BACKGROUND
the innate immune system provides a first level of defense against invading agents such as microbial pathogens and toxins and plays a role in maintaining a stable, healthy composition of the commensal microbiome.
in mammals, the important role of continuously surveying the environment is played by members of two protein families: extracellular toll-like receptors  and intracellular nod-like receptors , also referred to as nucleotide-binding domain- and leucine-rich repeat-containing proteins.
while individual receptors from both families have distinct specific roles, the overall function of the innate immune system requires their complex, synergistic interaction.
both tlr and nlr families belong to the oldest branch of the immune system, the innate immunity, and have complex evolutionary history that started in the simplest multicellular organisms and went through significant expansions and contractions in various lineages leading to present day vertebrates and mammals  <cit> .
for instance, tlr and nlr families in some invertebrates, such as amphioxus, have over  <dig> members.
in early branching vertebrates, such as sea lamprey and hagfish, a closely related family of variable lymphocyte receptors  provide a version of adaptive immunity, which was lost in higher vertebrates  <cit> .
in humans, the tlr family consists of ten proteins, which have been a subject of intense experimental studies during the past decade due to their critical role in immunity and involvement in diseases such as crohn's disease  <cit> , cystic fibrosis lung disease  <cit> , inflammatory bowel disease  <cit> , familial mediterranean fever  <cit> , septic joint disease  <cit> , and several others.
tlrs sense the presence of pathogen-associated molecular patterns  by specific binding sites in their extracellular "horseshoe"-shaped receptor domains consisting of multiple leucine-rich repeat  motifs.
binding-induced conformational change and/or dimerization provides a signal to the intracellular toll/interleukin receptor  domain, which in turn binds specific adaptor molecules, such as myd <dig>  leading eventually to nf-κb transcription factor activation and transcription of proinflammatory cytokine genes  <cit> .
from numerous experimental studies it is known that the lrr domains of different tlrs specifically interact with a rather diverse plethora of ligands, from bacterial flagellin and ss/dsrna to peptidoglycans to imidazoquinioline compounds.
by integrating these signals, the tlr receptors sense the microbial state of the immediate environment and define the appropriate level of innate immunity activity.
the nlr family receptors provide complementary, but less-studied and -understood, mechanisms of intracellular pamp detection and innate immunity regulation.
twenty-two genes coding for nlr proteins are found in the human genome, typically consisting of a c-terminal lrr domain , the nucleotide-binding oligomerization  domain, and the n-terminal effector domain.
several different effector domains can be found in human nlrs, including the caspase recruitment  domain ; the pyrin/paad  domain ; the baculovirus "inhibitor of apoptosis"  domain ; and an uncharacterized domain , thereby partitioning nlrs into at least four subgroups.
the variety of effector domains in other organisms, specifically invertebrates, is much larger  <cit> .
following binding of a ligand to the lrr domain, nalp <dig>  nalp <dig>  and ipaf are known to form multimer complexes termed "inflammasomes."
inflammasome formation leads to a further sequence of molecular events: activation of caspases and conversion of pro-interleukins into their active form.
recently, inflammasomes were shown  <cit>  to be structurally similar to apoptosomes  <cit> , highlighting the evolutionarily conserved mechanism of activated oligomerization, carried our by nucleotide binding aaa  <cit>  proteins belonging to two distinct families: the nacht family  and nbarc family .
after almost a decade since their discovery, information on ligand specificities of the nlr family proteins remains elusive.
nod <dig> and nod <dig> are known to sense bacterial cell wall-derived peptidoglycans γ-d-glutamyl-meso-diaminopimelic acid   <cit>  and muramyl dipeptide   <cit> , respectively.
the nalp <dig> inflammasome was shown to be activated by mdp in a two-step manner  <cit> .
the nalp <dig> inflammasome is activated by a wide range of ligands: bacterial rna, uric crystals, antiviral imidazoquinioline compounds r <dig> and r <dig>  and contact sensitizers  such as trinitro-chlorobenzene.
nalp <dig> mutations are known to correlate with a number of autoinflammatory disorders, e.g., muckle-wells syndrome  <cit> , and mutations in nod <dig> gene are linked to increased susceptibility to the chronic inflammatory disorders such as crohn's disease , psoriatic arthritis, and blau syndrome  <cit> .
ipaf is known to sense bacterial flagellin  <cit> , although whether flagellin is the direct and the only ipaf ligand is under debate  <cit> .
finally, ciita that serves as a master regulator of major histocompatibility complex genes transcription is known to bind many dna-binding and co-activator proteins.
its lrr domain was found to be involved in ciita self-association  <cit>  as well as in binding a novel zinc-finger protein zxdc  <cit> .
the specific roles of other nlrs in innate immunity  are at this point unknown.
at the same time, there is no direct structure information on lrr domains of any of the nlrs.
given the importance of understanding nlrs' function and the lack of information on their structure and agonists, we believed a theoretical study might provide some useful insights and help design experimental efforts.
the main subjects of this work, the ligand-binding domains of nlrs and tlrs, both consist of multiple leucine-rich repeat  motifs.
lrrs are extensively used by nature as building blocks for assembling scaffolds of protein interfaces, designed primarily for specific protein-protein interactions.
about  <dig> lrr-containing proteins with diverse  functions have been identified in the human genome.
due to the importance of lrr proteins, they have been a subject of intensive experimental work, with the first crystal structure of a ribonuclease inhibitor  <cit>  providing a major advance in the field.
these advances continue today, with over  <dig> structures of lrr-containing proteins, including three tlr structures, solved through  <dig>  much has been learned about structural principles of lrr proteins organization from theoretical and modeling perspectives  <cit> .
lastly, it is interesting to note that while lrr domains of tlr and nlr proteins are homologous, they belong to distinct sub-branches of the lrr family, and both have close homologs that are not innate immunity receptors.
for instance, the families of lrr- and immunoglobulin-containing proteins, involved in cell-cell adhesion, are closer related to tlrs than nlrs are, and on the other hand, ribonuclease inhibitor, a protein not involved in innate immunity, is closely related to nlrs but is much more distant from tlrs.
these observations suggest that nlr, tlr, and vlr families have a very complex evolutionary history with multiple duplication, domain swapping, and domain recruitment events.
while it is generally believed that lrr domains of most nlrs and tlrs are involved in direct binding of agonist molecules, for nlrs there is little experimental proof of this.
some evidence of direct agonist binding by nalp <dig> lrrs is given in  <cit> .
when referring to "lrr ligands" in this manuscript, we recognize that binding to lrr domains is an assumption at this point, and that "ligands" can be either the "danger signals" themselves, or the possible intermediate molecules  <cit> .
since the overall shape of lrr domains from different proteins is relatively conserved, the functional similarities and differences between them must be defined by specific features of their surfaces.
this would be true regardless of whether lrr domains interact with immunity "danger signals" directly or by means of intermediate molecules , or the functionality is conferred by lrr interactions with other  domains of nlrs.
the key point is that if lrr surface properties within a given nlr/tlr pair are similar, they are likely to bind the broadly similar type of molecules.
in this manuscript, we investigate surface features of the twenty-two human nlrs, and by comparing them to those of tlrs, attempt to predict likely similarities between their functions.
first, we have built homology models for lrr domains of twenty nlrs.
