Identification of novel families and classification of the C2 domain superfamily elucidate the origin and evolution of membrane targeting activities in eukaryotes

Abstract

Eukaryotes contain an elaborate membrane system, which bounds the cell itself, nuclei, organelles and transient intracellular structures, such as vesicles. The emergence of this system was marked by an expansion of a number of structurally distinct classes of lipid-binding domains that could throw light on the early evolution of eukaryotic membranes. The C2 domain is a useful model to understand these events because it is one of the most prevalent eukaryotic lipid-binding domains deployed in diverse functional contexts. Most studies have concentrated on C2 domains prototyped by those in protein kinase C (PKC-C2) isoforms that bind lipid in a calcium-dependent manner. While two other distinct families of C2 domains, namely those in PI3K-C2 and PTEN-C2 are also recognized, a complete picture of evolutionary relationships within the C2 domain superfamily is lacking. We systematically studied this superfamily using sequence profile searches, phylogenetic and phyletic-pattern analysis and structure-prediction. Consequently, we identified several distinct families of C2 domains including those respectively typified by C2 domains in the Aida (axin interactor, dorsalization associated) proteins, B9 proteins (e.g. Mks1 (Xbx-7), Stumpy (Tza-1) and Tza-2) involved in centrosome migration and ciliogenesis, Dock180/Zizimin proteins which are Rac/CDC42 GDP exchange factors, the EEIG1/Sym-3, EHBP1 and plant RPG/PMI1 proteins involved in endocytotic recycling and organellar positioning and an apicomplexan family. We present evidence that the last eukaryotic common ancestor (LECA) contained at least 10 C2 domains belonging to 6 well-defined families. Further, we suggest that this pre-LECA diversification was linked to the emergence of several quintessentially eukaryotic structures, such as membrane repair and vesicular trafficking system, anchoring of the actin and tubulin cytoskeleton to the plasma and vesicular membranes, localization of small GTPases to membranes and lipid-based signal transduction. Subsequent lineage-specific expansions of Zizimin-type C2 domains and functionally linked CDC42/Rac GTPases occurred independently in eukaryotes that evolved active amoeboid motility. While two lipid-binding regions are likely to be shared by majority of C2 domains, the actual constellation of lipid-binding residues (predominantly basic) are distinct in each family potentially reflective of the functional and biochemical diversity of these domains. Importantly, we show that the calcium-dependent membrane interaction is a derived feature limited to the PKC-C2 domains. Our identification of novel C2 domains offers new insights into interaction between both the microtubular and microfilament cytoskeleton and cellular membranes.

Fig. 1

Multiple sequence and structural alignment for seven families of the C2 domain. The sequences are represented by an abbreviation of species name followed by Genbank GI and PDB ID. The consensus in 65% of the sequences shown below the alignment is derived using the following amino acid classes: a, aromatic (FHWY, black on orange); b, big (EFHIKLMQRWY, green on grey); h, hydrophobic (ACFGHILMTVWY, yellow on dark grey); l, aliphatic (ILV, red on grey); p, polar (CDEHKNQRST, blue on grey); s, small (ACDGNPSTV, cyan on dark grey); t, tiny (AGS, cyan on dark grey). Secondary structures for these C2 families, that were derived from PDB structures or predicted using the Jpred program, are indicated above the alignment of each own family (E in blue, β sheet; H in red, α helix). For each C2 family, conserved or specific residues that are potentially involved in binding membrane lipid are labeled on the top of the alignment and highlighted with the blue background for basic residues, the red background for acidic residues, and the orange background for aromatic residues. Non-conserved or subfamily-specific are indicated in italic. The numbers in bracket are indicative of the excluded residues from sequences. For complete multiple sequence alignments of AIDA-C2, B9-C2, DOCK-C2 and NT-C2 families refer to supplemental Figs. S1 to S4. Species name abbreviations: Ag, Anopheles gambiae; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Cn, Cryptococcus neoformans; Cr, Chlamydomonas reinhardtii; Dd, Dictyostelium discoideum; Eh, Entamoeba histolytica; Hs, Homo sapiens; Lt, Lachancea thermotolerans; Mb, Monosiga brevicollis; Mi Micromonas sp.; Mt, Medicago truncatula; Nv, Nematostella vectensis; Ot, Ostreococcus tauri; Pi, Phytophthora infestans; Pm, Perkinsus marinus; Ps, Pichia stipitis; Pt, Paramecium tetraurelia; Sp, Strongylocentrotus purpuratus; Ta, Trichoplax adhaerens; Tb, Trypanosoma brucei; Tt, Tetrahymena thermophila; Tv, Trichomonas vaginalis.

