BACKGROUND
heat-shock proteins, also known as molecular chaperones, belong to a highly conserved set of protein families that perform essential functions to the cell in prokaryotes and eukaryotes  <cit> .
these functions include, but are not limited to, protein folding, assembly, and transport  <cit> .
while the folding function of groel has been extensively characterized, emerging literature uncover many alternative functions and structures for this protein .
mutations in this molecule that are responsible for the emergence of alternative functions remain uncharacterized.
therefore, the potential evolvability of this essential protein is largely unexplored.
groes and groel, also known as cpn <dig> and cpn <dig> respectively, are expressed at constitutive levels under physiological conditions and their expression increases at high temperatures, allowing the growth and survival of bacteria at a broad range of temperatures  <cit> .
both chaperonins are encoded by the operon groe and they form a homotetradecamer organized into two back-to-back oriented rings.
each of the rings comprises seven identical groel subunits, with each subunit being divided into three domains: the apical, which binds unfolded proteins and groes, the intermediate, which acts as a hinge allowing the movement of the apical domain as well as the transition between trans and cis conformations needed for groel function, and the equatorial which is responsible for the atpase and the folding activities that take place in the central cavity of the ringed complex  <cit> .
the main function of groel has been considered to be the folding of other proteins in the cell  <cit> , although evidence supports other folding-unrelated roles for groel, such as immune response in humans  <cit>  or growth and biofilm formation in bacteria, among others  <cit> .
these functions are context dependent and may vary from one organism to another.
alternative functions may emerge in proteins after the duplication and evolution of their encoding gene or through amino acid replacements that impinge on the protein structure.
the gene groel has undergone many duplications in bacteria  <cit> , adaptive evolution  <cit>  and functional divergence  <cit> .
moreover, structural evolutionary changes have been recently described for groel, according to which changes in the amino acid composition of its co-chaperonin groes can determine groel functioning as a single instead of double ring  <cit> .
the strong evolutionary sequence conservation of groel and the high number of interactions it establishes with other proteins in the cell  <cit>  contrast with groel´s functional and structural plasticity and its propensity to persist in duplicate in some bacteria.
particularly striking is the fact that, while performing essential functions in the cell, groel presents alternative functions  <cit> .
the trade-off between groel´s high conservation at the sequence and functional levels and its high propensity to evolve novel functions remains poorly understood.
researchers have attempted to uncover groel’s multi-functionality through the testing of the effects of directed mutagenesis of groel amino acids under laboratory-controlled conditions.
however, the multifunctional nature of groel suggests the existence of a reservoir of functionalities resulting from the interaction between distinct sets of amino acids in different bacteria.
here we propose the hypothesis that the functional plasticity of groel is mediated by an evolutionary plasticity of potentially functional amino acids.
in support of this hypothesis, bacteria growing under different physiological conditions present groel variants with functions alternative to folding and which involve different sets of amino acids.
the strong selective constraints acting on groel imply important functional and structural links between amino acids.
these links impose reciprocal selection pressures among amino acid sites.
therefore, changes on groel functions from one bacterial group to another should be reflected in strong coevolutionary signatures between linked amino acids whose evolvability is co-regulated by selection in a particular bacterial clade.
in this study we performed an exhaustive coevolutionary analysis using an extensive bacterial phylogeny to uncover the evolutionary, hence functional, dependencies among amino acid residues within groes, groel and between both these proteins.
the coevolutionary networks identified in these chaperonins from hundreds of bacteria reveal the complexity underlying the evolution of this essential protein and shed light on the functional importance of previously uncharacterized residues.
RESULTS
sequence data and coevolution analyses
to perform intra-protein coevolution analyses in groes and groel, we searched groe sequences amongst the major bacterial phyla and found that actinobacteria, cyanobacteria, bacteroidetes and chlorobi, firmicutes, proteobacteria, and spirochaetes comprised a number of groe homologs that would allow accurate inference of coevolution.
the number of sequences ranged between  <dig> and  <dig> for groes genes, and  <dig> and  <dig> for groel genes belonging to spirochaetes and proteobacteria groups, respectively .
in spite of the differences in the number of sequences, the mean amino acid sequence divergence was of the same order in all bacteria groups ranging between  <dig>  and  <dig> , and these divergence levels were not correlated with the number of sequences in the alignment.
these divergence levels are also within the levels ensuring robust results when using coevolution analyses.
inter-protein coevolution analyses between groes and groel were performed building pairs of files for each group of bacteria, both of which included the same bacterial strains.
accordingly, the size of the alignments used for the groes-l inter-coevolution analyses ranged between  <dig> in cyanobacteria and  <dig> in proteobacteria .
all coevolution analyses were performed with a phylogenetic tree built up function in caps and pairs of coevolving sites were further filtered through a novel bootstrap analysis .
therefore, the number of sequences in the alignment, level of sequence divergence and new introduced filters warranted minimizing false positives rate and increasing accuracy of our results.
for the individual intra-group analyses we chose those bacterial groups with more than  <dig> sequences.
for the overall cpn <dig> and cpn <dig> intra-group analyses we took all sequences .
evolutionary dependencies between functional sites within groes and groel
to determine the magnitude of the evolutionary plasticity of groel and groes, we first conducted a coevolutionary analysis to determine the network of residues dependencies in all bacteria.
we performed intra-protein coevolution analyses in a  <dig> sequences based groes alignment and  <dig> sequences based groel alignment, representing the  <dig> major bacterial groups.
we also calculated the support of each pair of coevolutionary sites taking into account the phylogenetic relationships using a non-parametric bootstrap approach .
all amino acid sites numbering and composition are referred throughout the text to the numbering in the crystal structure of groesl from e. coli .