second, we have investigated distributions of amino acid charge and hydrophobicity across lrr domain surfaces and by means of clustering analysis have quantitatively assessed their similarities.
additionally, we have analyzed distributions of consensus n-linked glycosylation sites within tlrs, in order to support application of our approach to  nlrs.
while the simplicity of our analyses requires care when interpreting the results, we believe they may be helpful in designing future experiments to shed light on the role of nlr-coding genes in human innate immunity.
methods
phylogenetic analyses
multiple alignments and phylogenetic trees  were built using clustalx <dig>  <cit> .
the most divergent sequence, naip, was used as an outgroup to root the tree.
the tree reliability assessment was conducted by means of the bootstrap method using  <dig>  tree samples.
several other multiple alignment and tree building strategies produced essentially identical results .
protein sequences and gene organization information were obtained from the ensembl database  <cit> .
homology modeling
in order to visualize structures and surface features of lrr domains from human nlrs , homology models were built based on pairwise alignments of nlr amino acid sequences with the sequence of porcine ribonuclease inhibitor , whose x-ray structure  was used as a template.
the two proteins for which the models were not built are nod <dig> and nalp <dig>  nod <dig> lrr domain consists of  <dig> lrrs, and all lrr-containing proteins with available structures are too small to be used as a template.
the second protein, nalp <dig>  lacks an lrr domain.
amino-acid sequence alignments were manually refined using the swiss-pdb viewer  <cit>  to account for additional information obtained from the gene structure and from structural comparisons between lrr proteins .
because of the high sequence similarity between the template and the prediction targets , homology modeling was performed by means of the swiss-model server  <cit> .
electrostatics and hydrophobicity calculations were carried out for nlr homology models as well as for experimental crystal structures of two tlrs.
they were conducted utilizing moe software .
lrr sequence-to-structure mapping
in order to conduct a quantitative comparative analysis of lrr domain surface features in the absence of experimental structure data, we mapped physicochemical features of residues on the surface using existing knowledge about lrr sequence-to structure-mapping  <cit> .
the typical lrr sequence contains a conserved motif lxxlxlxxnxl, where l is leucine, isoleucine, valine, or phenylalanine, and n is asparagine, threonine, serine, or cysteine.
in nlrs there is a striking correlation between their gene organization and amino acid sequence.
specifically, in nalps, two consecutive lrr motifs xlaanalaaaaaaaaaaaaaaaaalaalxlaanalaaaaaaaaaaaaaaaaaaiaal of length  <dig> +  <dig> =  <dig> residues are encoded by a single exon  <cit> , while we have found that in nods each lrr motif xlaanalaaaaaaaaaaaaaaaaalaal of length  <dig> residues is encoded by a single exon.
this unusual genomic structure has interesting structural consequences, which would be a subject of a separate publication  <cit> .
these two facts have allowed us to make reliable predictions for positions and boundaries of lrr motifs in human nlr sequences.
in tlrs such an unusual structure-exon correlation is lacking, and the whole lrr domain is typically encoded by one large exon.
finally, from the available structures of other lrr-containing proteins it is known that the aalxlaa pattern corresponds to a beta-strand in the inner concave surface, which is then followed by a c-terminal side loop, by an alpha-helix on the outside convex surface, and by the n-terminal side loop.
this property is very well conserved in numerous lrr-containing proteins, and we have utilized it to improve mapping between lrr sequence and structure.
the residue x in the aalxlaa motif was used as reference residue to define positions of all other residues on the lrr surface .
the next  <dig> residues are located on the c-terminal side , the next  <dig> residues are located in the outer convex part , and finally the next  <dig> residues represent the n-terminal side .
in tlrs, the typical length of the outer part is  <dig> residues, but there is much variation.
after we performed the sequence-to-structure mapping as described above, we were able to partition the surface into four parts and analyze distributions of amino acid properties such as charge and hydrophobicity within these parts separately.
distribution of potential glycosylation sites was also examined for extracellular tlrs.
quantitative comparison of charge and hydropathy distributions
amino acid charge and hydrophobicity distributions within lrr domains were calculated for each of the four surfaces shown in figure 3b.
the amino acid charge was calculated assuming the ph of  <dig> , and the hydrophobicity was quantified by hydropathy index ranging from - <dig>  to + <dig>   <cit>  with negative  values corresponding to hydrophilic  properties.
within a given protein, for each lrr motif i, we calculated average charge  and hydropathy  separately for each of the four surface regions.
for an lrr domain with n motifs, this produced  <dig> n values for charge and the same for hydropathy.
next, in order to characterize the distribution of these values within each surface, we have calculated the first four moments, mk,  of qi and hi distributions:
where fi is either qi or hi, and
with these definitions, the moment m <dig> is simply the average value, and the moment m <dig> is related to the square of the standard deviation.
the moments m <dig> and m <dig> reflect additional details of the distributions, in particular the directional trends .
the distributions of charge and hydropathy within one protein are thus each characterized by a sequence of  <dig> surface × moments =  <dig> values.
in order to quantify similarities of distributions within different proteins, we used spearman's rank-order correlation coefficient, rs .
the similarity between distributions was then defined as .
clustering analysis was then performed by a complete linkage method utilizing java treeview software  <cit> .
glycosylation distribution analysis
consensus n-linked glycosylation sites were predicted by searching for patterns nxxs and nxxt in tlr sequences.
in globular proteins, such predictions greatly overestimate the number of glycosylation sites, assuming that all sites matching the pattern above are accessible to oligosaccharyltransferases in order for glycosylation to occur, while in reality most of these sites are buried inside protein structure.
in our case, however, such predictions can be much more accurate because we could predict which of the potential glycosylation sites were exposed to solvent.
RESULTS
phylogenetic analysis of nlr and tlr leucine-rich repeat domain sequences
as a first step, we have performed a phylogenetic analysis of lrr domains in nlrs and tlrs.
extreme sequence length diversity among lrrs from these families makes conducting such an analysis in a meaningful way nontrivial.
this is further complicated by the fact that full length multiple alignment of complete sequences contains numerous large gap regions, which makes its truncation difficult.
to circumvent the latter problem, we built a multiple alignment using a 10-fold increased gap extension penalty and truncated it from n- and c-terminal ends to make its length similar to that of the shortest sequence .
the resulting alignment was then used to build a rooted phylogenetic tree .
among the nlr and tlr sequences, we also included a sequence of human ribonuclease inhibitor  consisting of a highly-regular lrr domain closely homologous to that of lrrs in the nrl family.
the sequences group into three distinct clusters:  nalps and ri;  card-containing proteins and nlrx1; and  tlrs.
surprisingly, the sequence similarity-based phylogeny of the c-terminal lrr domain well agrees with the classification of all these proteins based on the n-terminal effector domain.
within nalps, there are three distinct subgroups:  nalp <dig>  nalp <dig>  nalp <dig>  nalp <dig>  nalp14;  nalp <dig>  nalp <dig>  nalp <dig>  nalp13;  nalp <dig>  nalp <dig>  and ri.
it is interesting to note that ipaf and ciita, as well as nlrx <dig> whose effector domain lacks definitive classification, group together with card-containing nods.
this suggests that nlrx <dig> effector domain might be card-like.
this analysis supports known phylogenetic relationships between nlr and tlr families, but it does not permit us to draw any conclusions regarding similarities of surface features between tlrs with known ligands and nlrs.