Fig. 2

Domain architectures of representatives of the AIDA-C2, B9-C2, DOCK-C2 and NT-C2 domain-containing proteins. The sequences are indicated by species name with Genbank GI. Domains are drawn approximately to scale. Domain abbreviations are as follows: FAT, four-helical up-and-down bundle found in focal adhesion kinase and talin; AIDA-C2, Aida version of the C2 domain; Ank, Ankyrin repeat (PF00023); B9-C2, B9 version of the C2 domain (PF07162); CH, Calponin homology domain (PF00307); Coiled coil, coiled coil region; Ded_Cyto, Dedicator of cytokinesis (PF06920); DIL, DIL domain (PF01843); DOCK-C2, Dock180 version of the C2 domain; EC, EEIG1 C terminal region; EF, Eps15 homology domain or EF Hand (PF00036); Fil, Filamin/ABP280 repeat (PF00630); IQ, IQ calmodulin-binding motif (PF00612); Mg, MgtE family of Mg²⁺ transporter (PF01769); Myosin head, Myosin head motor domain (PF00063); NT-C2, N-terminal C2 domain (also known as EEIG1 domain in PFAM, PF10358); PH, Pleckstrin homology domain (PF00169); PMI1-C, PMI1 C-terminal region; SH3, Src homology 3 (PF00018); SYNC1-C, SYNC1 C-terminal region. In the case of a subset of insect B9-C2 proteins there has been a systematic mis-annotation of the gene model which has resulted in the B9-C2 gene being fused to an adjacent gene of the Tectonic family (PF07773). Hence this architecture is likely to be artefactual and is indicated with a question mark.

Fig. 3

Phylogenetic relationship between seven families of the C2 domain. An approximate maximum-likelihood method implemented in the FastTree 2.1 program was used to build the phylogenetic tree for seven versions of the C2 domain. Protein sequences are represented by species names following Genbank GI ID. Main nodes are shown with supporting bootstrap values. Four newly defined families of C2 domain and sub-groups within them are distinguished by different colors. L with a blue circle indicates that those C2 families or subfamilies can be traced to LECA, where question mark (?) with green circle indicates an unresolved evolutionary origination. Species name abbreviations refer to Fig. 1 legend and additional ones are as follows: An, Aspergillus niger; As, Aspergillus terreus; Bm, Brugia malayi; Cc, Coprinopsis cinerea; Ci, Ciona intestinalis; Cm, Cryptosporidium muris; Co, Coccidioides immitis; Cp, Cryptosporidium parvum; Da, Drosophila ananassae; Dr, Danio rerio; Lb, Leishmania braziliensis; Mc, Microsporum canis; Pb, Paracoccidioides brasiliensis; Pg, Pichia guilliermondii; Sc, Saccharomyces cerevisiae; So, Schizosaccharomyces pombe; Ss, Sclerotinia sclerotiorum; St, Strongylocentrotus purpuratus; Ts, Talaromyces stipitatus; Vp, Vanderwaltozyma polyspora; Vv, Vitis vinifera; Zm, Zea mays; Zr, Zygosaccharomyces rouxii.

Fig. 4

Electrostatic potential surface and cartoon diagrams of structures of seven C2 domains. All structures are depicted in the same orientation. Surface diagrams are colored by electrostatic potential as calculated by APBS in VMD program with positively charged regions (>10 mV) colored in blue and negatively charged regions (<-10 mV) in red. The α helices are shown in purple, β sheets in yellow, and loops in white. Conserved residues that are involved in membrane lipid-binding are highlighted in licorice drawing with basic residues (H, K, R) in blue, acidic residues in red, and aromatic residues (F, W, Y) in orange. The α helix 2 region of B9-C2 and DOCK-C2 domains is not modeled. 1E7U was remodeled for its loop region between beta strand 5 and 6 for which no X-ray data was available.

Fig. 5

Divergent evolution of the C2 domain superfamily in eukaryotes. Duplications of the domain and speciation are indicated by red lines whereas domain loss for B9-C2 domains is indicated by blue lines. Dashed red line with question mark (?) indicates unclear point of origination of AIDA-C2. A domain architecture linkage graph for the seven C2 versions is shown in the next column. The arrows indicate the direction of domain connection in a polypeptide with the arrowhead pointing to the C-terminus. Microfilament function-related domains are shown in yellow, phosphatase and phospholipase domains in orange, kinase domains in blue, whereas other domains with enzyme activities in violet. Additional domain abbreviations with PFAM domain ID are as follows: C1, Protein kinase C, C1 domain (PF00130); CalpainIII, Calpain large subunit, domain III (PF01067); DnaJ, DnaJ domain (PF00226); PIP3 phosphatase, including both dual specificity phosphatase, catalytic domain (PF00782) and protein-tyrosine phosphatase (PF00102); Thiored, is a thioredoxin-like domain called DUF547 in PFAM (PF04784); FH2, Formin homology 2 domain (PF02181); HECT, HECT-domain (ubiquitin-transferase) (PF00632); MACPF, MAC/Perforin domain (PF01823); MUNC13, Munc13 (mammalian uncoordinated) homology domain (PF10540); p85, PI3-kinase family, p85-binding domain (PF02192); Papain-like peptidase, calpain family cysteine protease (PF00648); PI34-K, Phosphatidylinositol 3- and 4-kinase (PF00454); PI3Ka, Phosphoinositide 3-kinase family, accessory domain (PF00613); PI-PLC-X, Phosphatidylinositol-specific phospholipase C, X domain (PF00388); PI-PLC-Y, Phosphatidylinositol-specific phospholipase C, Y domain (PF00387); Pkinase, Protein kinase domain (PF00069); PLA2B, Lysophospholipase catalytic domain (PF01735); PLDc, Phospholipase D C terminal (PF12357); PSDcarbxylase, Phosphatidylserine decarboxylase (PF02666); PTB, Phosphotyrosine-binding domain (PF08416); PX, PX domain (PF00787); RasGAP, GTPase-activator protein for Ras-like GTPase (PF00616); Rbd, PI3-kinase family, ras-binding domain (PF00794); SH2, Src homology 2 domain (PF00017); vWA, vWA-like copine domain (PF07002); WW, WW domain (PF00397).