we identified a single connected network of  <dig> coevolving amino acid sites in groes, with lys <dig>  leu <dig>  gly <dig>  thr <dig>  arg <dig>  glu <dig>  arg <dig> and lys <dig> establishing most of the evolutionary dependencies .
to determine the importance of each of the amino acid sites in the network  we applied network centrality measures to coevolving sites, typically used in networks biology: degree centrality, betweenness and closeness.
networks are a collection of points joined together in pairs by lines.
in the networks jargon, points are referred to as vertices or nodes while the links are referred to as edges.
centrality measures of nodes, including degree, betweenness and closeness, are typically used to determine the importance of these nodes in the network.
degree is the number of edges departing from a node in the network.
a node presents high closeness when its shortest distances to all other nodes in the network are low compared to the average closeness.
a node has high betweenness when the number of shortest paths between all pairs of nodes in a network that pass through it is high.
interestingly, leu <dig> and gly <dig>  two amino acids known to be involved in the interaction between groes and groel  <cit>  are the most central in the coevolution network .
the dependency of these two essential amino acids on other functionally uncharacterized ones hints possible functional links between both sets of amino acid sites.
indeed, lys <dig>  thr <dig>  arg <dig>  gly <dig>  arg <dig> and lys <dig>  while lacking apparent functions, they form a structural cluster establishing important contacts among groes subunits .
amino acid sites within each of the structural clusters were in close proximity to each other .
coevolution among structurally proximal amino acid sites is a general pattern  <cit>  and suggests compensatory relationships, hence functional or structural links, between amino acids  <cit> .
in groel, we identified  <dig> coevolving amino acid residues , of which leu <dig>  ala <dig>  ser <dig>  arg <dig>  lys <dig>  gln <dig>  arg <dig>  ala <dig>  and asn <dig> were the most central residues to the network .
arg <dig>  val <dig>  and lys <dig> are involved or close to  sites mediating substrate and groes binding.
other positions were either included or close to charged amino acid sites that were facing the central groel cavity .
finally, asn <dig> is located in the atp and mg2+ binding site, while other amino acid sites, such as ala <dig> and ala <dig>  are at the rings interface and likely involved in protein folding within the groes-l ring complex.
all  <dig> amino acids are distributed into two structural groups: one in the apical and another in the equatorial domains .
remarkably, coevolving sites are very close to sites involved in protein folding, substrate and groes binding, atp binding and hydrolysis, or inter-subunits contacts, thus, suggesting that changes at these amino acids may have important functional consequences .
coevolution of groes with groel
the interaction of groes and groel is essential to induce the conformational changes needed for the folding cycle.
these conformational changes may force coadaptation dynamics between groes and groel.
we performed coevolutionary analyses using the protein sequences of groes and groel from the same set of bacterial strains .
these sequences span all the different bacterial groups , with all these groups being well represented.
analysis of coevolution identified a group of amino acids from groes coevolving with groel .
the centrality measures of coevolving sites were also calculated .
coevolution did not affect groes sites involved in the groes-l interaction.
nonetheless, sites coevolving between both proteins had important functional roles and mapped to different functional domains of groel.
for example, two of the groel sites, ala <dig> and arg <dig>  are involved in the binding of substrates and overlap with sites involved in groes binding as well  <cit> .
in addition, glu <dig>  involved in the coevolution between ala <dig> and arg <dig>  has a role in stabilizing inter-ring contacts  <cit> .
since groes is heavily involved in determining the function of groel as a single or as a double ring  <cit> , the coevolution of glu <dig> from groel with groes amino acid sites may have implications in the structural stability of the double ring, and thus, groes-groel folding cycle.
in support of the structural and functional communication between the coevolving sites of groes and groel, coevolving amino acids formed structural clusters within groesl .
in addition to their clustering, coevolving sites were either functionally relevant or were close to sites with reported functional importance.
taken together, these results support the hypothesis that the coevolutionary relationships are the result of selective constraints on amino acid sites that are structurally or functionally linked in the groes-l complex.
shifts of groes-groel coevolutionary relationships during bacterial evolution
we tested whether the coevolutionary relationships among amino acid sites have changed among the different bacterial groups, which would indicate functional changes in groes-l. functional shifts in groel have been previously documented and linked to events of groel gene duplication  <cit>  and to changes in the organismal lifestyle  <cit> .
however, a precise analysis of the sites potentially driving groel functional changes in major bacterial groups has not been conducted before.
we identified evolutionary dependencies between amino acid sites that were specific to a particular bacterial group but not to others.
previous studies have shown that the number of sequences in the alignment may undermine the accuracy of coevolution-detection methods  <cit> .
to avoid such size-dependent effects, we performed bootstrap analyses of the coevolving pairs of sites .
amino acid sites identified as coevolving presented high bootstrap values .
amino acid sites detected in coevolution analyses between groes and groel  were not detected in intra-protein coevolution analyses, and thus, were not the result of indirect evolutionary dependencies.
amino acid sites from groel coevolving with sites from groes were centred in the apical and equatorial domains .
while this was the general pattern when analysing the full alignment, this distribution varied significantly between bacterial clades.
figure  <dig> represents the distribution of coevolving sites in groes and groel for each of the bacterial groups examined in this study.
a brief inspection of the graph allows identifying the sharp differences in the distribution of sites in the different domains of groel.
for example, in firmicutes coevolving sites  concentrated mainly in the apical domain, in good agreement with the distribution of such sites when analysing the entire set of bacteria .
proteobacteria  presented one set of coevolving sites in the apical domain and another in the c-terminal equatorial domain.
finally, in actinobacteria  all but one coevolving site were located in the c-terminal domain of groel.
the distribution of coevolving sites in groel secondary structures and domains also differed among bacterial groups.
figure  <dig> represents the distribution of the expected number and the number of coevolving sites observed in figure  <dig> in the alpha helices, beta-strands and extended strands.
