significant and novel with respect to the prosite database, they are filtered on the basis of an accurate functional annotation and manual checking in the scientific literature.
furthermore, the chosen motifs are biologically significant as they play an important role in elucidating the protein functions of specific families or they evolved independently and occur in different families, i.e.
convergent evolution.
only a few fragment-based clustering methods exist that can automatically identify motifs and relate them to function  <cit> .
these methods are based either on geometric features of the fragments or secondary structure patterns  <cit> .
in another study, espalader and co-workers identified loop motifs in proteins associated with specific functions by using the gene ontology function  <cit> .
in a recent study  <cit> , karuppasamy and co-workers used a clustering algorithm based on backbone torsion angles to find fragment clusters enriched in gene ontology function and associated with a significant biochemical function.
in this study, we cluster transmembrane protein fragments based on common structural features in order to generate a library of linear sequence motifs.
new structural motifs and their corresponding signals at sequence level are derived.
our analysis concentrates on α-helical transmembrane proteins as they are more abundant in the pdb  than beta-barrels .
the identified motifs are described in terms of sequence patterns , structural features, and functional relevance.
three of the most interesting motifs are discussed in detail.
RESULTS
biological potential of novel sequence motifs
three biologically interesting motifs from our library are discussed in the following section.
a motif is considered biologically significant if it adds further information, at a detailed structural level, that can help to enhance the understanding of protein function or shed light on mechanisms of structural stability.
the chosen motifs are statistically significant, novel with respect to the prosite database and have very low false positive rates.
new functional insights of these novel motifs are assessed by detailed automated functional annotation and manual literature check .
furthermore, the three examples presented below could not be identified by other structure-based methods such as profunc  <cit>  or topdom  <cit> .
a list of  <dig> non-redundant significant novel motifs, their specificity for membrane proteins and basic structural/functional annotation is provided in additional file  <dig>  these  <dig> motifs exhibit different structural features and locations with respect to the bilayer planes.
most of them  are regular helix motifs embedded in the hydrophobic membrane core.
about 32% are irregular helices or loops at the membrane-water interface, and among them, 20% are short helices parallel to the membrane planes.
only 8% of the motifs are structured loops located on the cytoplasmic or extracellular side with respect to the membrane.
about 6% of the motifs form reentrant loops.
other structural features associated to the motifs are: helix kinks, tilted helices, and π-bulges in 17% of the cases.
about 33% of the motifs occur in different families, i.e.
they are either structurally important or independently evolved motifs.
about 67% of the motifs seem to be associated with the function of a specific family: 53% of the family-specific motifs belong to cytochrome b .
the other families covered by the motifs are: cytochrome c and quinol oxidases  in 12% of the cases, bacterial opsins  in 12% of the cases, photosynthetic reaction center  in 9% of the cases and, in few cases, voltage-gated chloride channels , ammonium transporters , nadh-dehydrogenase  and g-protein coupled receptor-like superfamily .
from the functional annotation, it is worth to notice that about 70% of the motifs are found in cofactor/ligand/ion binding sites, suggesting that they are specific for the protein's function.
about 30% of the motifs are also found at protein-protein interaction interfaces of transmembrane complexes.
finally, 12% of the motifs are found to bind special kinds of lipids such as glycerol or cardiolipins, which are know to modulate protein function via specific protein-lipid interactions.
a- - - -a-p-l- -g: a novel dimer interface motif in voltage-gated chloride channels
the a- - - -a-p-l- -g motif is a pattern specific to voltage gated chloride channels .
clc channels are voltage-gated transmembrane proteins that catalyze the selective flow of cl- ions across cell membranes.
from the structural point of view, the motif corresponds to two reentrant regions of clc channels, i.e.
protein regions that partially dip in the lipid membrane without crossing it entirely .
no functional annotation about the protein regions carrying this motif is present in the prosite database or in the swiss-prot feature field.
the structure of a clc channel reveals two identical triangular subunits  related through a two-fold axis of symmetry perpendicular to the membrane plane and two parallel, independent pores.
each clc cl- channel subunit contains  <dig> α-helices and exhibits a complex topology: the transmembrane α-helices within a subunit are tilted and variable in length and five of them have the typical features of reentrant regions.
three of the five reentrant regions are brought together near the membrane centre to form the selectivity filter for cl- ions, with their n-terminus dipoles pointing towards the binding site and creating a favourable electrostatic environment  <cit> .
these regions are, for each subunit: gsgip , grewgp  and gifap , where residues ser <dig>  ile <dig>  phe <dig>  tyr <dig> and glu <dig>  are annotated as chloride binding sites  <cit> .
the a- - - -a-p-l- -g motif is not part of the selectivity filter and no functional annotation for this motif is available.
nevertheless, this motif, found in reentrant regions at the interaction interface of the two clc channel subunits forming the functional dimer, is highly conserved among bacterial voltage gated chloride channel sequences .
this evidence suggests that the motif must have a role in protein structural stability or dimerization and sheds light on novel functional aspects of voltage gated chloride channels.
the large, stable interface between the subunits is expected because clc cl- channels exist and function only as dimers  <cit> .
due to the electrical dipoles formed by the reentrant regions, the motif could contribute to provide a barrierless and energetically favourable environment for negatively charged particles present in the channel pore.
in fact, it has been shown that, although the pore residues dominate the interaction with cl-, other portions of the protein still contribute a significant fraction of the attractive interaction with cl-  <cit> .
detailed electrostatic calculations would be needed to assess if the reentrant regions at the dimer interface could contribute in a favourable way to the electric field around the cl- ions.
the motif suggests that some residues are important for the protein function and could be tested experimentally for example by site-directed mutagenesis experiments and by checking if dimerization still takes place .
the motif has been derived from the backbone torsion angle clustering of fragments of size  <dig> in the co-called reentrant region .
it contains the conserved hydrogen bond pattern of regular α-helices between main chain atoms in relative positions  <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>  the other residues in the fragment are not linked by hydrogen bonds between the backbone atoms, meaning that the regularity of the α-helix is broken to leave space for a small flexible loop characteristic of reentrant regions.
the motif is highly specific to transmembrane proteins, with  <dig> occurrences in the swiss-prot-tm dataset  and 0% false positive rate.
the motif is specific to bacterial voltage-gated chloride channels and enriched in go terms: voltage-gated chloride activity, antiporter activity, chloride transport, chloride ion binding.
furthermore, from the scoppi database  <cit>  it has been found that some residues of this motif are positioned at the interaction interface between the clc cl- channel subunits in the functional dimer .
statistics and basic structural/functional annotation for this motif are shown in fig.
2a.
