Evolution of an ancient protein function involved in organized multicellularity in animals

Abstract

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

Keywords: Salpingea rosetta; ancestral protein reconstruction; cell biology; evolutionary biochemistry; evolutionary biology; evolutionary cell biology; genomics; mitotic spindle; protein evolution.

Figure 1.. Function and phylogeny of the guanylate kinase (gk) and GK_PID protein family.

(A) The GK_PID of the protein Discs-large (Dlg, blue) serves as a scaffold for spindle orientation by physically linking the localized cortical protein Pins (green) to astral microtubules (red) via the motor protein KHC-73 (black). (B) Reduced phylogeny of the protein family containing gk enzymes (brown) and protein-binding GK_PIDs (blue). Parentheses show the number of sequences in each clade. Reconstructed proteins Anc-gk_dup (the preduplication ancestor of gk enzymes and GK_PIDs in animals/choanoflagellates), Anc-GK1_PID and Anc-GK2_PID (the GK_PID in the common ancestor of animals and choanoflagellates, and of animals, respectively) are marked as circles with approximate likelihood ratio support. Scale bar indicates number of substitutions per site. For unreduced phylogeny, see Figure 1—figure supplement 1. Characteristics of the reconstructed sequences are found in Figure 1—figure supplement 2. For sequences analyzed, see Figure 1—source data 1. For sequences and posterior probabilities of amino acid states, see Figure 1—source data 2, Figure 1—source data 3. DOI: http://dx.doi.org/10.7554/eLife.10147.003

Figure 1—figure supplement 1.. Complete phylogeny of 224 guanylate kinase enzyme and GK_PIDs.

Nodes are labeled with approximate likelihood ratio supports, and branch lengths are in substitutions per site (see scale bar). The tree is rooted on the bacterial GK enzymes. Major paralogs in the GK_PID family, with the number of sequences included in each, are labeled. See Figure 1—source data 1 for species and accessions used. See Supplementary files 1 and 2 for alignment and tree file. DOI: http://dx.doi.org/10.7554/eLife.10147.007

Figure 1—figure supplement 2.. Sequence characteristics of maximum likelihood reconstructions of Anc-gk_dup and Anc-GK1_PID.

(A,B) The histogram shows the distribution over sites of posterior probability support for maximum likelihood amino acid states (see Figure 1—source data 2, Figure 1—source data 3 for full sequences and support). (C) Sequence similarity of ancestral sequences to extant gk enzymes and GK_PIDs. The table shows the percent of residues identical between each pair of sequences. DOI: http://dx.doi.org/10.7554/eLife.10147.008

Figure 2.. Evolution of a novel spindle-orientation function in ancestral GK_PID.

(A) Anc-gk_dup (circles) is an active nucleotide kinase in a coupled enzyme assay for the reaction shown; Anc-GK1_PID (boxes) is inactive. Activity of the human gk enzyme (triangles) is shown for reference. Error bars show SEM for three replicates. (B) The more recent ancestral protein Anc-GK1_PID (boxes) binds a 20 amino-acid peptide (see methods) from the Pins protein in a fluorescent anisotropy assay, but Anc-gk_dup (cirlcles) does not. Pins binding by the GK_PID of the Drosophila melanogaster Dlg protein (triangles) is shown for reference. Error bars show SEM for three replicates. (C–F) Evolution of spindle orientation function as assayed in cultured S2 cells that do not express endogenous Dlg protein. Cells were transfected with a GK construct (C, –control: empty transfection vector; D, + control: GK_PID from extant Drosophila Dlg) and scored for alignment of the mitotic spindle (red, tubulin, visualized immunocytochemically) relative to the Pins cortical crescent (green, a GFP-tagged Pins-Ecd fusion). In the example images for each experiment, two cells are shown, the bottom one of which is dividing. The angle of the mitotic spindle relative to a line bisecting the Pins crescent (from 0°, precisely aligned, to 90°) was recorded in many dividing cells; the radial histogram (right) shows the distribution of observed angles among all cells scored with a given genotype. Cells transfected with Anc-gk_dup (E) do not display robust spindle orientation, but those transfected with Anc-GK1_PID do (F). SEM: Standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.10147.009

Figure 2—figure supplement 1.. Properties of ancestral protein AncGK2-PID.

(A) Distribution over sites of posterior probabilities of ML amino acid states in AncGK2_PID . Mean posterior probability = 0.87. (B) Anc-GK2_PID(squares) binds the Pins peptide ligand with high affinity in a fluorescence anisotropy assay. Binding by the Drosophila melanogaster Dlg GK_PID is shown for comparison (circles). Error bars who standard error of the mean of three replicates. (C) Anc-GK2_PID is capable of orienting the mitotic spindle. DOI: http://dx.doi.org/10.7554/eLife.10147.010

Figure 2—figure supplement 2.. Robustness of functional inferences about ancestral proteins to uncertainty about the sequence reconstruction.