the main differences in the distribution of coevolving sites among bacterial groups reside in the beta-strands.
beta-strands were significantly enriched for sites under coevolution in proteobacteria, non-enriched in other bacterial groups, and significantly impoverished in actinobacteria.
these data are in good agreement with the functional and structural differences in groel found between proteobacteria and actinobacteria  <cit> .
coevolving sites are three-dimensionally proximal in the structure of groes and groel.
for example, his <dig> and asn <dig> from actinobacteria that are strongly proximal in the structure  were coevolving with two sets of amino acids from groel.
one set included tyr <dig>  ala <dig> and cys <dig>  all three being very proximal to one another in the equatorial domain of groel, and another set comprised cys <dig> and his <dig>  which were proximal in the intermediate domain.
to determine the functional meaning of the groupings of coevolving sites in each bacterial clade, we performed two different analyses.
first, we followed a previously published approach to define functional sectors in groel and groes  <cit> .
in this study, sectors are characterized by statistical independence, structural continuity, biochemical independence and divergence independence.
halabi and colleagues  <cit>  showed that statistical protein sectors correspond to functional sectors.
we tested three of the sectors properties using computational means: statistical and divergence independences and structural continuity.
second, we mapped sites identified as coevolving in one bacterial group but not in other into those protein regions known to have shifted groel function to other folding unrelated functions in that bacterial group.
groups of coevolution form protein sectors statistically independent among bacteria
functional links between sites impose correlation in their entropies  <cit> .
to test this, we measured the amount of conservation  for the sites of each groel protein domain as a function of entropy .
then, we calculated the correlation entropy  for each group of coevolving sites .
to determine if the group of coevolving sites within a bacterial clade is independent from that of another bacterial clade, we compared the correlation entropy of groups of different bacterial clades for each of the groel domains.
three were the domains compared  between bacterial groups.
if the change in the sites composition of coevolution networks is the result of functional shifts between bacteria, sites within a network in a bacterial group  should correlate in their entropies  more than with any of the sites of the network of the other bacterial group .
that is, the entropy correlation of one group should be independent of that of the other group .
a main difference between our approach and that of the previous study  <cit>  is that sectors in our approach are defined based on coevolution analyses derived from caps, while those of halabi and colleagues  <cit>  were identified using statistical coupling analyses  to determine the contribution of correlations to conservation profiles.
analyses of correlation entropies showed that all groups of coevolving sites within the apical domain for a bacterial group were independent from those in other bacterial groups   from the real group with a set of  <dig> pseudorandom replicates yield no significant difference between the two groups ).
the same was inferred for the groups of coevolving sites from the intermediate domain of groel.
conversely, in the apical domain we found independent groups of coevolution for all bacterial groups with the exception of spirochaetes, in which ig1-g <dig> was much smaller than  .
comparison of the mean differences  indicates that equatorial domain showed the strongest signal of functional sectors independence among bacterial strains, followed by the intermediate and apical domains .
these differences were not, however, statistically significant under a wilcoxon ranked test.
groups of coevolution present structural continuity
to determine if the sites within a coevolution group were linked structurally within a bacterial clade, we plotted them into the crystal structure of e. coli groesl proteins complex.
figure  <dig> presents evidence of the structural clustering of sites within each of the bacterial groups in the three protein domains.
importantly, the coevolutionary shifts between bacterial groups are apparent and their structural mapping provides insights into the possible functional differences among the groups of coevolving residues.
a remarkable observation is that amino acids that coevolved in one group of bacteria are located in a completely different structure face to those detected in another group of bacteria, while both keeping structural continuity.
as a case in point, the alpha helices populated with coevolving amino acids in proteobacteria are independent from those in actinobacteria.
this rule applies to both, the equatorial and the apical domains .
in addition to the difference in structural patterns, proteobacteria present coevolving amino acids in regions involved in protein folding while actinobacteria are mostly affected in the surfaces of subunits mediating the inter-ring contacts.
this differential distribution supports functional shifts between both bacterial clades, with one having larger effect on folding while the other on the stability of the groel double ring complex.
another striking example of functional and structural differentiation is that of spirochaetes, with most of the coevolving amino acids mapping to the inter-ring regions of the equatorial domain .
coevolution of groel sites with folding-independent functions
groel regions responsible for functional differences among bacteria are reported in figure  <dig> of  <cit> .
we have compared the sites coevolving in one bacterial clade but not another and plotted these sites in the different domains known to confer groel alternative non-folding functions.
many of the sites involved in a coevolutionary relationship in a bacterial group have been reported to be involved in a groel function alternative to protein folding .
for example, two of the coevolving sites in actinobacteria are directly involved in monocyte modulation by the actinobacterium micobacterium tuberculosis .
moreover, a number of the amino acids identified as coevolving exclusively in proteobacteria map to a region from groel previously found to bind to potato leafroll virus and to facilitate its movement in the plant  <cit>  .
the extensive list of coevolving amino acid sites mapping within these folding-alternative functions  is testament to the important implications of groups of coevolution in the functional plasticity of groel.
discussion
complex coevolutionary networks in groesl define the functional boundaries of amino acid sites
our analyses of the coevolutionary dynamics within groes and groel as well as between both these interacting proteins uncover a complex network of evolutionary dependencies among amino acid sites.
these dependencies often involve sets of sites with known functional relevance but also comprise other sites with unknown importance.
however, the functional importance of these untested sites is supported by a number of observations and tests made in this study.
first, we show that most amino acids involved in coevolutionary dynamics are three-dimensionally clustered in the protein structure and closely located to functionally or structurally important sites.
as a case in point, functionally important sites in groes present the largest centrality values in groes coevolutionary network, indicating their greater evolutionary dependencies with other sites closely located in the protein structure.