-x-y-p-p-l: a membrane-water interface motif in heme-copper oxidases
the -x-y-p-p-l motif corresponds to a structured loop in the interface region, close to the extracellular side of heme-copper oxidases .
the heme-copper oxidases catalyse the reduction of molecular oxygen to water.
the chemical energy released in the reduction reaction is utilized to transfer protons across the membrane and to generate an electrochemical proton gradient.
they all have a low-spin heme, which is the initial electron acceptor, and a high-spin heme, which forms the catalytic site with a copper centre.
the  <dig> histidine residues ligating the cofactors are fully conserved in the superfamily of heme-copper oxidases  <cit> .
the heme-copper oxidase profiles are widely documented in the prosite database .
also the his residues that ligate the heme groups and the copper ion are annotated in the swiss-prot feature field.
the pattern we find is not documented in any protein motif databases, but it is worthwhile to investigate its function as it contains a tryptophane residue  which has been widely documented in literature.
it has been shown that trp <dig>  which is hydrogen bonded to Δ-propionate of heme a <dig> in the catalytic centre, is highly conserved  <cit> .
mutation studies of this residue suggest that it is involved in regulating proton transfer from the pumping site near heme a <dig> to the p-side of the membrane  <cit> .
we infer that the -x-y-p-p-l motif plays a special structural role in oxidases, creating the optimal environment to allow trp <dig>  perform its regulatory role in the proton transfer process.
furthermore, the motif suggests that tyr <dig> plays the same role in cytochrome ba <dig> as trp <dig> in cytochrome c. this evidence could guide future experiments in order to explore the detailed functional role of tyr <dig>  as, to our knowledge, no mutational studies of tyr <dig> in cytochrome ba <dig> oxidases have yet been carried out.
the motif has been derived from classes of fragments of size  <dig> and  <dig> clustered by backbone torsion angles.
the structural motif associated to the sequence pattern contains the highly conserved residues tyr <dig>  pro <dig>  pro <dig> and leu <dig>  which seem to form a highly structured cytoplasmic loop with the function of placing trp <dig> at the right distance from and orientation with respect to heme a <dig>  our protein-ligand analysis also reveals trp <dig> to be in close contact with the heme a <dig> group .
the -x-y-p-p-l motif has been found to be highly specific for transmembrane proteins, with  <dig> occurrences in the swiss-prot-tm dataset and enriched in the following go terms: oxidoreductase activity, heme binding, mitochondrial electron transport chain, iron ion binding, aerobic respiration, copper ion binding, electron transport, mitochondrion, metal ion binding, cytochrome-c oxidase activity.
statistics and basic structural/functional annotation for this motif are shown in fig.
2b.
l-x-s-i- : a convergently evolved interface helix motif
the l-x-s-i-  motif corresponds to a very short irregular helix almost parallel to the membrane plane  found across more than  <dig> protein families and derived from the structural clustering of fragments of length  <dig> in the reentrant region.
this motif was found in proteins with different functions and it is a case of convergent evolution, i.e.
proteins with different sequence and structure that share a common functional feature/mechanism.
the structures from which the motif is derived are: aspartate symporter , serine protease  and cytochrome b .
the aspartate symporter is a sodium-driven secondary transporter that catalyzes the uptake of aspartate from chemical synapses.
it exists in the membrane as a trimer and each subunit has eight transmembrane segments, two reentrant helical hairpins  and independent substrate translocation pathways  <cit> .
it is thought that the hp <dig> reentrant region, where the l-x-s-i-  motif is contained , acts as a gate, adopting a open conformation and allowing the aspartate to reach the binding site from the extracellular solution  <cit> .
the short parallel helix containing the l-x-s-i-  motif is also involved in the formation of one of the two sodium binding sites , as the transport of aspartate is highly coupled to sodium transport.
the serine protease, belonging to the rhomboid proteases family, is a protein whose function is to cleave the transmembrane domains of other proteins.
the crystal structure reveals six transmembrane segments and other two interesting features: an internal aqueous cavity that opens to the extracellular side and a long membrane-embedded loop between the first and the second helices  <cit> .
the opening of this loop, which also contains the irregular helix corresponding to the l-x-s-i-  motif , is thought to be the likely route by which the substrate enters the active site.
so, it has been postulated that this loop, and in particular the segment corresponding to the motif, functions as a gate and may change conformation when the substrate binds  <cit> .
finally, there is no evidence in literature that the same motif in cytochrome b  is associated to a gating function.
but it has been found, from ligand analysis, that the structural segment corresponding to the l-x-s-i-  motif is part of the q <dig> binding site and involved in non-covalent interactions with the substrate.
this suggests that the motif  is involved in conformational changes upon substrate binding, but this assumption needs experimental validation.
it can be concluded that these three proteins, unrelated in structure and biochemical function, share a convergently functional motif that, although not directly part of the core catalytic activity of the protein, modulates gate dynamics at the membrane-protein interface.
statistics and basic structural/functional annotation for this motif are shown in fig.
2c.
general results from the structure fragment clustering
consider fig.
<dig>  which describes the procedure of structural fragment clustering.
it consists of six steps.
in step  <dig> non-redundant sequences  and transmembrane protein structures with resolution less than  <dig>  Å are collected.
in step  <dig>  the protein structures are fragmented and fragments are labeled according to their location and topology.
in step  <dig>  the fragments are clustered based on their hydrogen bonding patterns and on torsion angles, respectively.
in step  <dig>  sequence motifs are derived from significant clusters of fragments and in step  <dig>  they are annotated regarding functional and structural features.
in the final step  <dig>  all motifs are filtered regarding their significance and novelty.
structural fragment clustering
in step  <dig> non-redundant sequences  and  but unspecific backbone torsion angle profiles.
motifs can also lack a specific hydrogen bond pattern but be detectable by means of their Φ and Ψ angle values .
a similarity measure based on common hydrogen bond profiles can reveal very stable structural motifs, as hydrogen bonds in membrane proteins are much stronger than in globular proteins .
this is due to the low dielectric constant of the membrane and lack of competing interactions with water molecules  <cit> .
on the other hand, it has been shown that a similarity measure based on differences in backbone torsion angle profiles is very sensitive to variations in local protein conformations and that active site torsion angles are usually highly conserved  <cit> .
in step  <dig> sequence motifs  were derived for each structural class, if possible.
if no significant motif could be associated with a cluster, a further sequence-based clustering step was performed to filter significant sequence patterns.
then, a library of regular expressions associated with specific structural features was compiled.
in step  <dig>  functional annotation of fragments was performed using multiple sources of information.
fragment clusters were annotated with shared go categories  <cit> .
each fragment in a cluster was associated to a swiss-prot feature ft field annotation, when this annotation existed.
fragments that belong to protein-protein interfaces or are part of ligand/substrate binding site were annotated by mapping them onto the scoppi  <cit>  and the pdb database  <cit>  for possible binding sites.
furthermore, residues in fragments that have been experimentally mutated and have a function reported in the literature were also annotated.
finally, for each cluster-derived sequence pattern, its total or partial overlap with a prosite  <cit>  pattern is checked and reported.
in step  <dig>  the final step, the motifs were filtered.
initially, there were  <dig> motifs.
first, we filtered by significance and specificity to membrane proteins, resulting in  <dig> motifs.
next, we filtered out motifs that are already documented in general motif databases, resulting in  <dig> novel motifs.
finally, we grouped overlapping motifs and thus remove redundancy, leaving  <dig> motifs.
these  <dig> motifs are described in detail regarding structural and functional features in additional file  <dig>
statistics of clusters and filtering
the total number of clustered fragments was  <dig>  for the hydrogen bond-based clustering and  <dig>  for the backbone angle-based one .
in table  <dig>  the statistics for both clusterings are summarized: number of fragments, structural clusters, outliers and size of the largest cluster for each region and fragment length.
plots of these numbers versus fragment length are presented and discussed in additional file  <dig>  the number of clusters and outliers  is low in the helix core region with respect to the cytoplasmic, extracellular and interface regions.
this is due to the low structural diversity of the helix core region, where 80% of the fragments fall into the regular α-helix structural class or irregular  <dig> helices .
in the cytoplasmic and extracellular regions, the number of classes increases and their distribution drastically changes since there is more structural variability compared to the helix core region.
length
percentage of structural classes covered by sequence patterns.
the first column shows the number of classes obtained for the hydrogen bond-based clustering before and after the sequence-clustering step.
the second column shows the percentage of clusters for which a sequence pattern could be derived by using the pratt program before and after the sequence-clustering step.
the third column shows the percentage of classes that could be assigned to a statistically significant motif before and after the sequence-clustering step.
colums four, five and six show the same numbers for the backbone torsion angle-based clustering.
in total,  <dig> non-redundant sequence motifs have been derived from both clusterings.
from this number, the statistically insignificant motifs and those motifs that are not specific to transmembrane proteins were filtered out.
a motif is considered statistically significant when its associated p-value, derived by randomly permutating the swiss-prot tm database, is smaller than  <dig>  .
furthermore, a motif is considered specific to transmembrane proteins if its false positive rate is low enough, i.e if the p-value of the hyper-geometric distribution is smaller than  <dig> × 10- <dig> .
fig.
