Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site

Abstract

Background: Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636.

Results: Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family.

Conclusion: After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Figure 1

Multiple sequence alignment of selected representatives of the extended COG4636+ family. The selection of representative sequences includes the modeled protein from Nostoc (motif H-PD-EXX-K, members from G. violaceus with different order of putative catalytic residues (Gv1: H-PD-EXK; Gv2: S-PD-EXD-K; Gv3: H-PD-EXD; Gv4: H-PD-EXX-N; Gv5: Q-PD-EXX-K), and members of mono-phyletic clusters from D. hafniense, C. aurantiacus, S. coelicolor, T. thermophilus, and G. violaceus). The positions of putative catalytic residues are labeled with "*". The variable termini, which could not be confidently aligned, are not shown; the number of omitted residues is indicated. A complete alignment of full-length sequences is available for download from . Amino acids are colored according to the physico-chemical properties of their side-chains (negatively charged: red, positively charged: blue, polar: magenta, hydrophobic: green). Conserved residues are highlighted. Elements of predicted secondary structure (helices and strands) are indicated by tubes and arrows, respectively.

Figure 2

Fold-recognition alignment between all3650 and structures of Hjc and Hje. Amino acids are colored according to the physico-chemical properties of their side-chains. Conserved residues are highlighted. Secondary structure elements experimentally identified in Hjc and Hje and predicted for all3650 are shown between the target and the template sequences. Known and predicted catalytic residues are indicated by "*" (above the alignment for the target, below the alignment for the templates).

Figure 3

Homology model of all3650. Helices and strands are shown in green and yellow, respectively. The predicted catalytic residues are shown in the wireframe representation and labeled. The termini are indicated.

Figure 4

Spatial conservation of the PD-(D/E)XK active site in all3650, Hjc, and NgoMVI. A) The predicted structure of all360 is shown in the same orientation as the crystal structures of the bona fide PD-(D/E)XK nucleases: B) Holliday junction resolvase Hje (1ob8 in PDB [9]) and C) REase NgoMIV (1fiu in PDB [78] to illustrate the spatial conservation of side-chains in the active site (the carboxylate residues in red and the Lys residue in blue), despite the lack of their conservation in the PD-EXX-K, PD-DXK, and PD-XXK-E variants of the sequence motif. Only the common core is shown, terminal regions and insertions have been omitted for clarity of the presentation.

Figure 5

Localization of COG4636+ family members in the chromosomes of Cyanobacteria with completed genomes. Circular chromosome maps of genomes with at least three genes encoding COG4636+ members (indicated by dots). Genes shown in dark blue are transcribed clockwise (positive reading frame) and those in red are transcribed anticlockwise (negative reading frame). Dots plotted inside the circle indicate that more than one gene is localized in the same region of the map (1/360 of the genome length).

Figure 6

The crystal structure of Tt1808 (1wdj in PDB). Tt1808 is shown in the same orientation and is colored and labeled in the same way as the homology model of all3650 on Figure 3. Two regions of differences between Tt1808 and the model of all3650 are indicated: the N-terminal subdomain has a similar fold, but different orientation (magenta line) and the C-terminal region folds as a β-harpin (cyan line) rather than as an α-helix.