the coevolution of sites surrounding important functional regions may compensate the effects of mutations at these functional sites or near functional and catalytic pockets, thereby maintaining an overall volume or shape for that pocket  <cit> .
our results on the proximity of coevolving sites to functional domains support previous studies claiming that covarying groups of amino acid sites are often identified at critical protein regions  <cit> .
second, covarying amino acid sites identified in this study are part of networks that correspond to structural clusters, that is, these sites fall close to each other in the protein structure.
in conclusion, the low number of sites identified in our coevolutionary analyses, their structural clustering, and their proximity to functional or proteins interface regions point to their functional or structural importance.
this is supported by previous studies indicating that sites coevolving with few others within the protein are likely to represent functional dependencies  <cit> .
most covarying amino acid sites in groel were identified in the equatorial and apical domains and only few sites were located in the intermediate domain.
apical and equatorial domains perform most functions in groel.
it is remarkable that many of the amino acids from the equatorial domain involved in coevolutionary relationships belong to the most carboxi-terminal groel tail.
indeed, the folding of substrates within the central groel cavity is favoured by the limited size and hydrophobicity of the cavity  <cit> .
the c-terminal tail of groel define the environment within the central cavity of groel with regards to its hydrophobicity, which would impact on both the size and nature of the substrate proteins folded by the chaperonin  <cit> .
collectively, our results uncover a list of amino acid sites that might have profound implications on the functions of groes and groel.
the evolutionary dependencies between groes and groel provide information on the structural consequences of their interaction
our coevolutionary analyses in groes and groel identified several sets of sites with apparently distinct roles.
first, groes amino acid regions coevolving with residues from groel are all located in the interface between the groes subunits.
second, groel residues coevolving with groes are distributed among the three domains, apical, intermediate and equatorial.
in the apical domain, two amino acid residues coevolving with groes are involved in substrate binding.
one site is located at the interface between the two groel heptameric rings and may be involved in the stabilization of these domains.
indeed, the folding reaction cycle requires the double ring of groel, in which the information passes between the rings to signal the atp hydrolysis progress in one ring and which causes important conformational changes in the opposite ring  <cit> .
one such change involves the weakening of groes-groel binding, which ends with the binding of an atp to the opposite ring  <cit> .
the inter-ring amino acid contacts are, therefore, essential for the folding cycle completion and release of groes from the cis ring once atp has been bound to the opposite ring.
arguably, coevolution between the interface of the rings and groes may be the result of the constraints to maintain the structural communication between the two groel rings upon the interaction with groes.
amino acids coevolution underlies the functional plasticity of groes and groel in bacteria
our results bring forward the controversial, although intuitive, suggestion that the function of a protein may change across an evolutionary scale leading to a plastic fitness landscape in which constraints on amino acids can vary dramatically.
against the static view of one protein one function, we propose that proteins have the potential to perform many alternative functions.
leaping from one function to another requires the correlated evolution of key amino acids in the protein.
groel, and its co-chaperonin groes, offer a unique system to test this hypothesis because, despite its essentiality to the cell, this protein has evolved many alternative functions in other bacteria  <cit> .
the performance of alternative functions is dependent on the fixation of mutations in genes.
since amino acids are constrained by their interactions with other amino acids, fixation of mutations at sites with functional relevance must be accompanied by mutations in other sites of the protein through molecular coadaptation dynamics—that is, amino acids that are structurally or functionally linked exercise reciprocal natural selection on one another  <cit> .
the groups of amino acids identified in the intra-protein and inter-protein coevolution analyses differed between bacterial groups, in good agreement with the apparent difference in functions of groel in these bacteria.
groups of coevolving amino acids in one domain of a bacterial group showed statistical and structural independence of that in the same domain from another bacterial group.
many of the coevolution groups found in one bacterial group map to regions of groel that are known to encode functions alternative to protein folding.
other coevolving amino acids could not be directly mapped to domains with known alternative functions, though their structural proximity to these domains hints potential roles for these sites.
remarkably, the set of amino acid sites involved in an evolutionary dependency in one bacterial group was close in the protein structure to the set of amino acids detected for another bacterial group.
in fact, in some cases, the same amino acid was detected as coevolving with different sets of amino acids in two bacterial groups, thereby acting as evolutionary hinges of alternative functional protein sectors.
for example, in the intra-groel coevolution analysis, met <dig> was detected in actinobacteria and bacteroidetes, but it was coevolving with different amino acids in these two groups.
the general trend was that alternative sets of coevolving sites identified in different bacteria were closely located in the structure.
this supports the plausible hypothesis that shifts in the selective constraints on amino acid sites of groel are subtle between bacteria, and affect the same structural regions; probably those regions undergoing conformational changes when groel interacts with groes.
to conclude, we provide evidence of the plasticity of the evolutionary relationships between the amino acid sites in an essential protein.
we also list a set of coevolving sites that might be worth testing for addressing important questions regarding the functional promiscuity of groel and its evolvability under different conditions.
experimental studies aimed at determining the importance of the amino acid sites listed in this study may aid the development of mechanistic models of protein folding in the cell and the evolution of alternative functions from highly conserved ones.
CONCLUSIONS
our results map genetic diversity in groesl to its functional promiscuity.
while different functional sectors in groesl can be assigned to distinct functions, the overlap in the amino acids sets of these sectors put forward the conclusion that functional leaps in proteins can be driven by subtle sequence compositional differences.
our results highlight the evolutionary plasticity of groel across the entire bacterial phylogeny.
evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins.