6a shows the distribution of motifs of different lengths.
the histogram shows that the strongest correlation between structural clusters and sequence preferences is obtained for motifs of length of  <dig> to  <dig> amino acids.
the number of sequence patterns associated to structural classes strongly decreases for fragment length greater than  <dig> amino acids.
fig.
6b shows the percentage of motifs associated with different false positive rates for three different fragment lengths .
an anti-correlation has been observed between false positive rate and motif length : the longer the sequence motif, the lower the false positive rate.
as shown in fig.
6a, for length  <dig>  100% of the motifs have false positive rate less than 20%; motifs of length  <dig> are very unspecific as most of them have false positive rate greater than 60%.
after the filtering step,  <dig> significant motifs were considered for further analysis.
the average resolution of the structural motifs after filtering is  <dig>  Å. this means that the clustering process automatically filtered out lower resolution fragments as outliers.
85% of the derived motifs were common to both hydrogen bond- and torsion angle-based clusters , especially in the helix core region.
this observation is a further proof of the reliability of the retrieved structural motifs.
in order to evaluate the ability of our structure-based method in finding novel motifs, which cannot be identified by sequence-based methods alone, an all-against-all comparison of our motifs against the prosite database was carried out.
the algorithm used for comparison with prosite patterns is described in detail in materials and methods.
it is necessary to stress that a direct comparison of our motif library with the prosite database is not straightforward for three main reasons.
first, as prosite patterns are derived at sequence level, from conserved regions in multiple alignments of homologous sequences, they are usually longer  than ours .
second, prosite patterns are derived by scanning all the sequences in the swiss-prot database, unlike our library that is derived only from transmembrane proteins of known structure.
for this reason the comparison is limited to those prosite patterns that hit anywhere in swiss-prot transmembrane proteins .
third, it has to be taken into account that the number of known structures for membrane proteins is considerably smaller than the number of known sequences.
this implies that a high coverage value of our motif library against prosite cannot be expected.
on the other hand, it is interesting to quantify and investigate the number of found linear motifs that do not have any match in prosite, as they provide a proof that structural information adds new knowledge about unannotated sequences and functional implication.
by analyzing the results from the comparison, it has been found that by varying the value of the cut-off for defining a match between two patterns, the number of matched/unmatched motifs strongly varies .
the cut-off for the similarity score was set to  <dig> .
the choice of the cut-off is based on a comparison to a random model .
by setting the cut-off for the similarity score to  <dig> , the percentage of significant novel motifs is about 10%,  <dig> motifs out of  <dig> non-redundant, statistically significant motifs.
although a comparison with prosite is not straightforward, it is clear that the  <dig> novel motifs represent new knowledge, which cannot be gained from sequence alone.
after grouping overlapping motifs and removing redundancy,  <dig> motifs were checked in the scientific literature for the first biological assessment and described in detail regarding structural and functional features, in additional file  <dig>
comparison with meme
in order to evaluate the capability of our structure-based method to survey motifs that cannot be found by sequence-based pattern searching tools alone, we compared our motifs with the motifs obtained by using the meme tool, a software package to discover motifs in groups of related dna or protein sequences  <cit> .
we derived motifs with meme on the same dataset of  <dig> protein chain sequences used for generating our motifs.
in total,  <dig> motifs were generated using meme, with the following options: motifs lenght ranging from  <dig> to  <dig> amino acids; motifs generated from a minimum of  <dig> sequences; e-value less that  <dig> .
in oder to compare our motifs with meme-generated motifs, we computeted the overlap between couples of motifs by counting the number of common transmembrane proteins in swiss-prot where the two motifs hit.
if the overlap was higher than 80% the motifs were considered as the same motif.
we find that less than 20% of our motifs could be found by meme.
in particular, the three motifs corresponding to the three examples discussed in the previous sub-section could not be detected from the meme tool.
out of the  <dig> motifs described in additional file  <dig>   <dig> could also be found by meme and they all correspond to family-specific motifs.
<dig> motifs out of  <dig> could only be found by means of our structure fragment clustering approach and represent new knowledge that cannot be derived by means of sequence-based pattern searching tools alone.
since a key finding of this paper is that motifs exist as modular building blocks across unrelated families, it is clear that purely sequence-based approached are not adequate due to the divergence of the different families.
family vs non-family specific motifs
the sequence homology of the dataset for deriving the motifs is reduced at 90% identity.
this is a quite permissive cut-off and the risk of deriving only homology-based patterns, representative of a limited sampling, is high.
to verify that not all the patterns we derive are obvious signals of homologous proteins, but that many of them are functionally or structurally of great importance , an analysis at family level was carried out by using the pfam database.
each motif can be assigned to a pfam family or to a set of pfam families by assigning a pfam family to the swiss-prot-tm sequences the motif matches.
surprisingly, we found that only 6% of the  <dig> statistically significant motifs are family-specific.
the rest of the motifs are found across different pfam families or clans .
novel family-specific motifs, not represented in the prosite database, are interesting because they can shed light on novel and different aspects of a protein's structure and function.
motifs across families can be important for structural stability, e.g.
transmembrane helix kinks, motifs at the membrane-water interface, protein-lipid interaction motifs, helix-helix packing motifs, or they can be cases of convergent evolution, i.e.
found in proteins that are not homology-related but share some functional mechanisms.
the a- - - -a-p-l- -g and -x-y-p-p-l motifs discussed in the previous sections are examples of motifs specific to the voltage-gated chloride channel family and the heme copper oxidase family, respectively.
the l-x-s-i-  parallel helix motif, also discussed in the previous section, is an example of motif found across different protein families and it is a potential convergent evolution motif.
other important motifs across families, such as helix kinks, helix distortions, interface helices, helix-helix packing and protein-lipid interaction motifs are described in additional file  <dig>
motifs help to identify membrane proteins in novel genomes
to demonstrate the capability of our motifs to identify new transmembrane proteins, we performed the following analysis: we determined the distribution of the number of motif hits in swiss-prot sequences for transmembrane proteins against globular proteins.
the two distributions are shown in fig.
<dig>  this analysis was performed for high-confidence motifs, i.e.
motifs with false positive rate less than 40% as defined in additional file  <dig>  fig.
<dig> shows that all globular proteins  contain five or less motif hits, in contrast to transmembrane proteins which can contain up to  <dig> motif hits.
this allows to define a simple rule: if a new sequence contains more than  <dig> of the motifs, then it is predicted to be a transmembrane protein.
applied to swiss-prot this rule does not make any false predictions.
however, since the motifs are based on structures, the coverage is not high and thus there will be still many membrane proteins among the sequences for which no prediction is made.
in this respect, our method can help identifying transmembrane proteins in novel genomes.
coverage of motifs in different genomes
the coverage of the  <dig> non-redundant motifs in different genomes has been estimated and the results presented in table  <dig>  table  <dig> shows the percentage of transmembrane proteins in the swiss-prot database, where at least one motif hits, for the top ten genomes, i.e.
the genomes with the highest number of motifs hits, ordered by the decreasing number of the motif hits.
in average, the coverage of the portion of genome encoding for membrane proteins is 9%.