homology models of lrr domains and sequence-to-structure mapping
in figure  <dig> we present homology models for lrr domains of twenty nlrs  along with experimentally determined structures of two tlrs.
the first observation is that there is much diversity in the lrr domain size - from  <dig> lrrs  to  <dig> lrrs .
an unusually large number of lrrs  is found in nod <dig>  whose model was not built because there is no large enough template structure available .
second, coloring of the models according to surface electrostatic potential and hydrophobicity reveals significant diversity of their surface features.
the models in figure  <dig> are helpful in visualizing lrr domain structures and could be used for explicit protein-ligand docking; however they are not suitable for quantitative assessment of surface similarity.
in order to quantitatively analyze the similarity of surface features of lrr domains, we resorted to their reduced-resolution representation.
first, we adopted a simple approach to implicitly infer lrr domain three-dimensional structures from their amino acid sequences .
second, we have partitioned the total surface of the lrr domain of each protein into four regions : concave inner surface , n-terminal side, c-terminal side, and convex outer surface .
before conducting a quantitative comparison of nlr and tlr surface features we had to consider possible perturbations to tlr surface properties due to glycosylation.
role of glycosylation of lrr domain surfaces
it is well known that lrr domains in all ten human tlrs are n-glycosylated due to their extracellular localization, and in some of them glycosylation is essential for proper function  <cit> .
this is not surprising as glycosylation can dramatically alter surface properties and thereby affect ligand binding.
since nlrs are all known to be cytosolic proteins their n-linked glycosylation is unlikely, which complicates a direct comparison of their surface features to those of tlrs.
to understand the potential role of tlr glycosylation in their ligand binding, we have performed a search for consensus n-linked glycosylation sites within tlr sequences, and analyzed their distribution over the domain surfaces.
the results are summarized in table  <dig>
the tlrs that contain many consensus glycosylation sites are tlr <dig>  tlr <dig>  tlr <dig>  and tlr <dig>  in tlr <dig>  all surfaces except the c-terminal side that is known to bind dsrna contain several glycosylation sites.
in tlr <dig>  inner and outer surfaces are likely to be glycosylated while side ones are not, which is consistent with binding of ssrna via one of its side surfaces.
glycosylation of tlr <dig> and tlr <dig> resembles that of tlr <dig> in that only the c-terminal side surface is glycan-free.
in the rest of the tlrs, each of the four surfaces contains no more then three potential glycosylation sites.
as an additional step, we have also analyzed nlr sequences for potential glycosylation , and found that they contain 0- <dig> consensus glycosylation sites per surface, with the only exception being an n-terminal surface of nod <dig>  which interestingly contains  <dig> such sites.
these findings indicate that while glycosylation of some tlrs certainly modulates their surface properties, in most cases the ligand binding sites are located in non-glycosylated regions and, in addition, the overall surface area affected by glycosylation is relatively small, so that global surface comparisons such as those performed here are not affected.
hydrophobicity and charge distributions: clustering analysis within full lrr surfaces
the two physico-chemical amino acid properties that critically affect ligand binding and whose distributions we have thus analyzed are charge and hydrophobicity.
figure  <dig> shows an example of our clustering analyses of hydropathy distribution moments within each of the four domain surfaces.
the similarity measure in this analysis is spearman's correlation .
based on clustering results, lrr domain pairs that have particularly similar hydropathy distributions and lrr numbers are: tlr <dig> and ipaf ; nod <dig>  and nalp <dig> ; ri  and nalp <dig> ; tlr <dig>  and naip ; nalp <dig>  and nalp <dig> ; tlr <dig>  and tlr <dig> ; tlr <dig>  and nalp <dig> .
hydrophobicity and charge distributions within individual lrr domain surfaces
in order to gain further insight into similarities of lrr surface features between nlrs and tlrs, we conducted a careful manual inspection of similarity patterns across individual biologically relevant surfaces, also taking into account results of our tlr glycosylation analysis .
we assessed similarities between lrr domains of all  <dig> proteins considered, as detailed below.
in nalp <dig>  which is known to sense mdp, the concave and side surfaces have both hydropathy and charge distributions that are the most similar to napl <dig>  which is known to bind a diverse set of ligands, including mdp.
nalp <dig> and nalp <dig> have similar hydropathy distributions, but dissimilar charge distributions.
nalp <dig> and nalp <dig> have hydropathy and charge distributions similar to nod <dig>  that binds ie-dap.
nalp <dig> has the shortest lrr domain, containing only  <dig> lrrs.
its surface properties are similar to those of tlr <dig>  which contains  <dig> lrrs.
because the hydropathy and charge distribution moments are normalized by the number of lrrs in a given domain, similarity of distributions still allows to hypothesize that nalp <dig> and tlr <dig> may bind the same type of ligands, rna motifs.
nalp <dig> charge and hydropathy are similar to nod <dig> and nalp <dig> both binding mdp, suggesting small molecules as probable ligands.
the high similarity of nalp <dig> hydropathy distribution and size to those of nod <dig> and also nod <dig>  suggests that they may share the same type of ligands--peptidoglycans.
this is also supported by similarities in their charge distributions, with the difference being a more positively charged convex surface of nalp <dig>   the hydropathy and charge of nalp <dig> are similar to ri, which binds rnase, thus suggesting that the nalp <dig> ligand may be protein-like.
nalp <dig> shows hydropathy similarity with tlr <dig> that binds ssrna, most likely by one of its side surfaces.
nalp <dig> charge distribution is similar to that of tlr <dig> and also tlr <dig>  over its side surfaces, suggesting that ss/dsrna is likely to be among possible nalp <dig> ligands.
nalp <dig> surface properties do not exhibit any obvious similarity to others.
the hydropathy distribution of nalp <dig> is similar to that of nalp <dig> and nod <dig>  but neither of them has known ligands.
nalp <dig> charge distribution on its concave surface is almost identical to that of ipaf, and distributions over other surfaces are similar to ipaf.
the hydropathy distributions of these two nlrs are similar within only its concave side.
assuming that ipaf binds flagellin similar to tlr <dig>  i.e., on its concave surface  <cit> , flagellin can be considered a likely ligand of nalp <dig>   nod <dig> hydropathy has some similarity to nalp <dig>  but charge distribution is unlike that in any other domain.
nod <dig> concave and side surfaces have hydrophobicity similar to nalp <dig>  but the charge distribution is different.
also, nod <dig> appears to have the largest ligand-binding domain of all nlrs and tlrs: it contains as many as  <dig> lrrs.
nlrx <dig> surface features do not exhibit similarity with any other domain.
naip hydropathy distribution is similar to ri, while naip charge distribution is similar to tlr <dig>
it is interesting to note that the functional similarities inferred based on comparison of molecular surfaces correlate well with experimental data where available, while hypotheses based on evolutionary relationships shown in figure  <dig> generally lack such correlation.
for example, while the surface comparison and the known experimental data suggest that nalp <dig> and nalp <dig> are activated by the same type of molecules, mdp, the phylogenetic reconstruction places nalp <dig> and nalp <dig> into two distinct subgroups in figure  <dig>  although both subgroups belong to the nalp group.
similarly, whereas ipaf and tlr <dig> are known from experiment to sense a common molecule  with which our surface analysis is in agreement, in the phylogenetic analysis they are placed into two distinct groups.
