Comparative genomics uncovers novel structural and functional features of the heterotrimeric GTPase signaling system

Abstract

Though the heterotrimeric G-proteins signaling system is one of the best studied in eukaryotes, its provenance and its prevalence outside of model eukaryotes remains poorly understood. We utilized the wealth of sequence data from recently sequenced eukaryotic genomes to uncover robust G-protein signaling systems in several poorly studied eukaryotic lineages such as the parabasalids, heteroloboseans and stramenopiles. This indicated that the Gα subunit is likely to have separated from the ARF-like GTPases prior to the last eukaryotic common ancestor. We systematically identified the structure and sequence features associated with this divergence and found that most of the neomorphic positions in Gα form a ring of residues centered on the nucleotide binding site, several of which are likely to be critical for interactions with the RGS domain for its GAP function. We also present evidence that in some of the potentially early branching eukaryotic lineages, like Trichomonas, Gα is likely to function independently of the Gβγ subunits. We were able to identify previously unknown Gγ subunits in Naegleria, suggesting that the trimeric version was already present by the time of the divergence of the heteroloboseans from the remaining eukaryotes. Evolution of Gα subunits is dominated by several independent lineage-specific expansions (LSEs). In most of these cases there are concomitant, independent LSEs of RGS proteins along with an extraordinary diversification of their domain architectures. The diversity of RGS domains from Naegleria in particular, which has the largest complement of Gα and RGS proteins for any eukaryote, provides new insights into RGS function and evolution. We uncovered a new class of soluble ligand receptors of bacterial origin with RGS domains and an extraordinary diversity of membrane-linked, redox-associated, adhesion-dependent and small molecule-induced G-protein signaling networks that evolved in early-branching eukaryotes, independently of parallel systems in animals. Furthermore, this newly characterized diversity of RGS domains helps in defining their ancestral conserved interfaces with Gα and also those interfaces that are prone to extensive lineage-specific diversification and are thereby responsible for selectivity in Gα-RGS interactions. Several mushrooms show LSEs of Gαs but not of RGS proteins pointing to the probable differentiation of Gαs in conjunction with mating-type diversity. When combined with the characterization of the 7TM receptors (GPCRs), it becomes apparent that, through much of eukaryotic evolution, cells contained both 7TM receptors that acted as GEFs and those as GAPs (with C-terminal RGS domains) for Gαs. Only in some lineages like animals and stramenopiles the 7TM receptors were restricted to GEF only roles, probably due to selection imposed by the rate-constants of the Gαs that underwent lineage-specific expansion in them. In the alveolate lineage the 7TM receptors occur independently of heterotrimeric G-proteins, suggesting the prevalence of G-protein-independent signaling in these organisms.

Figure 1

A) A cartoon representation of the heterotrimeric G-protein (PDB: 1got). Gα is colored in orange, whereas the α-helical insert is shown in grey color; Gβ is colored in green and Gγ in purple. The substrate GDP is shown as spheres. Interacting residues are colored as follows: Red: residues in Gα (N-term helix) interacting with Gβ; Blue: residues in Gβ interacting with Gα (N-term helix);Magenta: residues in Gα interacting with Gβ; Green: residues in Gβ interacting with Gα; Purple: residues in Gγ interacting with Gβ; Teal: residues in Gβ interacting with Gγ. B) Gα with RGS. A cartoon representation of the Gi-RGS4 complex (pdb: 1agr) is shown. Gα is colored in orange, with the insert shown in grey color and RGS is shown in blue color. Residues in RGS interacting with Gα are shown in red color. Interacting residues in Gα are shown in green and cyan colors. P-loop is marked by a box. N-terminal helix of Gα is only shown partially. C) A topological representation of Gα is shown. The active site motifs are labeled in yellow. The 45 conserved positions that are unique to Gα when compared with the ARFs are labeled in blue ovals. The numberings correspond to the “idealized” Gα (hybrid of transducin and Gi) represented by pdb: 1got.

Figure 2

The sequence logos were made using multiple sequence alignments of respective domains (Crooks et al., 2004). Consecutive gap-rich positions in the alignment are reduced to a single gap marked by grey numbers in the logo. Secondary structures are represented at the bottom of the logos. Various conserved motifs are marked with boxes below the alignment position numbers. A) Gα vs Arf: The red diamonds are conserved positions that are unique to Gα when compared with the ARFs. These are the same positions that are marked in Fig. 1. The blue diamonds indicate positions which are synapomorphies of Gαs and ARFs. The black box marks the alpha-helical insert in Gα and the red boxes mark the Switch III insert and the bi-helical insert after S5 in Gα. T is the “sensor threonine” and the G2 motif is centered on this residue. B) Gβ: Conserved residues that interact with Gα are shown with a red star on top of the sequence logo. Blue stars on top of the logo mark the conserved residues that interact with Gγ. The propellers are shown using discretely colored strands, with the first strand colored same as the last propeller to show circular permutation. C) Gγ: Conserved residues that interact with Gβ are shown with a red star on top of the sequence logo. D) RGS: Conserved residues that interact with Gα are shown with a red circle on top of the sequence logo.