<dig> transmembrane proteins were found to be associated with at least one of the  <dig> motifs in the human genome.
percentage of transmembrane proteins, containing at least one of the  <dig> non-redundnat, fully characterized sequence motifs, for each species.
only the top  <dig> mostly represented species, oredered by the number of motif hits in the dataset of transmembrane proteins are reported.
the first colomn specifies the protein species, the second colomn specifies, for each species, the number of motif hits in swiss-prot transmembrane sequences, the third colomn specifies, for each species, the number of corresponding transmembrane protiens in swiss-prot and the last colomn shows, for each species, the percentage of transmembrane protein that contains at least one motif.
more generally, we estimated also the coverage of the  <dig> motifs in three protein kingdoms.
it was found that 15% of eukaryotic proteins were covered by the  <dig> motifs, against 9% in bacteria and archaea.
CONCLUSIONS
in this work we introduced structural fragment clustering and derived  <dig> novel sequence motifs in membrane proteins together with their functional characterization the novel motifs appear across many families and therefore show that they form functional modules, which are re-used.
the majority of the motifs is found on binding sites to membrane, ligands, co-factors, other proteins or other helices, highlighting their functional role and the importance of the environment for these structural building blocks .
we discuss three novel motifs in detail.
two of them, a re-entrant region in voltage-gated chloride channels and a structured loop in the membrane-water interface region of heme-copper oxidases, are family-specific and help add precious details to the functional mechanisms of the proteins they are found in.
the third motif, an interface helix derived from the clustering of fragments in three different protein families, is an interesting case of convergent evolution, where three evolutionarily unrelated families with different functions share a common gating mechanism.
the three motifs discussed here have been chosen among  <dig> novel statistically significant and non-redundant motifs derived by means of an unsupervised learning method also described here.
the method uses structural information about protein fragments, like conserved hydrogen bond patterns and backbone torsion angle profiles, to derive short linear motifs in α-helical transmembrane proteins.
although the data set contains low-resolution structures with a resolution worse than  <dig>  Å, 98% of the clusters contain at least one high-resolution fragment from a structure of less than  <dig>  Å resolution.
this garantees reliability of the results.
furthermore, the distribution of the average resolution value for all clusters has a peak around  <dig>  Å .
due to intrinsic difficulties in experimental determination of membrane protein structures  <cit> , their average resolution in the pdb is worse than for globular proteins   <cit> .
removing structures of resolution worse than  <dig>  Å and filtering for redundancy would reduce our dataset from  <dig> to only  <dig> structures.
this would make it nearly impossible to perform clustering and to obtain statistically significant results.
the method, even though it is based on the information retrieved from the limited set of membrane proteins with known 3d structures, is able to find novel functionally or structurally important motifs that can complement and enrich information retrieved from sequence-based methods like prosite or meme.
while new protein domains or family signatures, such as those contained in pfam  <cit>  or prosite  <cit> , can be defined from alignments of evolutionarily related sequences, the identification of short sequence motifs, related to specific structural features and shared between transmembrane protein functional classes, is much harder.
to address this problem different sources of information are taken into account to elucidate the role played by short structural/sequential motifs in different transmembrane proteins classes.
these are: structural properties, location with respect to the membrane planes, go annotations, swiss-prot functional annotations, interaction interface information, and mutational analysis.
it is shown in the three examples that the method is able to predict new functional residues.
in future studies, the method will be applied to better characterize other important sequential/structural motifs in transmembrane proteins, like interfacial helices or patterns at helix-helix interaction interfaces.
our method, in contrast to the approach described in  <cit> , not only enables the discovery of 3d motifs associated with function, but makes use of regular expressions to allow searching for functional motifs in transmembrane proteins of unknown structure.
cluster signatures are an attractive way to annotate protein function at both the structure and the sequence levels.
indeed, it has been shown that protein annotation effort benefits immensely from the knowledge of functional signatures in both primary, secondary and tertiary structure.
in fact, sometimes the multifunctionality and overall structural diversity of even closely related proteins confounds efforts to assign function on the basis of overall sequence or structural similarity  <cit> .
approaches based on the identification of common small functional motifs can help to overcome this problem.
this is especially true for membrane proteins, where protein with the same topology, fold or signal at sequence level in the hydrophobic core, perform totally different functions, thanks to hotspot residues and small motifs that differentiate them, or share common functional mechanisms .
in addition, functional motifs pinpoint individual residues that play a crucial functional role and complement the information contained in alignments of homologous proteins, such as the ones contained in pfam, which focus on large functional signatures, at family level, but not on individual residues or small motifs, related to specific structural features.
our method generates structural motifs and associate them directly to function by mapping onto the protein fragments functional characteristics, such as swiss-prot 'features', protein-protein interface and protein-ligand binding sites associated residues and go annotation.
furthermore, fragments belonging to functional sites or containing hot-spot residues are ideal candidates for experimental validation.
possible experiments that can be done to validate the structural and functional role of a motif involve different experimental techniques.
first, force spectroscopy, which allows to measure the force necessary to pull proteins out of the native membrane, can be used to validate structural motifs important for protein stability  <cit> .
second, confocal microscopy can validate motifs relating to the protein's topology, such as interface helices, by labeling membrane and motif with different dyes and by determining the location of the motif seuence relative to the membrane.
third, mutants from membrane proteins can be tested by means of functional assays.
for example, for the dimerization motif in chloride channels, dimerization upon mutation of one of the conserved residues in the motif can be tested by means of imaging through atomic force microscopy .
if dimerization still takes place, it can be tested whether the dimer is a functional dimer, e.g.
by measuring the concentration of cl- and h+ exchanged.
to conclude, it is worthwhile to emphasize the power of the method and the results presented here in two other application fields.
first, motifs from our library can help identifying transmembrane proteins in novel genomes, as discussed in results and discussion.
second, structural motifs, such as reentrant regions, helix kinks and helix-helix contact motifs or functional motifs, such as the ones related to the protein binding sites or protein-lipid interactions, can be used as constraints while building more refined two-dimensional models of α-helical transmembrane proteins from sequence alone.
it has been shown that both two-dimensional tools  <cit>  and three-dimensional prediction algorithms benefit from the use of structure-sequence motifs as constraints.