the closest agreement between surface-based and phylogeny-based functional inferences appears to be between nalp <dig> and nalp <dig>
the above surface similarity considerations, which we believe are the most interesting results of this work, are summarized in table  <dig>  for each nlr with unknown ligands, we indicate to which other tlrs/nlrs its charge and hydropathy distributions are the most similar, and based on this infer the molecules that are likely to be its ligands.
we would like to stress that because of low resolution of our approach, the suggested similarity-based predictions for putative nlr ligands should be considered with care.
cases with correctly reproduced similarities are highlighted in bold.
in the "putative ligands" column, cases with known ligands are denoted by "n/a", and cases where no prediction was made are marked accordingly.
discussion
in this paper we use similarities between surface features of two families of innate immunity receptors to reason about their possible functional similarities.
the validity of this approach is supported by results presented here - for instance, ipaf and tlr <dig>  which despite distant phylogenetic relationship both respond to bacterial flagellin , show very similar distribution of hydrophobic surface residues .
other examples include similarities in binding specificities and hydrophobic distribution moments between tlr <dig> and tlr <dig> , between ciita and tlr <dig> , similar charge distribution between nod <dig> and nod <dig> , and between nalp <dig> and nalp <dig>  .
these correlations suggest that surface similarities between nlrs and tlrs found here may reflect possible but as yet unknown functional similarities.
for most nlr genes the binding specificities are unknown, and only indirect functional information, such as from rna and protein expression profiles, is available  <cit> .
in the following, we have attempted to put the possible predicted functional similarities within nlr families  into the context of what is known from such experimental studies.
we realize that the questions asked in most of those studies have quite distant relation to the nlr molecular surface features we focus on here, therefore such a comparison rarely allows for definitive conclusions.
specific cases of similarities found here include predicted functional similarity between nalp <dig> and nalp <dig>  but this doesn't allow for any inferences as the ligands for both are unknown; it is only known that nalp <dig> plays a role in spermatogenesis and its expression is restricted to testis  <cit> .
the similarity between nalp <dig> and nalp <dig> indirectly correlates with the fact that their transcripts are both detected in reproductive organs , but again neither of their binding specificities is known.
their surface similarity to nod <dig> indicates small molecules as potential binding partners, but in contrast to mdp it is unlikely that they are bacteria-derived.
it should be noted that nalp <dig> was also implicated as an autoantigen in autoimmune polyendocrine syndrome type  <dig>  <cit> , and also that transient expression of recombinant nalp <dig> or nalp <dig> in neurons was found to induce caspase- <dig> activation and apoptosis  <cit> .
nalp <dig>  were suggested to play a role in immunity by activating nf-κb signaling and caspase-1-dependent cytokine processing  <cit> .
here we find similarity between the very short nalp <dig> lrr domain and the tlr <dig>  which suggests nalp <dig> may respond to rna motifs.
an interesting observation is that nalp <dig>  which is implicated in recurrent hydatidiform moles and reproductive wastage in humans and is known to inhibit il-1beta upon stimulation with such small molecules as lps in vitro  <cit> , appears in our analysis to have surface features similar to nod <dig> that binds mdp.
on the contrary, nalp <dig>  whose expression profiles indicate its localization to testis and ovary similarly to nalp <dig> and nalp <dig>  in our analysis displayed most significant similarity to ri, which implies other proteins as possible binding partners.
nalp <dig> lacking the lrr domain was excluded from the analyses.
nalp <dig> showed charge distribution similarity with tlr <dig> and tlr <dig> implying potential to bind rna, but we are unaware of any reports on related experimental studies.
nalp <dig> has been shown to suppress the non-canonical nf-κb pathway by inducing degradation of nik   <cit> , but its ability to form an "inflammasome" still awaits experimental proof.
in our analysis nalp <dig> surface properties did not display similarity to any other proteins.
similarity of nalp <dig> to nalp <dig> and to nod <dig> did not allow us to draw any functional similarity inferences, and we are unaware of experimental studies of this receptor.
the last member of the nalp family, nalp <dig>  displays a controversial similarity to ipaf because it implies bacterial flagellin as potential ligand, while nalp <dig> expression is restricted to testis  <cit> ; this however may point to nalp <dig> potential role of responding to other proteins.
surface features of nod <dig>  nod <dig>  and nlrx <dig> did not provide us with an insight to make functional inferences.
two observations however need to be mentioned here.
first, nod <dig> contains unusually large number  of potential n-linked glycosylation sites on its n-terminal side, unlike in any other nlr and tlr.
this however likely has little relevance to possibility of actual nod <dig> glycosylation, as currently there are no reports of it being secreted.
second, as mentioned above, nod <dig> appears to have extremely large number of lrrs  making its lrr domain the largest among all nlrs and tlrs.
this implies that the nod <dig> lrr domain structure may possess some of the following properties untypical for nlrs:  in-plane circular shape with very large radius ;  nonplanar, torroid-like shape with regular radius ;  structure consists of two or more connected circular parts each having regular radius/curvature.
the actual three-dimensional shape of this domain thus represents a puzzle.
lastly, naip surface properties resemble those of ri and tlr <dig> binding ribonuclease and rna, respectively.
murine naip <dig>  was shown to be required for intracellular response to bacterial flagellin in mice  <cit> , which indirectly signifies naip's potential for binding protein fragments.
results in figures  <dig> and  <dig> also point to a significant diversity of lrr domain surface features within both tlr and especially nlr families.
this diversity is apparent even given the low resolution of our analyses.
we believe this is mostly a consequence of the need of the receptors to recognize a group of very diverse ligands  and their binding modes.
finally, in nlrs the interactions between card/pyr/bir, nacht, and lrr domains are likely to play important roles in their oligomerization and activation.
the relative three-dimensional orientation of the lrr, nacht, and effector domains is likely to vary within the nlr family, which is signified e.g.
by generally large and highly variable size of the linker domain .
we believe that this could be another possible explanation of the diversity of lrr domain surface properties within nlrs, which in turn points to the possibility of diverse ligand-induced inflammasome activation mechanisms.
CONCLUSIONS
we have built homology models and performed quantitative analyses of charge, hydrophobicity, and glycosylation distributions of lrr domains in almost all human nlrs and tlrs known today.
these analyses, while conducted without experimental knowledge of nlr structures, provide a low-resolution comparison of lrr surface features between the two protein families.
the validity of our approach is supported by several cases when receptors that bind similar ligands were found to have similar charge and hydrophobicity distributions.
comparison of nlrs with unknown functions to nlrs/tlrs that have known ligands allowed us to infer possible functional similarities, which can not be derived from conventional phylogenetic analyses.
we hope that our predictions will be helpful in design of future experiments to decipher roles of previously uncharacterized nlrs in human innate immunity.
we propose an experimental study to test whether the nlr macromolecular ligands we predict indeed modulate the activity of caspase- <dig> in the presence and absence of the corresponding nlrs in vitro.
because of the low resolution of our approach, our predictions should be more reliable in cases where tlr/nlr known ligands are macromolecular fragments, such as flagellin, ds/ssrna, or dna motifs.
improving the reliability of predictions and providing quantitative estimates for relative ligand binding affinities requires combined application of explicit structure-based approaches such as homology modeling and molecular docking.
work in this direction is in progress and will be reported elsewhere.
authors' contributions
ayi performed the calculations, analyzed the results, and drafted the manuscript.
ag helped to formulate and coordinated the study, and edited the manuscript.
both authors read and approved the final manuscript.
supplementary material
additional file 1
supplementary materials.
a multiple sequence alignment used for phylogenetic analysis in figure  <dig>
click here for file
acknowledgements
we would like to thank john c. reed for stimulating discussions and critical comments, and cindy cook for proofreading the manuscript.
this work was supported in part by nih grant p <dig> gm <dig> .