For each of Anc-gk_dup and Anc-GK1_PID, an alternate reconstruction (Alt-All) was synthesized, containing the next-best amino acid state at all sites with multiple plausible states ((PP>0.2). The enzyme activity of Anc-gk_dup in a coupled enzymatic assay for cofactor turnover (panel A) and the Pins-binding activity of Anc_GK1_PID (panel B) in a fluorescence anisotropy assay are shown for both maximum likelihood and Alt-All reconstructions. Affinities and maximal velocities differ quantitatively, but the presence/absence of each property is robust to incorporation of uncertainty about the ancestral sequence (see Figure 1F). DOI: http://dx.doi.org/10.7554/eLife.10147.011

Figure 3.. Taxonomic distribution of proteins in the spindle orientation complex.

(A) The genomes of the choanoflagellate Salpingoeca rosetta and the filasterean Capsaspora owczarzaki contain orthologs of human Dlg, Pins, and KHC-73. The domain architecture of each protein is shown, as inferred using the SMART database. Each group of proteins are reciprocal best BLAST hits (RBH) to the human query protein shown. For details, see Figure 3—source data 1. (B) Aligned sequences from the linker portion of Pins (see panel A), which binds to Dlg. Colors highlight identical or biochemically conservative residues. Asterisk, phosphorylated or negatively charged residue 436, which in the Drosophila melanogaster Pins protein anchors Dlg binding. For complete species names and accessions, see Figure 3—source data 2. DOI: http://dx.doi.org/10.7554/eLife.10147.012

Figure 4.. Spindle orientation and its molecular components in choanoflagellates.

(A) Purified GK_PID from the Salpingoeca rosetta Dlg ortholog (triangles) binds fluorescently labeled Drosophila melanogaster Pins. Binding by Anc-GK1_PID (squares) and lack of binding by Anc-gk_dup (circles) are shown for comparison. Error bars, SEM for three replicates. (B) Purified GK_PID of S. rosetta does not interact with Pins of S. rosetta; under the same conditions, the D. melanogaster orthologs of these proteins do bind. A GST-fusion of each Pins linker regions (visible on the coomassie stained gel) was incubated with an MBP-fusion of each species’ GK_PID and detected by anti-MBP western blot. As previously found, binding of D. melanogaster Pins requires phosphorylation by Aurora A kinase; S. rosetta GK_PID does not bind its own Pins whether or not the kinase is present. (C, D) Spindle orientation in colonial ( panel C, n = 12) and noncolonial (panel D, n = 7) S. rosetta. For each condition, the image shows one representative colony; the cell at bottom center is mitotic, as evidenced by condensed DNA (blue, DAPI) without a defined nuclear envelope (green, visualized using anti-nuclear pore complex). Red, mitotic spindle visualized using anti-tubulin. In colonial cells, the angle of the mitotic spindle (solid white line) was measured relative to a line perpendicular to the plane of the colony extending through the colony’s center (dashed line). The histogram shows the distribution of spindle angles among all dividing S. rosetta cells measured, with 90° representing perfect alignment relative to the colony ring. In noncolonial cells, the spindle angle was measured relative to a line extending from the midpoint between the bases of the two duplicate flagella to the cell body’s centroid (dotted line). An angle of 90° represents perfect alignment relative to the flagella. SEM: Standard error of the mean. DOI: http://dx.doi.org/10.7554/eLife.10147.015

Figure 5.. Evolution of the binding interface and hinge during the evolution of AncGK_PID spindle orientation functions.

(A) All gk/GK_PID family members share a common structural architecture, comprising a catalytic core, two binding lobes (the GMP-binding domain, GBD, shown in dark hue, and the ATP-binding lid), and a flexible hinge region, which connects the GBD to the core and comprises two segments of contiguous residues (magenta). Left: in gk enzymes bound to GMP (red spheres), the lobes adopt a closed conformation, bringing GMP and ATP (cyan spheres) adjacent to each other in the core. Right: the GK_PID has an open conformation, in which Pins (green spheres) binds to the surface of the GBD in the cleft between the two binding lobes. Structures shown are mouse gk enzyme (brown, PDB 1LVG) and the GK_PID from rat Dlg1 (blue, 3UAT). (B) Most residues in Anc-GK1_PID that bind Pins (blue boxes) are unchanged from the ancestral state in Anc-gk_dup. White surface, D. melanogaster Dlg GK1_PID (3TVT). Green, Pins peptide. Ancestral and derived amino acid states are shown; residues with historical amino acid replacements between the two ancestral proteins are outlined. (C) In the hinge region, two historical substitutions (outlined and colored, with side-chains shown as sticks) were conserved in the ancestral state in extant enzymes and in a different state in extant GK_PIDs. Colored bars above the sequence indicate position in the protein structure (right). Hinge segments are shown in pink and the GMP-binding lobe in gray. DOI: http://dx.doi.org/10.7554/eLife.10147.016

Figure 6.. A simple genetic basis for the evolution of spindle orientation functions in the ancestral GK_PID.