 integral membrane proteins play essential roles in living cells by transporting ions and small molecules across the membrane, participating in signal transduction and light harvesting. although they account for about 20-30% of the open reading frames of various sequenced genomes, they represent only less than 2% of the protein data bank, due to the difficulty to obtain high-resolution structures. many disease-linked point mutations, which can lead to misfolding and misfunction, occur in membrane proteins. there has been considerable research in the area of membrane protein structure and function, particularly with respect to sequences, topology, and the effect of mutations. even though the number of experimentally known membrane protein structures has increased in the last few years, an exhaustive analysis of structural features is still widely needed for enhancing the understanding of many basic phenomena underlying functions, for annotation of large scale genome sequencing data, modeling, and drug design. proteins in general are known to be rich in small 3d structural motifs important for protein folding and stability as well as for function. structural motifs are commonly occurring small sections in proteins that can characterise active sites, play a structural role in protein folding, and are involved in enzyme biological functions. sequence-structure correlation studies of small structural motifs have been carried out and several motif databases have been developed in the past few years, including the i-sites library, developed by baker and co-workers and the msdmotif database at the ebi, developed from thornton and co-workers. most of the documented 3d motifs show unique patterns of hydrogen bonds, patterns of highly conserved residues, and particular distributions of backbone torsion angles. when it is possible to associate sequence patterns with structural motifs, they can be used to predict the occurrence of the motifs in new sequences to improve structure prediction methods or help functional annotation such as prosite or profunc. although the structural roles of several small 3d motifs have been widely recognized, their functional roles are not always known. numerous experiments demonstrate the important role played by helix caps in stabilizing helical termini, and linking secondary/supersecondary structure elements. in some cases, structural motifs have been found to be functionally very important: beta-hairpins in specific protein-protein interactions and nest motifs, as part of small hydrogen-bonded motifs, are prominent in p-loops. integral α-helical membrane proteins are composed of a bundle of α-helices that completely span the membrane. besides motifs that are also common to globular proteins, α-helical transmembrane proteins are rich in reentrant regions, interfacial helices, irregular structures at the water-membrane interface, and structured extracellular or cytoplasmic loops. furthermore, helix-helix interaction motifs have been defined by walters and co-workers by few clusters of 3d helical pairs in transmembrane proteins. among these motifs, the most important are the gasleft and gasright motifs, characterized by high propensities of the small residues gly, ala and ser to occur at periodic positions in the helix-helix interfaces. although these transmembrane protein structure-sequence motifs are very important from a functional point of view, very few motif databases focus on transmembrane proteins. there are no comprehensive studies, to our knowledge, that focus on functional/structural motifs in transmembrane proteins. among recent studies on transmembrane protein motifs, the topdom database collects domains and transmembrane protein sequence motifs from different motif databases and organizes them by their location in the protein with respect to the lipid layer. in the present work we describe three novel motifs in transmembrane proteins and a novel computational approach, structural fragment clustering, which learns sequential motifs from 3d structural fragments. the motifs are a dimer interface motif in voltage-gated chloride channels, a proton transfer motif in heme-copper oxidases and a convergently evolved interface helix motif in aspartate symporter, serine protease and cytochrome b. these motifs were chosen from among a list of novel motifs specific to transmembrane proteins, because, besides being statistically significant and novel with respect to the prosite database, they are filtered on the basis of an accurate functional annotation and manual checking in the scientific literature. furthermore, the chosen motifs are biologically significant as they play an important role in elucidating the protein functions of specific families or they evolved independently and occur in different families, i.e. convergent evolution. only a few fragment-based clustering methods exist that can automatically identify motifs and relate them to function. these methods are based either on geometric features of the fragments or secondary structure patterns. in another study, espalader and co-workers identified loop motifs in proteins associated with specific functions by using the gene ontology function. in a recent study, karuppasamy and co-workers used a clustering algorithm based on backbone torsion angles to find fragment clusters enriched in gene ontology function and associated with a significant biochemical function. in this study, we cluster transmembrane protein fragments based on common structural features in order to generate a library of linear sequence motifs. new structural motifs and their corresponding signals at sequence level are derived. our analysis concentrates on α-helical transmembrane proteins as they are more abundant in the pdb than beta-barrels. the identified motifs are described in terms of sequence patterns, structural features, and functional relevance. three of the most interesting motifs are discussed in detail.  biological potential of novel sequence motifs three biologically interesting motifs from our library are discussed in the following section. a motif is considered biologically significant if it adds further information, at a detailed structural level, that can help to enhance the understanding of protein function or shed light on mechanisms of structural stability. the chosen motifs are statistically significant, novel with respect to the prosite database and have very low false positive rates. new functional insights of these novel motifs are assessed by detailed automated functional annotation and manual literature check. furthermore, the three examples presented below could not be identified by other structure-based methods such as profunc or topdom. a list of non-redundant significant novel motifs, their specificity for membrane proteins and basic structural/functional annotation is provided in additional file these motifs exhibit different structural features and locations with respect to the bilayer planes. most of them are regular helix motifs embedded in the hydrophobic membrane core. about 32% are irregular helices or loops at the membrane-water interface, and among them, 20% are short helices parallel to the membrane planes. only 8% of the motifs are structured loops located on the cytoplasmic or extracellular side with respect to the membrane. about 6% of the motifs form reentrant loops. other structural features associated to the motifs are: helix kinks, tilted helices, and π-bulges in 17% of the cases. about 33% of the motifs occur in different families, i.e. they are either structurally important or independently evolved motifs. about 67% of the motifs seem to be associated with the function of a specific family: 53% of the family-specific motifs belong to cytochrome b. the other families covered by the motifs are: cytochrome c and quinol oxidases in 12% of the cases, bacterial opsins in 12% of the cases, photosynthetic reaction center in 9% of the cases and, in few cases, voltage-gated chloride channels, ammonium transporters, nadh-dehydrogenase and g-protein coupled receptor-like superfamily. from the functional annotation, it is worth to notice that about 70% of the motifs are found in cofactor/ligand/ion binding sites, suggesting that they are specific for the protein's function. about 30% of the motifs are also found at protein-protein interaction interfaces of transmembrane complexes. finally, 12% of the motifs are found to bind special kinds of lipids such as glycerol or cardiolipins, which are know to modulate protein function via specific protein-lipid interactions. a- - - -a-p-l- -g: a novel dimer interface motif in voltage-gated chloride channels the a- - - -a-p-l- -g motif is a pattern specific to voltage gated chloride channels. clc channels are voltage-gated transmembrane proteins that catalyze the selective flow of cl- ions across cell membranes. from the structural point of view, the motif corresponds to two reentrant regions of clc channels, i.e. protein regions that partially dip in the lipid membrane without crossing it entirely. no functional annotation about the protein regions carrying this motif is present in the prosite database or in the swiss-prot feature field. the structure of a clc channel reveals two identical triangular subunits related through a two-fold axis of symmetry perpendicular to the membrane plane and two parallel, independent pores. each clc cl- channel subunit contains α-helices and exhibits a complex topology: the transmembrane α-helices within a subunit are tilted and variable in length and five of them have the typical features of reentrant regions. three of the five reentrant regions are brought together near the membrane centre to form the selectivity filter for cl- ions, with their n-terminus dipoles pointing towards the binding site and creating a favourable electrostatic environment. these regions are, for each subunit: gsgip, grewgp and gifap, where residues ser ile phe tyr and glu are annotated as chloride binding sites. the a- - - -a-p-l- -g motif is not part of the selectivity filter and no functional annotation for this motif is available. nevertheless, this motif, found in reentrant regions at the interaction interface of the two clc channel subunits forming the functional dimer, is highly conserved among bacterial voltage gated chloride channel sequences. this evidence suggests that the motif must have a role in protein structural stability or dimerization and sheds light on novel functional aspects of voltage gated chloride channels. the large, stable interface between the subunits is expected because