 the innate immune system provides a first level of defense against invading agents such as microbial pathogens and toxins and plays a role in maintaining a stable, healthy composition of the commensal microbiome. in mammals, the important role of continuously surveying the environment is played by members of two protein families: extracellular toll-like receptors and intracellular nod-like receptors, also referred to as nucleotide-binding domain- and leucine-rich repeat-containing proteins. while individual receptors from both families have distinct specific roles, the overall function of the innate immune system requires their complex, synergistic interaction. both tlr and nlr families belong to the oldest branch of the immune system, the innate immunity, and have complex evolutionary history that started in the simplest multicellular organisms and went through significant expansions and contractions in various lineages leading to present day vertebrates and mammals. for instance, tlr and nlr families in some invertebrates, such as amphioxus, have over members. in early branching vertebrates, such as sea lamprey and hagfish, a closely related family of variable lymphocyte receptors provide a version of adaptive immunity, which was lost in higher vertebrates. in humans, the tlr family consists of ten proteins, which have been a subject of intense experimental studies during the past decade due to their critical role in immunity and involvement in diseases such as crohn's disease, cystic fibrosis lung disease, inflammatory bowel disease, familial mediterranean fever, septic joint disease, and several others. tlrs sense the presence of pathogen-associated molecular patterns by specific binding sites in their extracellular "horseshoe"-shaped receptor domains consisting of multiple leucine-rich repeat motifs. binding-induced conformational change and/or dimerization provides a signal to the intracellular toll/interleukin receptor domain, which in turn binds specific adaptor molecules, such as myd leading eventually to nf-κb transcription factor activation and transcription of proinflammatory cytokine genes. from numerous experimental studies it is known that the lrr domains of different tlrs specifically interact with a rather diverse plethora of ligands, from bacterial flagellin and ss/dsrna to peptidoglycans to imidazoquinioline compounds. by integrating these signals, the tlr receptors sense the microbial state of the immediate environment and define the appropriate level of innate immunity activity. the nlr family receptors provide complementary, but less-studied and -understood, mechanisms of intracellular pamp detection and innate immunity regulation. twenty-two genes coding for nlr proteins are found in the human genome, typically consisting of a c-terminal lrr domain, the nucleotide-binding oligomerization domain, and the n-terminal effector domain. several different effector domains can be found in human nlrs, including the caspase recruitment domain; the pyrin/paad domain; the baculovirus "inhibitor of apoptosis" domain; and an uncharacterized domain, thereby partitioning nlrs into at least four subgroups. the variety of effector domains in other organisms, specifically invertebrates, is much larger. following binding of a ligand to the lrr domain, nalp nalp and ipaf are known to form multimer complexes termed "inflammasomes." inflammasome formation leads to a further sequence of molecular events: activation of caspases and conversion of pro-interleukins into their active form. recently, inflammasomes were shown to be structurally similar to apoptosomes, highlighting the evolutionarily conserved mechanism of activated oligomerization, carried our by nucleotide binding aaa proteins belonging to two distinct families: the nacht family and nbarc family. after almost a decade since their discovery, information on ligand specificities of the nlr family proteins remains elusive. nod and nod are known to sense bacterial cell wall-derived peptidoglycans γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. the nalp inflammasome was shown to be activated by mdp in a two-step manner. the nalp inflammasome is activated by a wide range of ligands: bacterial rna, uric crystals, antiviral imidazoquinioline compounds r and r and contact sensitizers such as trinitro-chlorobenzene. nalp mutations are known to correlate with a number of autoinflammatory disorders, e.g., muckle-wells syndrome, and mutations in nod gene are linked to increased susceptibility to the chronic inflammatory disorders such as crohn's disease, psoriatic arthritis, and blau syndrome. ipaf is known to sense bacterial flagellin, although whether flagellin is the direct and the only ipaf ligand is under debate. finally, ciita that serves as a master regulator of major histocompatibility complex genes transcription is known to bind many dna-binding and co-activator proteins. its lrr domain was found to be involved in ciita self-association as well as in binding a novel zinc-finger protein zxdc. the specific roles of other nlrs in innate immunity are at this point unknown. at the same time, there is no direct structure information on lrr domains of any of the nlrs. given the importance of understanding nlrs' function and the lack of information on their structure and agonists, we believed a theoretical study might provide some useful insights and help design experimental efforts. the main subjects of this work, the ligand-binding domains of nlrs and tlrs, both consist of multiple leucine-rich repeat motifs. lrrs are extensively used by nature as building blocks for assembling scaffolds of protein interfaces, designed primarily for specific protein-protein interactions. about lrr-containing proteins with diverse functions have been identified in the human genome. due to the importance of lrr proteins, they have been a subject of intensive experimental work, with the first crystal structure of a ribonuclease inhibitor providing a major advance in the field. these advances continue today, with over structures of lrr-containing proteins, including three tlr structures, solved through much has been learned about structural principles of lrr proteins organization from theoretical and modeling perspectives. lastly, it is interesting to note that while lrr domains of tlr and nlr proteins are homologous, they belong to distinct sub-branches of the lrr family, and both have close homologs that are not innate immunity receptors. for instance, the families of lrr- and immunoglobulin-containing proteins, involved in cell-cell adhesion, are closer related to tlrs than nlrs are, and on the other hand, ribonuclease inhibitor, a protein not involved in innate immunity, is closely related to nlrs but is much more distant from tlrs. these observations suggest that nlr, tlr, and vlr families have a very complex evolutionary history with multiple duplication, domain swapping, and domain recruitment events. while it is generally believed that lrr domains of most nlrs and tlrs are involved in direct binding of agonist molecules, for nlrs there is little experimental proof of this. some evidence of direct agonist binding by nalp lrrs is given in. when referring to "lrr ligands" in this manuscript, we recognize that binding to lrr domains is an assumption at this point, and that "ligands" can be either the "danger signals" themselves, or the possible intermediate molecules. since the overall shape of lrr domains from different proteins is relatively conserved, the functional similarities and differences between them must be defined by specific features of their surfaces. this would be true regardless of whether lrr domains interact with immunity "danger signals" directly or by means of intermediate molecules, or the functionality is conferred by lrr interactions with other domains of nlrs. the key point is that if lrr surface properties within a given nlr/tlr pair are similar, they are likely to bind the broadly similar type of molecules. in this manuscript, we investigate surface features of the twenty-two human nlrs, and by comparing them to those of tlrs, attempt to predict likely similarities between their functions. first, we have built homology models for lrr domains of twenty nlrs. second, we have investigated distributions of amino acid charge and hydrophobicity across lrr domain surfaces and by means of clustering analysis have quantitatively assessed their similarities. additionally, we have analyzed distributions of consensus n-linked glycosylation sites within tlrs, in order to support application of our approach to nlrs. while the simplicity of our analyses requires care when interpreting the results, we believe they may be helpful in designing future experiments to shed light on the role of nlr-coding genes in human innate immunity. methods phylogenetic analyses multiple alignments and phylogenetic trees were built using clustalx. the most divergent sequence, naip, was used as an outgroup to root the tree. the tree reliability assessment was conducted by means of the bootstrap method using tree samples. several other multiple alignment and tree building strategies produced essentially identical