 heat-shock proteins, also known as molecular chaperones, belong to a highly conserved set of protein families that perform essential functions to the cell in prokaryotes and eukaryotes. these functions include, but are not limited to, protein folding, assembly, and transport. while the folding function of groel has been extensively characterized, emerging literature uncover many alternative functions and structures for this protein. mutations in this molecule that are responsible for the emergence of alternative functions remain uncharacterized. therefore, the potential evolvability of this essential protein is largely unexplored. groes and groel, also known as cpn and cpn respectively, are expressed at constitutive levels under physiological conditions and their expression increases at high temperatures, allowing the growth and survival of bacteria at a broad range of temperatures. both chaperonins are encoded by the operon groe and they form a homotetradecamer organized into two back-to-back oriented rings. each of the rings comprises seven identical groel subunits, with each subunit being divided into three domains: the apical, which binds unfolded proteins and groes, the intermediate, which acts as a hinge allowing the movement of the apical domain as well as the transition between trans and cis conformations needed for groel function, and the equatorial which is responsible for the atpase and the folding activities that take place in the central cavity of the ringed complex. the main function of groel has been considered to be the folding of other proteins in the cell, although evidence supports other folding-unrelated roles for groel, such as immune response in humans or growth and biofilm formation in bacteria, among others. these functions are context dependent and may vary from one organism to another. alternative functions may emerge in proteins after the duplication and evolution of their encoding gene or through amino acid replacements that impinge on the protein structure. the gene groel has undergone many duplications in bacteria, adaptive evolution and functional divergence. moreover, structural evolutionary changes have been recently described for groel, according to which changes in the amino acid composition of its co-chaperonin groes can determine groel functioning as a single instead of double ring. the strong evolutionary sequence conservation of groel and the high number of interactions it establishes with other proteins in the cell contrast with groel´s functional and structural plasticity and its propensity to persist in duplicate in some bacteria. particularly striking is the fact that, while performing essential functions in the cell, groel presents alternative functions. the trade-off between groel´s high conservation at the sequence and functional levels and its high propensity to evolve novel functions remains poorly understood. researchers have attempted to uncover groel’s multi-functionality through the testing of the effects of directed mutagenesis of groel amino acids under laboratory-controlled conditions. however, the multifunctional nature of groel suggests the existence of a reservoir of functionalities resulting from the interaction between distinct sets of amino acids in different bacteria. here we propose the hypothesis that the functional plasticity of groel is mediated by an evolutionary plasticity of potentially functional amino acids. in support of this hypothesis, bacteria growing under different physiological conditions present groel variants with functions alternative to folding and which involve different sets of amino acids. the strong selective constraints acting on groel imply important functional and structural links between amino acids. these links impose reciprocal selection pressures among amino acid sites. therefore, changes on groel functions from one bacterial group to another should be reflected in strong coevolutionary signatures between linked amino acids whose evolvability is co-regulated by selection in a particular bacterial clade. in this study we performed an exhaustive coevolutionary analysis using an extensive bacterial phylogeny to uncover the evolutionary, hence functional, dependencies among amino acid residues within groes, groel and between both these proteins. the coevolutionary networks identified in these chaperonins from hundreds of bacteria reveal the complexity underlying the evolution of this essential protein and shed light on the functional importance of previously uncharacterized residues.  sequence data and coevolution analyses to perform intra-protein coevolution analyses in groes and groel, we searched groe sequences amongst the major bacterial phyla and found that actinobacteria, cyanobacteria, bacteroidetes and chlorobi, firmicutes, proteobacteria, and spirochaetes comprised a number of groe homologs that would allow accurate inference of coevolution. the number of sequences ranged between and for groes genes, and and for groel genes belonging to spirochaetes and proteobacteria groups, respectively. in spite of the differences in the number of sequences, the mean amino acid sequence divergence was of the same order in all bacteria groups ranging between and, and these divergence levels were not correlated with the number of sequences in the alignment. these divergence levels are also within the levels ensuring robust results when using coevolution analyses. inter-protein coevolution analyses between groes and groel were performed building pairs of files for each group of bacteria, both of which included the same bacterial strains. accordingly, the size of the alignments used for the groes-l inter-coevolution analyses ranged between in cyanobacteria and in proteobacteria. all coevolution analyses were performed with a phylogenetic tree built up function in caps and pairs of coevolving sites were further filtered through a novel bootstrap analysis. therefore, the number of sequences in the alignment, level of sequence divergence and new introduced filters warranted minimizing false positives rate and increasing accuracy of our results. for the individual intra-group analyses we chose those bacterial groups with more than sequences. for the overall cpn and cpn intra-group analyses we took all sequences. evolutionary dependencies between functional sites within groes and groel to determine the magnitude of the evolutionary plasticity of groel and groes, we first conducted a coevolutionary analysis to determine the network of residues dependencies in all bacteria. we performed intra-protein coevolution analyses in a sequences based groes alignment and sequences based groel alignment, representing the major bacterial groups. we also calculated the support of each pair of coevolutionary sites taking into account the phylogenetic relationships using a non-parametric bootstrap approach. all amino acid sites numbering and composition are referred throughout the text to the numbering in the crystal structure of groesl from e. coli. we identified a single connected network of coevolving amino acid sites in groes, with lys leu gly thr arg glu arg and lys establishing most of the evolutionary dependencies. to determine the importance of each of the amino acid sites in the network we applied network centrality measures to coevolving sites, typically used in networks biology: degree centrality, betweenness and closeness. networks are a collection of points joined together in pairs by lines. in the networks jargon, points are referred to as vertices or nodes while the links are referred to as edges. centrality measures of nodes, including degree, betweenness and closeness, are typically used to determine the importance of these nodes in the network. degree is the number of edges departing from a node in the network. a node presents high closeness when its shortest distances to all other nodes in the network are low compared to the average closeness. a node has high betweenness when the number of shortest paths between all pairs of nodes in a network that pass through it is high. interestingly, leu and gly two amino acids known to be