clc cl- channels exist and function only as dimers. due to the electrical dipoles formed by the reentrant regions, the motif could contribute to provide a barrierless and energetically favourable environment for negatively charged particles present in the channel pore. in fact, it has been shown that, although the pore residues dominate the interaction with cl-, other portions of the protein still contribute a significant fraction of the attractive interaction with cl-. detailed electrostatic calculations would be needed to assess if the reentrant regions at the dimer interface could contribute in a favourable way to the electric field around the cl- ions. the motif suggests that some residues are important for the protein function and could be tested experimentally for example by site-directed mutagenesis experiments and by checking if dimerization still takes place. the motif has been derived from the backbone torsion angle clustering of fragments of size in the co-called reentrant region. it contains the conserved hydrogen bond pattern of regular α-helices between main chain atoms in relative positions and and and and the other residues in the fragment are not linked by hydrogen bonds between the backbone atoms, meaning that the regularity of the α-helix is broken to leave space for a small flexible loop characteristic of reentrant regions. the motif is highly specific to transmembrane proteins, with occurrences in the swiss-prot-tm dataset and 0% false positive rate. the motif is specific to bacterial voltage-gated chloride channels and enriched in go terms: voltage-gated chloride activity, antiporter activity, chloride transport, chloride ion binding. furthermore, from the scoppi database it has been found that some residues of this motif are positioned at the interaction interface between the clc cl- channel subunits in the functional dimer. statistics and basic structural/functional annotation for this motif are shown in fig. 2a. -x-y-p-p-l: a membrane-water interface motif in heme-copper oxidases the -x-y-p-p-l motif corresponds to a structured loop in the interface region, close to the extracellular side of heme-copper oxidases. the heme-copper oxidases catalyse the reduction of molecular oxygen to water. the chemical energy released in the reduction reaction is utilized to transfer protons across the membrane and to generate an electrochemical proton gradient. they all have a low-spin heme, which is the initial electron acceptor, and a high-spin heme, which forms the catalytic site with a copper centre. the histidine residues ligating the cofactors are fully conserved in the superfamily of heme-copper oxidases. the heme-copper oxidase profiles are widely documented in the prosite database. also the his residues that ligate the heme groups and the copper ion are annotated in the swiss-prot feature field. the pattern we find is not documented in any protein motif databases, but it is worthwhile to investigate its function as it contains a tryptophane residue which has been widely documented in literature. it has been shown that trp which is hydrogen bonded to Δ-propionate of heme a in the catalytic centre, is highly conserved. mutation studies of this residue suggest that it is involved in regulating proton transfer from the pumping site near heme a to the p-side of the membrane. we infer that the -x-y-p-p-l motif plays a special structural role in oxidases, creating the optimal environment to allow trp perform its regulatory role in the proton transfer process. furthermore, the motif suggests that tyr plays the same role in cytochrome ba as trp in cytochrome c. this evidence could guide future experiments in order to explore the detailed functional role of tyr as, to our knowledge, no mutational studies of tyr in cytochrome ba oxidases have yet been carried out. the motif has been derived from classes of fragments of size and clustered by backbone torsion angles. the structural motif associated to the sequence pattern contains the highly conserved residues tyr pro pro and leu which seem to form a highly structured cytoplasmic loop with the function of placing trp at the right distance from and orientation with respect to heme a our protein-ligand analysis also reveals trp to be in close contact with the heme a group. the -x-y-p-p-l motif has been found to be highly specific for transmembrane proteins, with occurrences in the swiss-prot-tm dataset and enriched in the following go terms: oxidoreductase activity, heme binding, mitochondrial electron transport chain, iron ion binding, aerobic respiration, copper ion binding, electron transport, mitochondrion, metal ion binding, cytochrome-c oxidase activity. statistics and basic structural/functional annotation for this motif are shown in fig. 2b. l-x-s-i-: a convergently evolved interface helix motif the l-x-s-i- motif corresponds to a very short irregular helix almost parallel to the membrane plane found across more than protein families and derived from the structural clustering of fragments of length in the reentrant region. this motif was found in proteins with different functions and it is a case of convergent evolution, i.e. proteins with different sequence and structure that share a common functional feature/mechanism. the structures from which the motif is derived are: aspartate symporter, serine protease and cytochrome b. the aspartate symporter is a sodium-driven secondary transporter that catalyzes the uptake of aspartate from chemical synapses. it exists in the membrane as a trimer and each subunit has eight transmembrane segments, two reentrant helical hairpins and independent substrate translocation pathways. it is thought that the hp reentrant region, where the l-x-s-i- motif is contained, acts as a gate, adopting a open conformation and allowing the aspartate to reach the binding site from the extracellular solution. the short parallel helix containing the l-x-s-i- motif is also involved in the formation of one of the two sodium binding sites, as the transport of aspartate is highly coupled to sodium transport. the serine protease, belonging to the rhomboid proteases family, is a protein whose function is to cleave the transmembrane domains of other proteins. the crystal structure reveals six transmembrane segments and other two interesting features: an internal aqueous cavity that opens to the extracellular side and a long membrane-embedded loop between the first and the second helices. the opening of this loop, which also contains the irregular helix corresponding to the l-x-s-i- motif, is thought to be the likely route by which the substrate enters the active site. so, it has been postulated that this loop, and in particular the segment corresponding to the motif, functions as a gate and may change conformation when the substrate binds. finally, there is no evidence in literature that the same motif in cytochrome b is associated to a gating function. but it has been found, from ligand analysis, that the structural segment corresponding to the l-x-s-i- motif is part of the q binding site and involved in non-covalent interactions with the substrate. this suggests that the motif is involved in conformational changes upon substrate binding, but this assumption needs experimental validation. it can be concluded that these three proteins, unrelated in structure and biochemical function, share a convergently functional motif that, although not directly part of the core catalytic activity of the protein, modulates gate dynamics at the membrane-protein interface. statistics and basic structural/functional annotation for this motif are shown in fig. 2c. general results from the structure fragment clustering consider fig. which describes the procedure of structural fragment clustering. it consists of six steps. in step non-redundant sequences and transmembrane protein structures with resolution less than Å are collected. in step the protein structures are fragmented and fragments are labeled according to their location and topology. in step the fragments are clustered based on their hydrogen bonding patterns and on torsion angles, respectively. in step sequence motifs are derived from significant clusters of fragments and in step they are annotated regarding functional and structural features. in the final step all motifs are filtered regarding their significance and novelty. structural fragment clustering in step non-redundant sequences and but unspecific backbone torsion angle profiles. motifs can also lack a specific hydrogen bond pattern but be detectable by means of their Φ and Ψ angle values. a similarity measure based on common hydrogen bond profiles can reveal very stable structural motifs, as hydrogen bonds in membrane proteins are much stronger than in globular proteins. this is due to the low dielectric constant of the membrane and lack of competing interactions with water molecules. on the other hand, it has been shown that a similarity measure based on differences in backbone torsion angle profiles is very sensitive to variations in local protein conformations and that active site torsion angles are usually highly conserved. in step sequence motifs were derived for each structural class, if possible. if no significant motif could be associated with a cluster, a further sequence-based clustering step was performed to filter significant sequence patterns. then, a library of regular expressions associated with specific structural features was compiled. in step functional annotation of fragments was performed using multiple sources of information. fragment clusters were annotated with shared go categories. each fragment in a cluster was associated to a swiss-prot feature ft field annotation, when this annotation existed. fragments that belong to protein-protein interfaces or are part of ligand/substrate binding site were annotated by mapping them onto the scoppi and the pdb database for possible binding sites. furthermore, residues in fragments that have been experimentally mutated and have a function reported in the literature were also annotated. finally, for each cluster-derived sequence pattern, its total or partial overlap with a prosite pattern is checked and reported. in step the final step, the motifs were filtered. initially, there were motifs. first, we filtered by significance and specificity to membrane proteins, resulting in motifs. next, we filtered out motifs that are already documented in general motif databases, resulting in novel motifs. finally, we grouped overlapping motifs and thus remove redundancy, leaving motifs. these motifs are