results. protein sequences and gene organization information were obtained from the ensembl database. homology modeling in order to visualize structures and surface features of lrr domains from human nlrs, homology models were built based on pairwise alignments of nlr amino acid sequences with the sequence of porcine ribonuclease inhibitor, whose x-ray structure was used as a template. the two proteins for which the models were not built are nod and nalp nod lrr domain consists of lrrs, and all lrr-containing proteins with available structures are too small to be used as a template. the second protein, nalp lacks an lrr domain. amino-acid sequence alignments were manually refined using the swiss-pdb viewer to account for additional information obtained from the gene structure and from structural comparisons between lrr proteins. because of the high sequence similarity between the template and the prediction targets, homology modeling was performed by means of the swiss-model server. electrostatics and hydrophobicity calculations were carried out for nlr homology models as well as for experimental crystal structures of two tlrs. they were conducted utilizing moe software. lrr sequence-to-structure mapping in order to conduct a quantitative comparative analysis of lrr domain surface features in the absence of experimental structure data, we mapped physicochemical features of residues on the surface using existing knowledge about lrr sequence-to structure-mapping. the typical lrr sequence contains a conserved motif lxxlxlxxnxl, where l is leucine, isoleucine, valine, or phenylalanine, and n is asparagine, threonine, serine, or cysteine. in nlrs there is a striking correlation between their gene organization and amino acid sequence. specifically, in nalps, two consecutive lrr motifs xlaanalaaaaaaaaaaaaaaaaalaalxlaanalaaaaaaaaaaaaaaaaaaiaal of length + = residues are encoded by a single exon, while we have found that in nods each lrr motif xlaanalaaaaaaaaaaaaaaaaalaal of length residues is encoded by a single exon. this unusual genomic structure has interesting structural consequences, which would be a subject of a separate publication. these two facts have allowed us to make reliable predictions for positions and boundaries of lrr motifs in human nlr sequences. in tlrs such an unusual structure-exon correlation is lacking, and the whole lrr domain is typically encoded by one large exon. finally, from the available structures of other lrr-containing proteins it is known that the aalxlaa pattern corresponds to a beta-strand in the inner concave surface, which is then followed by a c-terminal side loop, by an alpha-helix on the outside convex surface, and by the n-terminal side loop. this property is very well conserved in numerous lrr-containing proteins, and we have utilized it to improve mapping between lrr sequence and structure. the residue x in the aalxlaa motif was used as reference residue to define positions of all other residues on the lrr surface. the next residues are located on the c-terminal side, the next residues are located in the outer convex part, and finally the next residues represent the n-terminal side. in tlrs, the typical length of the outer part is residues, but there is much variation. after we performed the sequence-to-structure mapping as described above, we were able to partition the surface into four parts and analyze distributions of amino acid properties such as charge and hydrophobicity within these parts separately. distribution of potential glycosylation sites was also examined for extracellular tlrs. quantitative comparison of charge and hydropathy distributions amino acid charge and hydrophobicity distributions within lrr domains were calculated for each of the four surfaces shown in figure 3b. the amino acid charge was calculated assuming the ph of, and the hydrophobicity was quantified by hydropathy index ranging from - to + with negative values corresponding to hydrophilic properties. within a given protein, for each lrr motif i, we calculated average charge and hydropathy separately for each of the four surface regions. for an lrr domain with n motifs, this produced n values for charge and the same for hydropathy. next, in order to characterize the distribution of these values within each surface, we have calculated the first four moments, mk, of qi and hi distributions: where fi is either qi or hi, and with these definitions, the moment m is simply the average value, and the moment m is related to the square of the standard deviation. the moments m and m reflect additional details of the distributions, in particular the directional trends. the distributions of charge and hydropathy within one protein are thus each characterized by a sequence of surface × moments = values. in order to quantify similarities of distributions within different proteins, we used spearman's rank-order correlation coefficient, rs. the similarity between distributions was then defined as. clustering analysis was then performed by a complete linkage method utilizing java treeview software. glycosylation distribution analysis consensus n-linked glycosylation sites were predicted by searching for patterns nxxs and nxxt in tlr sequences. in globular proteins, such predictions greatly overestimate the number of glycosylation sites, assuming that all sites matching the pattern above are accessible to oligosaccharyltransferases in order for glycosylation to occur, while in reality most of these sites are buried inside protein structure. in our case, however, such predictions can be much more accurate because we could predict which of the potential glycosylation sites were exposed to solvent.  phylogenetic analysis of nlr and tlr leucine-rich repeat domain sequences as a first step, we have performed a phylogenetic analysis of lrr domains in nlrs and tlrs. extreme sequence length diversity among lrrs from these families makes conducting such an analysis in a meaningful way nontrivial. this is further complicated by the fact that full length multiple alignment of complete sequences contains numerous large gap regions, which makes its truncation difficult. to circumvent the latter problem, we built a multiple alignment using a 10-fold increased gap extension penalty and truncated it from n- and c-terminal ends to make its length similar to that of the shortest sequence. the resulting alignment was then used to build a rooted phylogenetic tree. among the nlr and tlr sequences, we also included a sequence of human ribonuclease inhibitor consisting of a highly-regular lrr domain closely homologous to that of lrrs in the nrl family. the sequences group into three distinct clusters: nalps and ri; card-containing proteins and nlrx1; and tlrs. surprisingly, the sequence similarity-based phylogeny of the c-terminal lrr domain well agrees with the classification of all these proteins based on the n-terminal effector domain. within nalps, there are three distinct subgroups: nalp nalp nalp nalp nalp14; nalp nalp nalp nalp13; nalp nalp and ri. it is interesting to note that ipaf and ciita, as well as nlrx whose effector domain lacks definitive classification, group together with card-containing nods. this suggests that nlrx effector domain might be card-like. this analysis supports known phylogenetic relationships between nlr and tlr families, but it does not permit us to draw any conclusions regarding similarities of surface features between tlrs with known ligands and nlrs. homology models of lrr domains and sequence-to-structure mapping in figure we present homology models for lrr domains of twenty nlrs along with experimentally determined structures of two tlrs. the first observation is that there is much diversity in the lrr domain size - from lrrs to lrrs. an unusually large number of lrrs is found in nod whose model was not built because there is no large enough template structure available. second, coloring of the models according to surface electrostatic potential and hydrophobicity reveals significant diversity of their surface features. the models in figure are helpful in visualizing lrr domain structures and could be used for explicit protein-ligand docking; however they are not suitable for quantitative assessment of surface similarity. in order to quantitatively analyze the similarity of surface features of lrr domains, we resorted to their reduced-resolution representation. first, we adopted a simple approach to implicitly infer lrr domain three-dimensional structures from their amino acid sequences. second, we have partitioned the total surface of the lrr domain of each protein into four regions: concave inner surface, n-terminal side, c-terminal side, and convex outer surface. before conducting a quantitative comparison of nlr and tlr surface features we had to consider possible perturbations to tlr surface properties due to glycosylation. role of glycosylation of lrr domain surfaces it is well known that lrr domains in all ten human tlrs are n-glycosylated due to their extracellular localization, and in some of them glycosylation is essential for proper function. this is not surprising as glycosylation can dramatically alter surface