Functional significance may underlie the taxonomic utility of single amino acid substitutions in conserved proteins

Abstract

We hypothesized that some amino acid substitutions in conserved proteins that are strongly fixed by critical functional roles would show lineage-specific distributions. As an example of an archetypal conserved eukaryotic protein we considered the active site of beta-tubulin. Our analysis identified one amino acid substitution--beta-tubulin F224--which was highly lineage specific. Investigation of beta-tubulin for other phylogenetically restricted amino acids identified several with apparent specificity for well-defined phylogenetic groups. Intriguingly, none showed specificity for "supergroups" other than the unikonts. To understand why, we analysed the beta-tubulin Neighbor-Net and demonstrated a fundamental division between core beta-tubulins (plant-like) and divergent beta-tubulins (animal and fungal). F224 was almost completely restricted to the core beta-tubulins, while divergent beta-tubulins possessed Y224. Thus, our specific example offers insight into the restrictions associated with the co-evolution of beta-tubulin during the radiation of eukaryotes, underlining a fundamental dichotomy between F-type, core beta-tubulins and Y-type, divergent beta-tubulins. More broadly our study provides proof of principle for the taxonomic utility of critical amino acids in the active sites of conserved proteins.

Fig. 1

Key nucleotide–protein interactions at the GTP-binding site of bovine β-tubulin. Red GTP; dark blue Y224; light blue N228, yellow S174, D179, and E183. Other residues are omitted for clarity. The hydrogen bond between Y224 and the ribose 2´-OH is indicated in light green (after Lowe et al. 2001)

Fig. 2

Phylogenetic distribution of F224 and Y224. The Mesquite software package was used to describe the phylogenetic distribution of F224 figuratively (Maddison and Maddison 2009). Distribution of 224 substitution amongst the major lineages of the eukaryotic tree is shown. Tree is based on the five supergroup model with modification (Keeling et al. 2005)

Fig. 3

Only T260 shows co-variation with F224. Close scrutiny of the β-tubulins from Paxillus fumigatus (P) and Suillus bovinus (S) reveal they possess several β-tubulin amino acids which are normally associated with plant rather than fungal lineages. P. fumigatus possesses only two amino acids which are plant-like and are not possessed by S. bovinus F224 and T260 implicating the latter as a requirement for the 224 Y to F transition

Fig. 4

Core and divergent groups of β-tubulins. A Neighbor-Net of the considered β-tubulins (excluding E. histolytica: with a LSFit 99.77). The underpinning maximum likelihood distances were computed using the WAG + Gamma model (shape parameter is 0.6) (Huson and Bryant 2006). Species names in red correspond to tubulins which carry the Y-type version of 224, while the ones in light blue carry the F-type version of 224. Deletion of the band of parallel branches marked by an arrow labelled ‘A’ segregates the network and thus the β-tubulins into the core and divergent tubulin groups. Both groups are indicated by correspondingly labelled arcs