described in detail regarding structural and functional features in additional file statistics of clusters and filtering the total number of clustered fragments was for the hydrogen bond-based clustering and for the backbone angle-based one. in table the statistics for both clusterings are summarized: number of fragments, structural clusters, outliers and size of the largest cluster for each region and fragment length. plots of these numbers versus fragment length are presented and discussed in additional file the number of clusters and outliers is low in the helix core region with respect to the cytoplasmic, extracellular and interface regions. this is due to the low structural diversity of the helix core region, where 80% of the fragments fall into the regular α-helix structural class or irregular helices. in the cytoplasmic and extracellular regions, the number of classes increases and their distribution drastically changes since there is more structural variability compared to the helix core region. length percentage of structural classes covered by sequence patterns. the first column shows the number of classes obtained for the hydrogen bond-based clustering before and after the sequence-clustering step. the second column shows the percentage of clusters for which a sequence pattern could be derived by using the pratt program before and after the sequence-clustering step. the third column shows the percentage of classes that could be assigned to a statistically significant motif before and after the sequence-clustering step. colums four, five and six show the same numbers for the backbone torsion angle-based clustering. in total, non-redundant sequence motifs have been derived from both clusterings. from this number, the statistically insignificant motifs and those motifs that are not specific to transmembrane proteins were filtered out. a motif is considered statistically significant when its associated p-value, derived by randomly permutating the swiss-prot tm database, is smaller than. furthermore, a motif is considered specific to transmembrane proteins if its false positive rate is low enough, i.e if the p-value of the hyper-geometric distribution is smaller than × 10-. fig. 6a shows the distribution of motifs of different lengths. the histogram shows that the strongest correlation between structural clusters and sequence preferences is obtained for motifs of length of to amino acids. the number of sequence patterns associated to structural classes strongly decreases for fragment length greater than amino acids. fig. 6b shows the percentage of motifs associated with different false positive rates for three different fragment lengths. an anti-correlation has been observed between false positive rate and motif length: the longer the sequence motif, the lower the false positive rate. as shown in fig. 6a, for length 100% of the motifs have false positive rate less than 20%; motifs of length are very unspecific as most of them have false positive rate greater than 60%. after the filtering step, significant motifs were considered for further analysis. the average resolution of the structural motifs after filtering is Å. this means that the clustering process automatically filtered out lower resolution fragments as outliers. 85% of the derived motifs were common to both hydrogen bond- and torsion angle-based clusters, especially in the helix core region. this observation is a further proof of the reliability of the retrieved structural motifs. in order to evaluate the ability of our structure-based method in finding novel motifs, which cannot be identified by sequence-based methods alone, an all-against-all comparison of our motifs against the prosite database was carried out. the algorithm used for comparison with prosite patterns is described in detail in materials and methods. it is necessary to stress that a direct comparison of our motif library with the prosite database is not straightforward for three main reasons. first, as prosite patterns are derived at sequence level, from conserved regions in multiple alignments of homologous sequences, they are usually longer than ours. second, prosite patterns are derived by scanning all the sequences in the swiss-prot database, unlike our library that is derived only from transmembrane proteins of known structure. for this reason the comparison is limited to those prosite patterns that hit anywhere in swiss-prot transmembrane proteins. third, it has to be taken into account that the number of known structures for membrane proteins is considerably smaller than the number of known sequences. this implies that a high coverage value of our motif library against prosite cannot be expected. on the other hand, it is interesting to quantify and investigate the number of found linear motifs that do not have any match in prosite, as they provide a proof that structural information adds new knowledge about unannotated sequences and functional implication. by analyzing the results from the comparison, it has been found that by varying the value of the cut-off for defining a match between two patterns, the number of matched/unmatched motifs strongly varies. the cut-off for the similarity score was set to. the choice of the cut-off is based on a comparison to a random model. by setting the cut-off for the similarity score to, the percentage of significant novel motifs is about 10%, motifs out of non-redundant, statistically significant motifs. although a comparison with prosite is not straightforward, it is clear that the novel motifs represent new knowledge, which cannot be gained from sequence alone. after grouping overlapping motifs and removing redundancy, motifs were checked in the scientific literature for the first biological assessment and described in detail regarding structural and functional features, in additional file comparison with meme in order to evaluate the capability of our structure-based method to survey motifs that cannot be found by sequence-based pattern searching tools alone, we compared our motifs with the motifs obtained by using the meme tool, a software package to discover motifs in groups of related dna or protein sequences. we derived motifs with meme on the same dataset of protein chain sequences used for generating our motifs. in total, motifs were generated using meme, with the following options: motifs lenght ranging from to amino acids; motifs generated from a minimum of sequences; e-value less that. in oder to compare our motifs with meme-generated motifs, we computeted the overlap between couples of motifs by counting the number of common transmembrane proteins in swiss-prot where the two motifs hit. if the overlap was higher than 80% the motifs were considered as the same motif. we find that less than 20% of our motifs could be found by meme. in particular, the three motifs corresponding to the three examples discussed in the previous sub-section could not be detected from the meme tool. out of the motifs described in additional file could also be found by meme and they all correspond to family-specific motifs. motifs out of could only be found by means of our structure fragment clustering approach and represent new knowledge that cannot be derived by means of sequence-based pattern searching tools alone. since a key finding of this paper is that motifs exist as modular building blocks across unrelated families, it is clear that purely sequence-based approached are not adequate due to the divergence of the different families. family vs non-family specific motifs the sequence homology of the dataset for deriving the motifs is reduced at 90% identity. this is a quite permissive cut-off and the risk of deriving only homology-based patterns, representative of a limited sampling, is high. to verify that not all the patterns we derive are obvious signals of homologous proteins, but that many of them are functionally or structurally of great importance, an analysis at family level was carried out by using the pfam database. each motif can be assigned to a pfam family or to a set of pfam families by assigning a pfam family to the swiss-prot-tm sequences the motif matches. surprisingly, we found that only 6% of the statistically significant motifs are family-specific. the rest of the motifs are found across different pfam families or clans. novel family-specific motifs, not represented in the prosite database, are interesting because they can shed light on novel and different aspects of a protein's structure and function. motifs across families can be important for structural stability, e.g. transmembrane helix kinks, motifs at the membrane-water interface, protein-lipid interaction motifs, helix-helix packing motifs, or they can be cases of convergent evolution, i.e. found in proteins that are not homology-related but share some functional mechanisms. the a- - - -a-p-l- -g and -x-y-p-p-l motifs discussed in the previous sections are examples of motifs specific to the voltage-gated chloride channel family and the heme copper oxidase family, respectively. the l-x-s-i- parallel helix motif, also discussed in the previous section, is an example of motif found across different protein families and it is a potential convergent evolution motif. other important motifs across families, such as helix kinks, helix distortions, interface helices, helix-helix packing and protein-lipid interaction motifs are described in additional file motifs help to identify membrane proteins in novel genomes to demonstrate the capability of our motifs to identify new transmembrane proteins, we performed the following analysis: we determined the distribution of the number of motif hits in swiss-prot sequences for transmembrane proteins against globular proteins. the two distributions are shown in fig. this analysis was performed for high-confidence motifs, i.e. motifs with false positive rate less than 40% as defined in additional file fig. shows that all globular proteins contain five or less motif hits, in contrast to transmembrane proteins which can contain up to motif hits. this allows to define a simple rule: if a new sequence contains more than of the motifs, then it is predicted to be a transmembrane protein. applied to swiss-prot this rule does not make any false predictions. however, since the motifs are based on structures, the coverage is not high and thus there will be still many membrane proteins among the sequences for which no prediction is made. in this respect, our method can help identifying transmembrane proteins in novel genomes. coverage of motifs in different genomes the coverage of the non-redundant motifs in different genomes has been estimated and the results presented in table table shows the percentage of transmembrane proteins in the swiss-prot database, where at least one motif hits, for the top ten genomes, i.e. the genomes with the highest number of motifs hits, ordered by the decreasing number of the motif hits. in average, the coverage of the portion of genome encoding for membrane proteins is 9%. transmembrane proteins were found to be associated with at least one of the motifs in the human genome. percentage of transmembrane proteins, containing at least one of the non-redundnat, fully characterized sequence motifs, for each species. only the top mostly represented species, oredered by the number of motif hits in the dataset of transmembrane proteins are reported. the first colomn specifies the protein species, the second colomn specifies, for each species, the number of motif hits in swiss-prot transmembrane sequences, the third colomn specifies, for each species, the number of corresponding transmembrane protiens in swiss-prot and the last colomn shows, for each species, the percentage of transmembrane protein that contains at least one motif. more generally, we estimated also the coverage of the motifs in three protein kingdoms. it was found that 15% of eukaryotic proteins were covered by the motifs, against 9% in bacteria and archaea.  