(A,B) Introducing either of two historical substitutions s36P or f33S into Anc-gk_dup confers Pins binding and reduces ancestral guanylate kinase activity. Error bars are SEM of 3 replicates. Combining s36P and f33S does not further affect the derived functions beyond the effects of the single substitutions. (C) Introducing s36P into Anc-gk_dup is sufficient to confer the full capacity to drive orientation of the mitotic spindle (compare Figure 1). As shown in Figure 6—figure supplement 1, historical substitutions in the binding interface do not recapitulate the evolution of Pins binding and the loss of gk activity. DOI: http://dx.doi.org/10.7554/eLife.10147.017

Figure 6—figure supplement 1.. Introducing other historical substitutions into AncGK_du does not confer GK_PID-like function.

(A) The hinge substitution s34C (purple) does not abolish guanylate kinase activity in a coupled enzyme activity assay; Anc-gk_dup is shown for comparison. (B) s34C (purple) does not confer substantial Pins-binding affinity on Anc-gk_dup. Pins-binding activity relative to s36P (orange) in a fluorescence anisotropy assay. (C,D) Anc-GK1_PID Pins interface substitutions a73G and f75Y reduce gunaylate kinase enzyme activity (D) but do not confer Pins binding (E) when introduced into Anc-gk_dup. Error bars are standard error of the mean with three replicates. DOI: http://dx.doi.org/10.7554/eLife.10147.018

Figure 7.. Evolution of GK_PID’s new function by unveiling a latent protein-binding site.

(A) The binding surface for Pins in GK_PIDs is derived from the GMP-binding surface of gk enzymes. Homology models of Anc-gk_dup (left) and Anc-GK1_PID (right) are shown as white surface, with all side chains that contact either GMP or Pins as yellow sticks. Pink sticks show GMP; green ribbon shows Pins backbone, with the side chains of all Pins residues that contact the GK protein shown as sticks. The phosphate group on GMP and on Pins residue 436 are shown as orange and red sticks. Black dotted lines, protein-ligand hydrogen bonds. In the AncGK1_PID structure , substitutions at sites in the binding interface are shaded red, including key substitution s36P. The binding modes of extant gk enzymes and GK_PIDs are similar and support the same conclusions (see Figure 7—figure supplement 1). (B) The structure of the hinge and GMP/Pins-binding lobes is conserved between the Pins-bound GK_PID (blue, rat Dlg, 3UAT), the apo-gk enzyme (brown, S. cerevisiae guanylate kinase 1EX6), and the apo-gk-s36P mutant (gray, 4F4J), all in the open conformation. DOI: http://dx.doi.org/10.7554/eLife.10147.019

Figure 7—figure supplement 1.. Structural context of key historical mutations.

(A) Location of historical hinge substitutions s36P and f33S. Residues Pro36 (orange) and Ser33 (purple) are shown on the crystal structure of the GK_PID from rat Dlg (3UAT, white surface). Pins peptide ligand is shown in green. (B,C) Similarity of GMP binding site in extant guanylate kinase enzyme to Pins binding site in extant GK_PID. The guanylate binding domains (GBDs) of yeast gk enzyme (PDB 1GKY, panel B) and of rat Dlg GK_PID (PDB 3UAT, panel C) is shown as white surface, with all side chains that contact either GMP or Pins as yellow sticks. Pink sticks show GMP; green ribbon shows Pins backbone, with the side chains of all Pins residues that contact the GK_PID protein shown as sticks. The phosphate group on GMP and on Pins residue S436 are indicated. Black dotted lines, protein-ligand hydrogen bonds. Key substitution s36P is highlighted in pink. DOI: http://dx.doi.org/10.7554/eLife.10147.020

Figure 8.. Historical evolution of GK_PID-mediated spindle orientation complex.

The center portion shows the phylogeny of Metazoa and closely related taxa. The origin of cell differentiation and spatially organized tissues is marked. The left portion shows major events in the evolution of the components of the spindle orientation complex reconstructed in this study. Duplication of an ancestral gk enzyme (brown) and the key mutations that led to the origin of a GK_PID (blue) that could bind other molecules in the complex are shown relative to the phylogeny’s time scale. The apparent date of origin of KHC-73 (black) and Pins (green) are also shown. Dotted green line shows the origin of Pins in a form not yet bound by GK_PID. Solid green line shows GK_PID-binding form. Horizontal lines indicate binding between proteins. The right portion shows a schematic of the spindle orientation machinery in metazoans, which allows orientation relative to external cues from nearby cells, as well as spindle orientation relative to the internal cell axis as marked by the flagella in both solitary and colonial choanoflagellates. DOI: http://dx.doi.org/10.7554/eLife.10147.021