involved in the interaction between groes and groel are the most central in the coevolution network. the dependency of these two essential amino acids on other functionally uncharacterized ones hints possible functional links between both sets of amino acid sites. indeed, lys thr arg gly arg and lys while lacking apparent functions, they form a structural cluster establishing important contacts among groes subunits. amino acid sites within each of the structural clusters were in close proximity to each other. coevolution among structurally proximal amino acid sites is a general pattern and suggests compensatory relationships, hence functional or structural links, between amino acids. in groel, we identified coevolving amino acid residues, of which leu ala ser arg lys gln arg ala and asn were the most central residues to the network. arg val and lys are involved or close to sites mediating substrate and groes binding. other positions were either included or close to charged amino acid sites that were facing the central groel cavity. finally, asn is located in the atp and mg2+ binding site, while other amino acid sites, such as ala and ala are at the rings interface and likely involved in protein folding within the groes-l ring complex. all amino acids are distributed into two structural groups: one in the apical and another in the equatorial domains. remarkably, coevolving sites are very close to sites involved in protein folding, substrate and groes binding, atp binding and hydrolysis, or inter-subunits contacts, thus, suggesting that changes at these amino acids may have important functional consequences. coevolution of groes with groel the interaction of groes and groel is essential to induce the conformational changes needed for the folding cycle. these conformational changes may force coadaptation dynamics between groes and groel. we performed coevolutionary analyses using the protein sequences of groes and groel from the same set of bacterial strains. these sequences span all the different bacterial groups, with all these groups being well represented. analysis of coevolution identified a group of amino acids from groes coevolving with groel. the centrality measures of coevolving sites were also calculated. coevolution did not affect groes sites involved in the groes-l interaction. nonetheless, sites coevolving between both proteins had important functional roles and mapped to different functional domains of groel. for example, two of the groel sites, ala and arg are involved in the binding of substrates and overlap with sites involved in groes binding as well. in addition, glu involved in the coevolution between ala and arg has a role in stabilizing inter-ring contacts. since groes is heavily involved in determining the function of groel as a single or as a double ring, the coevolution of glu from groel with groes amino acid sites may have implications in the structural stability of the double ring, and thus, groes-groel folding cycle. in support of the structural and functional communication between the coevolving sites of groes and groel, coevolving amino acids formed structural clusters within groesl. in addition to their clustering, coevolving sites were either functionally relevant or were close to sites with reported functional importance. taken together, these results support the hypothesis that the coevolutionary relationships are the result of selective constraints on amino acid sites that are structurally or functionally linked in the groes-l complex. shifts of groes-groel coevolutionary relationships during bacterial evolution we tested whether the coevolutionary relationships among amino acid sites have changed among the different bacterial groups, which would indicate functional changes in groes-l. functional shifts in groel have been previously documented and linked to events of groel gene duplication and to changes in the organismal lifestyle. however, a precise analysis of the sites potentially driving groel functional changes in major bacterial groups has not been conducted before. we identified evolutionary dependencies between amino acid sites that were specific to a particular bacterial group but not to others. previous studies have shown that the number of sequences in the alignment may undermine the accuracy of coevolution-detection methods. to avoid such size-dependent effects, we performed bootstrap analyses of the coevolving pairs of sites. amino acid sites identified as coevolving presented high bootstrap values. amino acid sites detected in coevolution analyses between groes and groel were not detected in intra-protein coevolution analyses, and thus, were not the result of indirect evolutionary dependencies. amino acid sites from groel coevolving with sites from groes were centred in the apical and equatorial domains. while this was the general pattern when analysing the full alignment, this distribution varied significantly between bacterial clades. figure  represents the distribution of coevolving sites in groes and groel for each of the bacterial groups examined in this study. a brief inspection of the graph allows identifying the sharp differences in the distribution of sites in the different domains of groel. for example, in firmicutes coevolving sites concentrated mainly in the apical domain, in good agreement with the distribution of such sites when analysing the entire set of bacteria. proteobacteria presented one set of coevolving sites in the apical domain and another in the c-terminal equatorial domain. finally, in actinobacteria all but one coevolving site were located in the c-terminal domain of groel. the distribution of coevolving sites in groel secondary structures and domains also differed among bacterial groups. figure  represents the distribution of the expected number and the number of coevolving sites observed in figure  in the alpha helices, beta-strands and extended strands. the main differences in the distribution of coevolving sites among bacterial groups reside in the beta-strands. beta-strands were significantly enriched for sites under coevolution in proteobacteria, non-enriched in other bacterial groups, and significantly impoverished in actinobacteria. these data are in good agreement with the functional and structural differences in groel found between proteobacteria and actinobacteria. coevolving sites are three-dimensionally proximal in the structure of groes and groel. for example, his and asn from actinobacteria that are strongly proximal in the structure were coevolving with two sets of amino acids from groel. one set included tyr ala and cys all three being very proximal to one another in the equatorial domain of groel, and another set comprised cys and his which were proximal in the intermediate domain. to determine the functional meaning of the groupings of coevolving sites in each bacterial clade, we performed two different analyses. first, we followed a previously published approach to define functional sectors in groel and groes. in this study, sectors are characterized by statistical independence, structural continuity, biochemical independence and divergence independence. halabi and colleagues showed that statistical protein sectors correspond to functional sectors. we tested three of the sectors properties using computational means: statistical and divergence independences and structural continuity. second, we mapped sites identified as coevolving in one bacterial group but not in other into those protein regions known to have shifted groel function to other folding unrelated functions in that bacterial group. groups of coevolution form protein sectors statistically independent among bacteria functional links between sites impose correlation in their entropies. to test this, we measured the amount of conservation for the sites of each groel protein domain as a function of entropy. then, we calculated the correlation entropy for each group of coevolving sites. to determine if the group of coevolving sites within a bacterial clade is independent from that of another bacterial clade, we compared the correlation entropy of groups of different