Clustering of protein families into functional subtypes using Relative Complexity Measure with reduced amino acid alphabets

Abstract

Background: Phylogenetic analysis can be used to divide a protein family into subfamilies in the absence of experimental information. Most phylogenetic analysis methods utilize multiple alignment of sequences and are based on an evolutionary model. However, multiple alignment is not an automated procedure and requires human intervention to maintain alignment integrity and to produce phylogenies consistent with the functional splits in underlying sequences. To address this problem, we propose to use the alignment-free Relative Complexity Measure (RCM) combined with reduced amino acid alphabets to cluster protein families into functional subtypes purely on sequence criteria. Comparison with an alignment-based approach was also carried out to test the quality of the clustering.

Results: We demonstrate the robustness of RCM with reduced alphabets in clustering of protein sequences into families in a simulated dataset and seven well-characterized protein datasets. On protein datasets, crotonases, mandelate racemases, nucleotidyl cyclases and glycoside hydrolase family 2 were clustered into subfamilies with 100% accuracy whereas acyl transferase domains, haloacid dehalogenases, and vicinal oxygen chelates could be assigned to subfamilies with 97.2%, 96.9% and 92.2% accuracies, respectively.

Conclusions: The overall combination of methods in this paper is useful for clustering protein families into subtypes based on solely protein sequence information. The method is also flexible and computationally fast because it does not require multiple alignment of sequences.

Figure 1

Protocol Overview. For RCM, the original sequences and sequences recoded with reduced alphabets are used to calculate RCM-based distances which are then inputted sequentially to the Neighbor-Joining and Retree programs of the PHYLIP v3.68 package. For MSA, first, alignments are carried out using ClustalW2 with substitution matrices corresponding to each amino acid alphabet. Following bootstrap analysis with ClustalW2, Retree program is used to root the trees with midpoint rooting and to discard branch lengths. Each phylogenetic tree is then inputted to the TBC algorithm along with its attribute file that shows the expert assignment of each sequence to each family to calculate the TBC error.

Figure 2

Tree topology of the simulated dataset. The identical topology of the three phylogenetic trees (i.e., RCM tree, bootstrap tree and true tree) for the simulated dataset is shown.

Figure 3

Phylogenetic trees of protein families. RCM trees were drawn using ML15 alphabet. For each family, the taxa corresponding to different subfamilies are colored differently. (A) Crotonases (B) Mandelate racemases (C) Vicinal oxygen chelates (D) Haloacid dehalogenase (E) Nucleotidyl cyclases (F) Acyl transferases (G) GH2 hydrolases