properties and thereby affect ligand binding. since nlrs are all known to be cytosolic proteins their n-linked glycosylation is unlikely, which complicates a direct comparison of their surface features to those of tlrs. to understand the potential role of tlr glycosylation in their ligand binding, we have performed a search for consensus n-linked glycosylation sites within tlr sequences, and analyzed their distribution over the domain surfaces. the results are summarized in table the tlrs that contain many consensus glycosylation sites are tlr tlr tlr and tlr in tlr all surfaces except the c-terminal side that is known to bind dsrna contain several glycosylation sites. in tlr inner and outer surfaces are likely to be glycosylated while side ones are not, which is consistent with binding of ssrna via one of its side surfaces. glycosylation of tlr and tlr resembles that of tlr in that only the c-terminal side surface is glycan-free. in the rest of the tlrs, each of the four surfaces contains no more then three potential glycosylation sites. as an additional step, we have also analyzed nlr sequences for potential glycosylation, and found that they contain 0- consensus glycosylation sites per surface, with the only exception being an n-terminal surface of nod which interestingly contains such sites. these findings indicate that while glycosylation of some tlrs certainly modulates their surface properties, in most cases the ligand binding sites are located in non-glycosylated regions and, in addition, the overall surface area affected by glycosylation is relatively small, so that global surface comparisons such as those performed here are not affected. hydrophobicity and charge distributions: clustering analysis within full lrr surfaces the two physico-chemical amino acid properties that critically affect ligand binding and whose distributions we have thus analyzed are charge and hydrophobicity. figure shows an example of our clustering analyses of hydropathy distribution moments within each of the four domain surfaces. the similarity measure in this analysis is spearman's correlation. based on clustering results, lrr domain pairs that have particularly similar hydropathy distributions and lrr numbers are: tlr and ipaf; nod and nalp; ri and nalp; tlr and naip; nalp and nalp; tlr and tlr; tlr and nalp. hydrophobicity and charge distributions within individual lrr domain surfaces in order to gain further insight into similarities of lrr surface features between nlrs and tlrs, we conducted a careful manual inspection of similarity patterns across individual biologically relevant surfaces, also taking into account results of our tlr glycosylation analysis. we assessed similarities between lrr domains of all proteins considered, as detailed below. in nalp which is known to sense mdp, the concave and side surfaces have both hydropathy and charge distributions that are the most similar to napl which is known to bind a diverse set of ligands, including mdp. nalp and nalp have similar hydropathy distributions, but dissimilar charge distributions. nalp and nalp have hydropathy and charge distributions similar to nod that binds ie-dap. nalp has the shortest lrr domain, containing only lrrs. its surface properties are similar to those of tlr which contains lrrs. because the hydropathy and charge distribution moments are normalized by the number of lrrs in a given domain, similarity of distributions still allows to hypothesize that nalp and tlr may bind the same type of ligands, rna motifs. nalp charge and hydropathy are similar to nod and nalp both binding mdp, suggesting small molecules as probable ligands. the high similarity of nalp hydropathy distribution and size to those of nod and also nod suggests that they may share the same type of ligands--peptidoglycans. this is also supported by similarities in their charge distributions, with the difference being a more positively charged convex surface of nalp the hydropathy and charge of nalp are similar to ri, which binds rnase, thus suggesting that the nalp ligand may be protein-like. nalp shows hydropathy similarity with tlr that binds ssrna, most likely by one of its side surfaces. nalp charge distribution is similar to that of tlr and also tlr over its side surfaces, suggesting that ss/dsrna is likely to be among possible nalp ligands. nalp surface properties do not exhibit any obvious similarity to others. the hydropathy distribution of nalp is similar to that of nalp and nod but neither of them has known ligands. nalp charge distribution on its concave surface is almost identical to that of ipaf, and distributions over other surfaces are similar to ipaf. the hydropathy distributions of these two nlrs are similar within only its concave side. assuming that ipaf binds flagellin similar to tlr i.e., on its concave surface, flagellin can be considered a likely ligand of nalp nod hydropathy has some similarity to nalp but charge distribution is unlike that in any other domain. nod concave and side surfaces have hydrophobicity similar to nalp but the charge distribution is different. also, nod appears to have the largest ligand-binding domain of all nlrs and tlrs: it contains as many as lrrs. nlrx surface features do not exhibit similarity with any other domain. naip hydropathy distribution is similar to ri, while naip charge distribution is similar to tlr it is interesting to note that the functional similarities inferred based on comparison of molecular surfaces correlate well with experimental data where available, while hypotheses based on evolutionary relationships shown in figure generally lack such correlation. for example, while the surface comparison and the known experimental data suggest that nalp and nalp are activated by the same type of molecules, mdp, the phylogenetic reconstruction places nalp and nalp into two distinct subgroups in figure although both subgroups belong to the nalp group. similarly, whereas ipaf and tlr are known from experiment to sense a common molecule with which our surface analysis is in agreement, in the phylogenetic analysis they are placed into two distinct groups. the closest agreement between surface-based and phylogeny-based functional inferences appears to be between nalp and nalp the above surface similarity considerations, which we believe are the most interesting results of this work, are summarized in table for each nlr with unknown ligands, we indicate to which other tlrs/nlrs its charge and hydropathy distributions are the most similar, and based on this infer the molecules that are likely to be its ligands. we would like to stress that because of low resolution of our approach, the suggested similarity-based predictions for putative nlr ligands should be considered with care. cases with correctly reproduced similarities are highlighted in bold. in the "putative ligands" column, cases with known ligands are denoted by "n/a", and cases where no prediction was made are marked accordingly. discussion in this paper we use similarities between surface features of two families of innate immunity receptors to reason about their possible functional similarities. the validity of this approach is supported by results presented here - for instance, ipaf and tlr which despite distant phylogenetic relationship both respond to bacterial flagellin, show very similar distribution of hydrophobic surface residues. other examples include similarities in binding specificities and hydrophobic distribution moments between tlr and tlr, between ciita and tlr, similar charge distribution between nod and nod, and between nalp and nalp. these correlations suggest that surface similarities between nlrs and tlrs found here may reflect possible but as yet unknown functional similarities. for most nlr genes the binding specificities are unknown, and only indirect functional information, such as from rna and protein expression profiles, is available. in the following, we have attempted to put the possible predicted functional similarities within nlr families into the context of what is known from such experimental studies. we realize that the questions asked in most of those studies have quite distant relation to the nlr molecular surface features we focus on here, therefore such a comparison rarely allows for definitive conclusions. specific cases of similarities found here include predicted functional similarity between nalp and nalp but this doesn't allow for any inferences as the ligands for both are unknown; it is only known that nalp plays a role in spermatogenesis and its expression is restricted to testis. the similarity between nalp and nalp indirectly correlates with the fact that their transcripts are both detected in reproductive organs, but again neither of their binding specificities is known. their surface similarity to nod indicates small molecules as potential binding partners, but in contrast to mdp it is unlikely that they are bacteria-derived. it should be noted that nalp was also implicated as an autoantigen in autoimmune polyendocrine syndrome type, and also that transient expression of recombinant nalp or nalp in neurons was found to induce caspase- activation and apoptosis. nalp