Diversity of opisthokont septin proteins reveals structural constraints and conserved motifs

Abstract

Background: Septins are cytoskeletal proteins important in cell division and in establishing and maintaining cell polarity. Although septins are found in various eukaryotes, septin genes had the richest history of duplication and diversification in the animals, fungi and protists that comprise opisthokonts. Opisthokont septin paralogs encode modular proteins that assemble into heteropolymeric higher order structures. The heteropolymers can create physical barriers to diffusion or serve as scaffolds organizing other morphogenetic proteins. How the paralogous septin modules interact to form heteropolymers is still unclear. Through comparative analyses, we hoped to clarify the evolutionary origin of septin diversity and to suggest which amino acid residues were responsible for subunit binding specificity.

Results: Here we take advantage of newly sequenced genomes to reconcile septin gene trees with a species phylogeny from 22 animals, fungi and protists. Our phylogenetic analysis divided 120 septins representing the 22 taxa into seven clades (Groups) of paralogs. Suggesting that septin genes duplicated early in opisthokont evolution, animal and fungal lineages share septin Groups 1A, 4 and possibly also 1B and 2. Group 5 septins were present in fungi but not in animals and whether they were present in the opisthokont ancestor was unclear. Protein homology folding showed that previously identified conserved septin motifs were all located near interface regions between the adjacent septin monomers. We found specific interface residues associated with each septin Group that are candidates for providing subunit binding specificity.

Conclusions: This work reveals that duplication of septin genes began in an ancestral opisthokont more than a billion years ago and continued through the diversification of animals and fungi. Evidence for evolutionary conservation of ~ 49 interface residues will inform mutagenesis experiments and lead to improved understanding of the rules guiding septin heteropolymer formation and from there, to improved understanding of development of form in animals and fungi.

Keywords: Ancestral state reconstruction; Evolution; Gene tree-species tree reconciliation; Modelling; Opisthokont; Protein-protein interaction; Septin; Subunit.

Fig. 1

– Organization of a septin protein. a) Three-dimensional crystal structure of human septin Sept2 (PDB: 2QA5) produced with pymol showing three orientations, each rotated 90°. Position of GTP binding is shown by solid arrow. Structures are coloured according to regions in b. Note that C-terminal extensions are not resolved in the crystal structure. b) Linear representation of a septin protein, indicating the arrangements of the major septin elements as described in the text. b) Reproduced with permission from Pan et al., [15]

Fig. 2

Analysis of early-diverging lineages provides evidence of ancestral septin duplications. a) Shaded area of pie charts indicates the proportional likelihood that a specific ancestor had a member of a septin group, when reconstructed under maximum likelihood. As Group 2 may or may not be monophyletic, reconstructions resulting from these two alternatives are illustrated. b) Gene copy number reconstructed within the species phylogeny. Branch thickness represents the average NOTUNG inferred number of septin gene copies based on the jPRIME septin gene phylogeny. Stars indicate a change in morphology of organisms in a lineage. Note: This species phylogeny was used to guide the jPRIME analysis. c) Cell shading indicates copy numbers of genes representing each septin group, classified by each of three phylogenetic methods. Classification of septins from an organism sometimes differed depending on the analysis method, reflecting uncertainty in phylogenetic placement of divergent sequences

Fig. 3

Highly conserved septin residues are involved in GTP-binding and interactions at G- and NC-interfaces. a) Conserved residues correspond to predicted interacting residues in interfaces. Solid line represents Shannon-Jensen sequence conservation; shaded curves indicate values above 0.5. Red columns: proportion of taxa where a residue interacts in the NC interface. Blue columns: proportion that interact in the G interface. GTP-binding residues are indicated with black arrows. The generalized diagram of S. cerevisiae CDC3 from Pan et al., [15] is shown to scale. b) Diagram of a septin monomer showing the organization of interface residues at the NC and G interfaces. The curved line at the top represents a coiled-coil. c) Model showing how monomers interact to form heterooligomers. The interacting group (ig) residues, colored as in b), indicate predicted residue interactions between septin partners. Not to scale

Fig. 4

Patterns of diversity and conservation across septin groups. WebLogo showing patterns of amino acid conservation and diversity across aligned motifs and domains of the septin Groups. Interacting residues are split into the G and the NC interfaces, and subdivisions into interacting groups are shown below. Residues are numbered according to the COBALT aligned position with S. cerevisiae CDC3. In parentheses below each Group number is the number of sequences used to build the WebLogo