bacterial clades for each of the groel domains. three were the domains compared between bacterial groups. if the change in the sites composition of coevolution networks is the result of functional shifts between bacteria, sites within a network in a bacterial group should correlate in their entropies more than with any of the sites of the network of the other bacterial group. that is, the entropy correlation of one group should be independent of that of the other group. a main difference between our approach and that of the previous study is that sectors in our approach are defined based on coevolution analyses derived from caps, while those of halabi and colleagues were identified using statistical coupling analyses to determine the contribution of correlations to conservation profiles. analyses of correlation entropies showed that all groups of coevolving sites within the apical domain for a bacterial group were independent from those in other bacterial groups from the real group with a set of pseudorandom replicates yield no significant difference between the two groups ). the same was inferred for the groups of coevolving sites from the intermediate domain of groel. conversely, in the apical domain we found independent groups of coevolution for all bacterial groups with the exception of spirochaetes, in which ig1-g was much smaller than. comparison of the mean differences indicates that equatorial domain showed the strongest signal of functional sectors independence among bacterial strains, followed by the intermediate and apical domains. these differences were not, however, statistically significant under a wilcoxon ranked test. groups of coevolution present structural continuity to determine if the sites within a coevolution group were linked structurally within a bacterial clade, we plotted them into the crystal structure of e. coli groesl proteins complex. figure  presents evidence of the structural clustering of sites within each of the bacterial groups in the three protein domains. importantly, the coevolutionary shifts between bacterial groups are apparent and their structural mapping provides insights into the possible functional differences among the groups of coevolving residues. a remarkable observation is that amino acids that coevolved in one group of bacteria are located in a completely different structure face to those detected in another group of bacteria, while both keeping structural continuity. as a case in point, the alpha helices populated with coevolving amino acids in proteobacteria are independent from those in actinobacteria. this rule applies to both, the equatorial and the apical domains. in addition to the difference in structural patterns, proteobacteria present coevolving amino acids in regions involved in protein folding while actinobacteria are mostly affected in the surfaces of subunits mediating the inter-ring contacts. this differential distribution supports functional shifts between both bacterial clades, with one having larger effect on folding while the other on the stability of the groel double ring complex. another striking example of functional and structural differentiation is that of spirochaetes, with most of the coevolving amino acids mapping to the inter-ring regions of the equatorial domain. coevolution of groel sites with folding-independent functions groel regions responsible for functional differences among bacteria are reported in figure  of. we have compared the sites coevolving in one bacterial clade but not another and plotted these sites in the different domains known to confer groel alternative non-folding functions. many of the sites involved in a coevolutionary relationship in a bacterial group have been reported to be involved in a groel function alternative to protein folding. for example, two of the coevolving sites in actinobacteria are directly involved in monocyte modulation by the actinobacterium micobacterium tuberculosis. moreover, a number of the amino acids identified as coevolving exclusively in proteobacteria map to a region from groel previously found to bind to potato leafroll virus and to facilitate its movement in the plant. the extensive list of coevolving amino acid sites mapping within these folding-alternative functions is testament to the important implications of groups of coevolution in the functional plasticity of groel. discussion complex coevolutionary networks in groesl define the functional boundaries of amino acid sites our analyses of the coevolutionary dynamics within groes and groel as well as between both these interacting proteins uncover a complex network of evolutionary dependencies among amino acid sites. these dependencies often involve sets of sites with known functional relevance but also comprise other sites with unknown importance. however, the functional importance of these untested sites is supported by a number of observations and tests made in this study. first, we show that most amino acids involved in coevolutionary dynamics are three-dimensionally clustered in the protein structure and closely located to functionally or structurally important sites. as a case in point, functionally important sites in groes present the largest centrality values in groes coevolutionary network, indicating their greater evolutionary dependencies with other sites closely located in the protein structure. the coevolution of sites surrounding important functional regions may compensate the effects of mutations at these functional sites or near functional and catalytic pockets, thereby maintaining an overall volume or shape for that pocket. our results on the proximity of coevolving sites to functional domains support previous studies claiming that covarying groups of amino acid sites are often identified at critical protein regions. second, covarying amino acid sites identified in this study are part of networks that correspond to structural clusters, that is, these sites fall close to each other in the protein structure. in conclusion, the low number of sites identified in our coevolutionary analyses, their structural clustering, and their proximity to functional or proteins interface regions point to their functional or structural importance. this is supported by previous studies indicating that sites coevolving with few others within the protein are likely to represent functional dependencies. most covarying amino acid sites in groel were identified in the equatorial and apical domains and only few sites were located in the intermediate domain. apical and equatorial domains perform most functions in groel. it is remarkable that many of the amino acids from the equatorial domain involved in coevolutionary relationships belong to the most carboxi-terminal groel tail. indeed, the folding of substrates within the central groel cavity is favoured by the limited size and hydrophobicity of the cavity. the c-terminal tail of groel define the environment within the central cavity of groel with regards to its hydrophobicity, which would impact on both the size and nature of the substrate proteins folded by the chaperonin. collectively, our results uncover a list of amino acid sites that might have profound implications on the functions of groes and groel. the evolutionary dependencies between groes and groel provide information on the structural consequences of their interaction our coevolutionary analyses in groes and groel identified several sets of sites with apparently distinct roles. first, groes amino acid regions coevolving with residues from groel are all located in the interface between the groes subunits. second, groel residues coevolving with groes are distributed among the three domains, apical, intermediate and equatorial. in the apical domain, two amino acid residues coevolving with groes are involved in substrate binding. one site is located at the interface between the two groel heptameric rings and may be involved in the stabilization of these domains. indeed, the folding reaction cycle requires the double ring of groel, in which the information passes between the rings to signal the atp hydrolysis progress