were suggested to play a role in immunity by activating nf-κb signaling and caspase-1-dependent cytokine processing. here we find similarity between the very short nalp lrr domain and the tlr which suggests nalp may respond to rna motifs. an interesting observation is that nalp which is implicated in recurrent hydatidiform moles and reproductive wastage in humans and is known to inhibit il-1beta upon stimulation with such small molecules as lps in vitro, appears in our analysis to have surface features similar to nod that binds mdp. on the contrary, nalp whose expression profiles indicate its localization to testis and ovary similarly to nalp and nalp in our analysis displayed most significant similarity to ri, which implies other proteins as possible binding partners. nalp lacking the lrr domain was excluded from the analyses. nalp showed charge distribution similarity with tlr and tlr implying potential to bind rna, but we are unaware of any reports on related experimental studies. nalp has been shown to suppress the non-canonical nf-κb pathway by inducing degradation of nik, but its ability to form an "inflammasome" still awaits experimental proof. in our analysis nalp surface properties did not display similarity to any other proteins. similarity of nalp to nalp and to nod did not allow us to draw any functional similarity inferences, and we are unaware of experimental studies of this receptor. the last member of the nalp family, nalp displays a controversial similarity to ipaf because it implies bacterial flagellin as potential ligand, while nalp expression is restricted to testis; this however may point to nalp potential role of responding to other proteins. surface features of nod nod and nlrx did not provide us with an insight to make functional inferences. two observations however need to be mentioned here. first, nod contains unusually large number of potential n-linked glycosylation sites on its n-terminal side, unlike in any other nlr and tlr. this however likely has little relevance to possibility of actual nod glycosylation, as currently there are no reports of it being secreted. second, as mentioned above, nod appears to have extremely large number of lrrs making its lrr domain the largest among all nlrs and tlrs. this implies that the nod lrr domain structure may possess some of the following properties untypical for nlrs: in-plane circular shape with very large radius; nonplanar, torroid-like shape with regular radius; structure consists of two or more connected circular parts each having regular radius/curvature. the actual three-dimensional shape of this domain thus represents a puzzle. lastly, naip surface properties resemble those of ri and tlr binding ribonuclease and rna, respectively. murine naip was shown to be required for intracellular response to bacterial flagellin in mice, which indirectly signifies naip's potential for binding protein fragments. results in figures and also point to a significant diversity of lrr domain surface features within both tlr and especially nlr families. this diversity is apparent even given the low resolution of our analyses. we believe this is mostly a consequence of the need of the receptors to recognize a group of very diverse ligands and their binding modes. finally, in nlrs the interactions between card/pyr/bir, nacht, and lrr domains are likely to play important roles in their oligomerization and activation. the relative three-dimensional orientation of the lrr, nacht, and effector domains is likely to vary within the nlr family, which is signified e.g. by generally large and highly variable size of the linker domain. we believe that this could be another possible explanation of the diversity of lrr domain surface properties within nlrs, which in turn points to the possibility of diverse ligand-induced inflammasome activation mechanisms.  we have built homology models and performed quantitative analyses of charge, hydrophobicity, and glycosylation distributions of lrr domains in almost all human nlrs and tlrs known today. these analyses, while conducted without experimental knowledge of nlr structures, provide a low-resolution comparison of lrr surface features between the two protein families. the validity of our approach is supported by several cases when receptors that bind similar ligands were found to have similar charge and hydrophobicity distributions. comparison of nlrs with unknown functions to nlrs/tlrs that have known ligands allowed us to infer possible functional similarities, which can not be derived from conventional phylogenetic analyses. we hope that our predictions will be helpful in design of future experiments to decipher roles of previously uncharacterized nlrs in human innate immunity. we propose an experimental study to test whether the nlr macromolecular ligands we predict indeed modulate the activity of caspase- in the presence and absence of the corresponding nlrs in vitro. because of the low resolution of our approach, our predictions should be more reliable in cases where tlr/nlr known ligands are macromolecular fragments, such as flagellin, ds/ssrna, or dna motifs. improving the reliability of predictions and providing quantitative estimates for relative ligand binding affinities requires combined application of explicit structure-based approaches such as homology modeling and molecular docking. work in this direction is in progress and will be reported elsewhere. authors' contributions ayi performed the calculations, analyzed the results, and drafted the manuscript. ag helped to formulate and coordinated the study, and edited the manuscript. both authors read and approved the final manuscript. supplementary material additional file 1 supplementary materials. a multiple sequence alignment used for phylogenetic analysis in figure click here for file acknowledgements we would like to thank john c. reed for stimulating discussions and critical comments, and cindy cook for proofreading the manuscript. this work was supported in part by nih grant p gm.
the human innate immune system uses a system of extracellular toll-like receptors and intracellular nod-like receptors to match the appropriate level of immune response to the level of threat from the current environment. the specific functional roles of lrr domains in nlrs and tlrs are thus defined by their detailed surface features. we report a quantitative, comparative analysis of the surface features of lrr domains in human nlrs and tlrs, using predicted three-dimensional structures for nlrs. while experimental crystal structures of four human tlrs have been solved, no structure data are available for nlrs. despite differences in structural and genomic organization, comparison of lrr surface features in nlrs and tlrs allowed us to hypothesize about their possible functional similarities. we find agreement between predicted surface similarities and similar functional roles in nlrs and tlrs with known agonists, and suggest possible binding partners for uncharacterized nlrs. our results illustrate diversity of surface features of innate immunity receptors and provide hints for function of nlrs whose specific role in innate immunity is yet unknown. specifically, we calculated amino acid hydrophobicity, charge, and glycosylation distributions within lrr domain surfaces and assessed their similarity by clustering.
 the human innate immune system uses a system of extracellular toll-like receptors and intracellular nod-like receptors to match the appropriate level of immune response to the level of threat from the current environment. almost all nlrs and tlrs have a domain consisting of multiple leucine-rich repeats, which is believed to be involved in ligand binding. lrrs, found also in thousands of other proteins, form a well-defined "horseshoe"-shaped structural scaffold that can be used for a variety of functions, from binding specific ligands to performing a general structural role. the specific functional roles of lrr domains in nlrs and tlrs are thus defined by their detailed surface features. while experimental crystal structures of four human tlrs have been solved, no structure data are available for nlrs.  we report a quantitative, comparative analysis of the surface features of lrr domains in human nlrs and tlrs, using predicted three-dimensional structures for nlrs. specifically, we calculated amino acid hydrophobicity, charge, and glycosylation distributions within lrr domain surfaces and assessed their similarity by clustering. despite differences in structural and genomic organization, comparison of lrr surface features in nlrs and tlrs allowed us to hypothesize about their possible functional similarities. we find agreement between predicted surface similarities and similar functional roles in nlrs and tlrs with known agonists, and suggest possible binding partners for uncharacterized nlrs.  despite its low resolution, our approach permits comparison of molecular surface features in the absence of crystal structure data. our results illustrate diversity of surface features of innate immunity receptors and provide hints for function of nlrs whose specific role in innate immunity is yet unknown. 