in this work we introduced structural fragment clustering and derived novel sequence motifs in membrane proteins together with their functional characterization the novel motifs appear across many families and therefore show that they form functional modules, which are re-used. the majority of the motifs is found on binding sites to membrane, ligands, co-factors, other proteins or other helices, highlighting their functional role and the importance of the environment for these structural building blocks. we discuss three novel motifs in detail. two of them, a re-entrant region in voltage-gated chloride channels and a structured loop in the membrane-water interface region of heme-copper oxidases, are family-specific and help add precious details to the functional mechanisms of the proteins they are found in. the third motif, an interface helix derived from the clustering of fragments in three different protein families, is an interesting case of convergent evolution, where three evolutionarily unrelated families with different functions share a common gating mechanism. the three motifs discussed here have been chosen among novel statistically significant and non-redundant motifs derived by means of an unsupervised learning method also described here. the method uses structural information about protein fragments, like conserved hydrogen bond patterns and backbone torsion angle profiles, to derive short linear motifs in α-helical transmembrane proteins. although the data set contains low-resolution structures with a resolution worse than Å, 98% of the clusters contain at least one high-resolution fragment from a structure of less than Å resolution. this garantees reliability of the results. furthermore, the distribution of the average resolution value for all clusters has a peak around Å. due to intrinsic difficulties in experimental determination of membrane protein structures, their average resolution in the pdb is worse than for globular proteins. removing structures of resolution worse than Å and filtering for redundancy would reduce our dataset from to only structures. this would make it nearly impossible to perform clustering and to obtain statistically significant results. the method, even though it is based on the information retrieved from the limited set of membrane proteins with known 3d structures, is able to find novel functionally or structurally important motifs that can complement and enrich information retrieved from sequence-based methods like prosite or meme. while new protein domains or family signatures, such as those contained in pfam or prosite, can be defined from alignments of evolutionarily related sequences, the identification of short sequence motifs, related to specific structural features and shared between transmembrane protein functional classes, is much harder. to address this problem different sources of information are taken into account to elucidate the role played by short structural/sequential motifs in different transmembrane proteins classes. these are: structural properties, location with respect to the membrane planes, go annotations, swiss-prot functional annotations, interaction interface information, and mutational analysis. it is shown in the three examples that the method is able to predict new functional residues. in future studies, the method will be applied to better characterize other important sequential/structural motifs in transmembrane proteins, like interfacial helices or patterns at helix-helix interaction interfaces. our method, in contrast to the approach described in, not only enables the discovery of 3d motifs associated with function, but makes use of regular expressions to allow searching for functional motifs in transmembrane proteins of unknown structure. cluster signatures are an attractive way to annotate protein function at both the structure and the sequence levels. indeed, it has been shown that protein annotation effort benefits immensely from the knowledge of functional signatures in both primary, secondary and tertiary structure. in fact, sometimes the multifunctionality and overall structural diversity of even closely related proteins confounds efforts to assign function on the basis of overall sequence or structural similarity. approaches based on the identification of common small functional motifs can help to overcome this problem. this is especially true for membrane proteins, where protein with the same topology, fold or signal at sequence level in the hydrophobic core, perform totally different functions, thanks to hotspot residues and small motifs that differentiate them, or share common functional mechanisms. in addition, functional motifs pinpoint individual residues that play a crucial functional role and complement the information contained in alignments of homologous proteins, such as the ones contained in pfam, which focus on large functional signatures, at family level, but not on individual residues or small motifs, related to specific structural features. our method generates structural motifs and associate them directly to function by mapping onto the protein fragments functional characteristics, such as swiss-prot'features', protein-protein interface and protein-ligand binding sites associated residues and go annotation. furthermore, fragments belonging to functional sites or containing hot-spot residues are ideal candidates for experimental validation. possible experiments that can be done to validate the structural and functional role of a motif involve different experimental techniques. first, force spectroscopy, which allows to measure the force necessary to pull proteins out of the native membrane, can be used to validate structural motifs important for protein stability. second, confocal microscopy can validate motifs relating to the protein's topology, such as interface helices, by labeling membrane and motif with different dyes and by determining the location of the motif seuence relative to the membrane. third, mutants from membrane proteins can be tested by means of functional assays. for example, for the dimerization motif in chloride channels, dimerization upon mutation of one of the conserved residues in the motif can be tested by means of imaging through atomic force microscopy. if dimerization still takes place, it can be tested whether the dimer is a functional dimer, e.g. by measuring the concentration of cl- and h+ exchanged. to conclude, it is worthwhile to emphasize the power of the method and the results presented here in two other application fields. first, motifs from our library can help identifying transmembrane proteins in novel genomes, as discussed in results and discussion. second, structural motifs, such as reentrant regions, helix kinks and helix-helix contact motifs or functional motifs, such as the ones related to the protein binding sites or protein-lipid interactions, can be used as constraints while building more refined two-dimensional models of α-helical transmembrane proteins from sequence alone. it has been shown that both two-dimensional tools and three-dimensional prediction algorithms benefit from the use of structure-sequence motifs as constraints. 
we describe three novel motifs in detail: a dimer interface motif found in voltage-gated chloride channels, a proton transfer motif found in heme-copper oxidases, and a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. from these motifs, of them were assigned to function and checked in the scientific literature for a biological assessment. the sequence motifs can be applied to identify and characterize membrane proteins in novel genomes. a large proportion of an organism's genome encodes for membrane proteins. membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. with the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. from over fragments, we obtain statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. the vast majority of motifs appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. we introduce a technique called structural fragment clustering, which learns sequential motifs from 3d structural fragments.
 a large proportion of an organism's genome encodes for membrane proteins. membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. with the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology.  we introduce a technique called structural fragment clustering, which learns sequential motifs from 3d structural fragments. from over fragments, we obtain statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. from these motifs, of them were assigned to function and checked in the scientific literature for a biological assessment. seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. the vast majority of motifs appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. we describe three novel motifs in detail: a dimer interface motif found in voltage-gated chloride channels, a proton transfer motif found in heme-copper oxidases, and a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b.  our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. the sequence motifs can be applied to identify and characterize membrane proteins in novel genomes. 