Figure 3

The eukaryotic phylogenetic tree is shown with the absolute protein counts for each organism. The expansions are shown in pink and absences are shown in grey. The 7TM counts are only the lower bounds because certain families could have eluded detection due to their great lineage specific diversity. The following organisms were analyzed for this study: Afum: Aspergillus fumigatus; Anid: Aspergillus nidulans; Cgla: Candida glabrata; Calb: Candida albicans; Cglo: Chaetomium globosum; Dhan: Debaryomyces hansenii; Egos: Ashbya gossypii; Gzea: Gibberella zeae; Klac: Kluyveromyces lactis; Mgri: Magnaporthe oryzae; Ncra: Neurospora crassa; Psti: Scheffersomyces stipitis; Pnod: Phaeosphaeria nodorum; Scer: Saccharomyces cerevisiae; Spom: Schizosaccharomyces pombe; Ylip: Yarrowia lipolytica; Cneo: Cryptococcus neoformans; Ccin: Coprinopsis cinerea; Ppla: Postia placenta; Umay: Ustilago maydis; Lbic: Laccaria bicolor; Bden: Batrachochytrium dendrobatidis; Ecun: Encephalitozoon cuniculi; Ebie: Enterocytozoon bieneusi; Pbla: Phycomyces blakesleeanus; Amel: Apis mellifera; Dmel: Drosophila melanogaster; Apis: Acyrthosiphon pisum; Dpul: Daphnia pulex; Tcas: Tribolium castaneum; Nvit: Nasonia vitripennis; Agam: Anopheles gambiae; Hmag: Hydra magnipapillata; Nvec: Nematostella vectensis; Cele: Caenorhabditis elegans; Caps: Capitella sp; Hrob: Helobdella robusta; Drer: Danio rerio; Tnig: Tetraodon nigroviridis; Hsap: Homo sapiens; Mmus: Mus musculus; Lgig: Lottia gigantea; Bflo: Branchiostoma floridae; Cint: Ciona intestinalis; Spur: Strongylocentrotus purpuratus; Bmal: Brugia malayi; Tadh: Trichoplax adhaerens; Sman: Schistosoma mansoni; Mbre: Monosiga brevicollis; Ehis: Entamoeba histolytica; Edis: Entamoeba dispar; Einv: Entamoeba invadens; Ddis: Dictyostelium discoideum; Ppal: Polysphondylium pallidum; Dpur: Dictyostelium purpureum; Crei: Chlamydomonas reinhardtii; Mpus: Micromonas pusilla; Micsp: Micromonas sp; Chlor: Chlorella NC64A; Vcar: Volvox carteri; Otau: Ostreococcus tauri; Atha: Arabidopsis thaliana; Ppat: Physcomitrella patens; Cmer: Cyanidioschyzon merolae; Smoe: Selaginella moellendorffii; Ptri: Phaeodactylum tricornutum; Psoj: Phytophthora sojae; Pram: Phytophthora ramorum; Tpse: Thalassiosira pseudonana; Aano: Aureococcus anophagefferens; Tthe: Tetrahymena thermophila; Ptet: Paramecium tetraurelia; Tgon: Toxoplasma gondii; Tpar: Theileria parva; Tann: Theileria annulata; Cpar: Cryptosporidium parvum; Cmur: Cryptosporidium muris; Pfal: Plasmodium falciparum; Bbov: Babesia bovis; Pmar: Perkinsus marinus; Tcru: Trypanosoma cruzi; Tbru: Trypanosoma brucei; Lmaj: Leishmania major; Linf: Leishmania infantum; Ngru: Naegleria gruberi; Glam: Giardia lamblia; Tvag: Trichomonas vaginalis; Gthe: Guillardia theta; Ehux: Emiliania huxleyi.

Figure 4

A) Domain network graph of RGS-containing proteins. The network is an ordered graph representing the connection between RGS and the domains it is fused to in various polypeptides. The direction of the arrow denotes the relative positions of the domain in the polypeptide from the N-terminus to the C-terminus. The networks are shown for the different clades. B) Domain architectures of RGS and Gα containing proteins are shown. The 7TM domain is shown as 7 connected TMs. The gene, organism name and gi are shown for proteins with gene names, while only the gi and the organism name are shown for draft genomes with non-standardized gene names. The domain abbreviations are TM – Transmembrane helix; B-Propeller – β-propeller; ACYC – Adenylyl Cyclase; ANK – Ankyrin repeats; ACB- Axin beta-catenin binding domain; BB- B-box zinc finger; B-Propeller - β-propeller; BLUF - Sensors of blue-light using FAD; BLec – BULBLECTIN; CS - Cysteine String; EF – EFHAND; FB – FBOX; Gg – Gγ; GLC – GoLoco; IG – Immunoglobulin domain; Ngru_X – Extra cellular domain specific to Naegleria 7TM+RGS proteins; RA - Ras association (RalGDS/AF-6) domain; RBD - Raf-like Ras-binding domain; RCC1 - Regulator of chromosome condensation (RCC1) repeat, a variety of β–propeller; SH – S-helix; SIG – Signal peptide; STYKIN – S/T/Y Kinase.

Figure 5

(A) Linear scaling of total number of RGS proteins against Gα proteins in eukaryotes along with the best-fit line without the outlying organisms (red). (B) Scatter plot of the number of 7TM receptor containing proteins with Gα proteins in eukaryotes. (C) Scatter plot of the number of 7TM receptor containing proteins with RGS proteins in eukaryotes. (D) Complexity quotient plot for RGS proteins. The complexity quotient for an organism is defined as the product of two values: the number of different types of domains that co-occurs in signaling proteins, and the average number of domains detected in these proteins. The complexity quotient is plotted against the total number of RGS-containing proteins in a given organism. A saturation curve fitting the distribution without the outlying organisms is shown in red. Organism abbreviations are as in Fig. 3.