Coevolution analyses illuminate the dependencies between amino acid sites in the chaperonin system GroES-L

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Coevolution analyses within GroES and GroEL. The network of coevolving amino acid sites within GroES is shown using the three-letter amino acid code (a) Sites coevolving within GroES were divided into two main structure clusters (b) One cluster includes two amino acid sites (blue spheres), which are involved in the interaction with GroEL. The second cluster includes residues (yellow spheres) mapping to the inter-GroES subunit faces. The network of coevolution in GroEL (c) identifies amino acid sites which are involved in the interaction with GroES and protein substrates (blue spheres in the structure of GroEL: d) sites involved in the inter-subunit GroEL contacts and and substrate folding in the ring cavity (red spheres), residues with a role in ATP hydrolysis (green sphere) and those mapping to the inter-ring interfaces (black spheres).

Figure 2

Coevolution between GroES and GroEL. The network of residues involved in the evolutionary dependency between GroES and GroEL identifies 7 residues from GroES and 8 from GroEL (a) Structural mapping of coevolving residues reveals the functional importance of coevolving residues (b) residues coevolving between both proteins belong to substrate binding regions, inter-subunit and inter-ring contacts.

Figure 3

Identifying shifts in coevolutionary links between amino acids in the different bacterial clades. We have analysed the coevolution of GroES and GroEL in the different bacterial clades (colour coded circles) and compared involved residues with those identified across the entire bacterial phylogeny (stars). The distribution of the coevolving residues along GroEL is shown in the X-axis, while this distribution in GroES is shown in the Y-axis. The continuous bar at the very bottom of the figure represents the three different major domains of GroEL (Apical: blue, Intermediate: yellow and Equatorial: red). On top of the continuous bar we have also identified regions reported to be involved in folding-independet functions. These regions are color-coded as in [10]: 1, 3 and 11, orange: binding to mouse adipocytes; 2 and 12, binding to potato leafroll virus; 4, insecticidal neurotoxin; 5, Monocytes and T-cell activators; 6, Binding to primary mouse macrophages; 7 and 9, binding to lipopolysaccharides; 8, insecticidal toxin; 10 and 13, binding to cell surface of J774A.1 cells; 14, monocytes modulation activity.

Figure 4

Distribution of coevolving sites amongst secondary structures in GroEL. The observed number of sites within each structure (colour coded bars according to the bacterial group) was compared to the expected number of such sites using a χ² distribution. Significant values (P < 0.05) are indicated with black stars.

Figure 5

Groups of coevolving sites correlate in their entropies forming independent protein sectors. We measured entropy and correlation entropies for each pair of groups belonging to different bacteria using the equations 1 to 4 from the text. Compared groups were taken from the same protein domain (Apical, Intermediate or Equatorial). Bacteria groups compared included Actinobacteria (a) Bacteroidetes (b) Cyanobacteria (c) Spirochaetes (d) Firmicutes (e) and Proteobacteria (f). Two groups of coevolution (g1 and g2) were considered independent when the joined correlation entropy for the groups (I_S(g1,g2)) was approximately equal to the sum of correlation entropies (I_S(g1)) and (I_S(g2)). The significance of the difference between these two parameters [Θ = I_S(g1,g2) – (I_S(g1) + I_S(g2))] was tested against a null distribution of Θ drawn from a 1000 groups built by randomly sampling sites from the same protein domain. Significant Θ values under a normal test (P < 0.05) are indicated with *.

Figure 6

Distribution of groups of coevolving sites within the three domains of crystal structure of GroEL (1AON.pdb). We compared these distributions using a dimer GroEL₂-GroES₂. The groups of bacteria represented are Actinobacteria (a) Bacteroidetes (b) Cyanobacteria (c) Spirochaetes (d) Firmicutes (e) and Proteobacteria (f). Sites under coevolution are highlighted as solid spheres, with those belonging to the same group colour-coded. Sites falling within the apical, intermediate and equatorial domains are coded with the colours blue, yellow and red, respectively.