DOI: 10.5061/dryad.cvdncjt2b
title: Comment on “Ancient origins of allosteric activation in a Ser-Thr kinase”

description: Hadzipasic et al. used ancestral sequence reconstruction to identify
 historical sequence substitutions that putatively caused Aurora kinases to
 evolve allosteric regulation. We show that their results arise from an
 implausible phylogeny and sparse sequence sampling. Addressing either
 problem reverses their inferences: allostery and the amino acids that
 confer it were not gained during the diversification of eukaryotes but
 were lost in a subgroup of Fungi.
description: Ancestral sequence reconstruction using Hadzipasic et al.
 sequences under congruence constraint (Fig. 1E, G) We
 acquired the AURK and PLK sequences analyzed by Hadzipasic et al. We
 aligned them using MUSCLE (v3.8.425) (1), removed sequence-specific
 insertions and ambiguously aligned sites, and trimmed the N- and
 C-termini, matching the sequence boundaries set by Hadzipasic et al. We
 used RAxML (v8.2.12) (2) to infer the constrained ML phylogeny, imposing
 the constraint shown in Fig. 1B, and used PAML (v4.8) (3) to perform ASR.
 For both phylogenetics and ASR, we used the same model of sequence
 evolution as used by Hadzipasic et al. (LG + G + X, four gamma rate
 categories). Phylogenetics and ASR with improved
 sequence sampling (Fig. 2) To obtain a broad sample of
 eukaryotic AURK and PLK sequences, we used a reciprocal best-hit protein
 BLAST strategy using the NCBI protein database (4). Human AURKA and PLK4
 were used as query sequences. Taxonomically restricted BLAST searches were
 conducted that together encompassed all species within the five major
 eukaryotic kingdoms/subkingdoms (Fungi, Holozoa, Amoebozoa,
 Archaeplastida, and SAR). BLAST hits of anomalous length (&lt;250 or
 &gt;600 amino acids for AURK, &lt;250 or &gt;1000 amino acids
 for PLK) were discarded. Redundant sequences were eliminated at similarity
 cutoff 0.85 using CD-HIT (v4.8.1) (5). Each remaining BLAST hit was then
 used as query in a reciprocal BLAST search against human proteins, and all
 sequences for which the best hit in humans was an AURK or PLK were
 retained. Sequence alignment of these hits was
 performed hierarchically using MUSCLE software. We first aligned sequences
 from within defined profile groups of species (each usually a superphylum
 or phylum). We trimmed the N- and C-termini, leaving sites corresponding
 to human AURKA sites 133 to 383, and then removed sites representing
 species-specific insertions and ambiguously aligned sites. We discarded
 sequences that were missing 10 or more consecutive amino acids present in
 the majority of other sequences. We then inferred the phylogeny of the
 profile group using FastTree (v2.1.11) (6). To minimize long branch
 attraction, we removed all sequences or groups of sequences subtended by
 branches of length &gt;0.5. We also removed sequences/small groups
 that were assigned to entirely different phyla (e.g., annelid sequences
 placed inside the molluscs, or green algae sequences placed inside land
 plants), as well as taxon-specific paralogs with long branches that were
 pulled outside of the entire profile group being aligned. We then used
 profile-profile alignment in MUSCLE to progressively align the
 group-specific alignments to each other, yielding a global AURK/PLK
 alignment. We used RAxML to infer the ML AURK-PLK
 phylogeny from this global alignment, using the best-fit model of
 evolution (LG + G + X). For all RAxML analyses, we iterated topology
 search 50 times using different random number seeds, and chose the
 iteration with the highest likelihood. On the ML phylogeny, AURKs from a
 few lower-level groups of Ecdysozoa and Platyhelminthes subtended by long
 branches were placed in kingdoms other than the animals; drastic
 long-branch misplacements also moved a few small groups of AURKs from
 Fungi and Alveolates into other kingdoms/superphyla and affected some PLK
 sequences. These sequences were removed to yield the final alignment, and
 the analysis was repeated to infer the final ML phylogeny. Approximate
 likelihood ratio test was performed using PhyML (v3.3) (7). For the
 maximum congruence constraint analysis, we imposed the topological
 constraint shown in Fig. 2A and used RAxML to perform phylogenetic
 analysis to find the ML tree, branch lengths, and other parameters given
 this constraint. We used a similar approach to find the ML tree consistent
 with the Fungi-out constraint (the same constraint in Fig. 2A, except that
 Fungi are the most basally branching group). Ancestral sequences were
 inferred using the marginal reconstruction algorithm in PAML using LG + G
 and the amino acid frequencies inferred on the ML tree by RAxML.
 The Shimodaira-Hasegawa test was used to evaluate relative
 support for the ML vs. MC trees. We used the R package phangorn to execute
 the SH test (8), comparing the ML tree found in the unconstrained ML
 search to the ML tree from the MC-constrained search (lnL -121973.5 and -
 121992.0, respectively); this returned a nonsignificant result
 (p-value=0.36). The heuristic searches may not have identified the
 globally optimal tree in each case, so we also compared the tree from the
 search iteration with the highest likelihood in the unconstrained ML
 analysis to the tree recovered from the iteration with the lowest
 likelihood in the MC-constrained analysis (lnL=-122032.7,
 p-value=0.21).
description: See File_descriptions.xlsx