in one ring and which causes important conformational changes in the opposite ring. one such change involves the weakening of groes-groel binding, which ends with the binding of an atp to the opposite ring. the inter-ring amino acid contacts are, therefore, essential for the folding cycle completion and release of groes from the cis ring once atp has been bound to the opposite ring. arguably, coevolution between the interface of the rings and groes may be the result of the constraints to maintain the structural communication between the two groel rings upon the interaction with groes. amino acids coevolution underlies the functional plasticity of groes and groel in bacteria our results bring forward the controversial, although intuitive, suggestion that the function of a protein may change across an evolutionary scale leading to a plastic fitness landscape in which constraints on amino acids can vary dramatically. against the static view of one protein one function, we propose that proteins have the potential to perform many alternative functions. leaping from one function to another requires the correlated evolution of key amino acids in the protein. groel, and its co-chaperonin groes, offer a unique system to test this hypothesis because, despite its essentiality to the cell, this protein has evolved many alternative functions in other bacteria. the performance of alternative functions is dependent on the fixation of mutations in genes. since amino acids are constrained by their interactions with other amino acids, fixation of mutations at sites with functional relevance must be accompanied by mutations in other sites of the protein through molecular coadaptation dynamics—that is, amino acids that are structurally or functionally linked exercise reciprocal natural selection on one another. the groups of amino acids identified in the intra-protein and inter-protein coevolution analyses differed between bacterial groups, in good agreement with the apparent difference in functions of groel in these bacteria. groups of coevolving amino acids in one domain of a bacterial group showed statistical and structural independence of that in the same domain from another bacterial group. many of the coevolution groups found in one bacterial group map to regions of groel that are known to encode functions alternative to protein folding. other coevolving amino acids could not be directly mapped to domains with known alternative functions, though their structural proximity to these domains hints potential roles for these sites. remarkably, the set of amino acid sites involved in an evolutionary dependency in one bacterial group was close in the protein structure to the set of amino acids detected for another bacterial group. in fact, in some cases, the same amino acid was detected as coevolving with different sets of amino acids in two bacterial groups, thereby acting as evolutionary hinges of alternative functional protein sectors. for example, in the intra-groel coevolution analysis, met was detected in actinobacteria and bacteroidetes, but it was coevolving with different amino acids in these two groups. the general trend was that alternative sets of coevolving sites identified in different bacteria were closely located in the structure. this supports the plausible hypothesis that shifts in the selective constraints on amino acid sites of groel are subtle between bacteria, and affect the same structural regions; probably those regions undergoing conformational changes when groel interacts with groes. to conclude, we provide evidence of the plasticity of the evolutionary relationships between the amino acid sites in an essential protein. we also list a set of coevolving sites that might be worth testing for addressing important questions regarding the functional promiscuity of groel and its evolvability under different conditions. experimental studies aimed at determining the importance of the amino acid sites listed in this study may aid the development of mechanistic models of protein folding in the cell and the evolution of alternative functions from highly conserved ones.  our results map genetic diversity in groesl to its functional promiscuity. while different functional sectors in groesl can be assigned to distinct functions, the overlap in the amino acids sets of these sectors put forward the conclusion that functional leaps in proteins can be driven by subtle sequence compositional differences. our results highlight the evolutionary plasticity of groel across the entire bacterial phylogeny. evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins. 
describing this plasticity can shed light on the functional diversity of groel. evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins. our results highlight the evolutionary plasticity of groel across the entire bacterial phylogeny. we conducted the first coevolution analyses in an extensive bacterial phylogeny, revealing complex networks of evolutionary dependencies between residues in groesl. functions alternative to folding have been previously described for groel in different bacterial groups, supporting enormous functional and structural plasticity for this molecule and the existence of a hidden combinatorial code in the protein sequence enabling such functions. these networks differed among bacterial groups and involved amino acid sites with functional importance and others with previously unsuspected functional potential. coevolutionary networks formed statistically independent units among bacterial groups and map to structurally continuous regions in the protein, suggesting their functional link. the folding activity proceeds through strong conformational changes mediated by the co-chaperonin groes and atp. shifts in these coevolutionary relationships may inevitably lead to evolution of alternative functions. moreover, coevolving sites within a bacterial group mapped to regions previously identified as involved in folding-unrelated functions, and thus, coevolution may mediate alternative functions. sites involved in coevolution fell within narrow structural regions, supporting dynamic combinatorial functional links involving similar protein domains.
 groesl is a heat-shock protein ubiquitous in bacteria and eukaryotic organelles. this evolutionarily conserved protein is involved in the folding of a wide variety of other proteins in the cytosol, being essential to the cell. the folding activity proceeds through strong conformational changes mediated by the co-chaperonin groes and atp. functions alternative to folding have been previously described for groel in different bacterial groups, supporting enormous functional and structural plasticity for this molecule and the existence of a hidden combinatorial code in the protein sequence enabling such functions. describing this plasticity can shed light on the functional diversity of groel. we hypothesize that different overlapping sets of amino acids coevolve within groel, groes and between both these proteins. shifts in these coevolutionary relationships may inevitably lead to evolution of alternative functions.  we conducted the first coevolution analyses in an extensive bacterial phylogeny, revealing complex networks of evolutionary dependencies between residues in groesl. these networks differed among bacterial groups and involved amino acid sites with functional importance and others with previously unsuspected functional potential. coevolutionary networks formed statistically independent units among bacterial groups and map to structurally continuous regions in the protein, suggesting their functional link. sites involved in coevolution fell within narrow structural regions, supporting dynamic combinatorial functional links involving similar protein domains. moreover, coevolving sites within a bacterial group mapped to regions previously identified as involved in folding-unrelated functions, and thus, coevolution may mediate alternative functions.  our results highlight the evolutionary plasticity of groel across the entire bacterial phylogeny. evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins. 