to GALA-LRR and GL-LRR proteins, contain F-box domains (Figure 1A). Hence they can share functional similarity in that they recruit proteins, via their LRRs, to the SCF-type E3-ubiquitin ligase complex [6]. On the assumption of the membership of GALA and GL-LRRs in the CC-LRR subfamily, the previously proposed CC-LRR consensus [3] requires modifications. The updated CC-LRR consensus sequence is shown on Figure 1C. In this motif, Cys is not the only residue that occurs in positions 3 and 16: the other frequently occurring residues are Thr and Asn in position 3 and Leu, Asn and Ser in position 16. The updated CC-LRR often has Gly in addition to Thr in position 6. Finally, position 9 is frequently occupied by Gly. Database searches with the updated CC-LRR generalized profiles were able to detect such domains in heterogeneous group of about thousand proteins. Among these newly defined CC-LRR proteins there are not only proteins of animal or plant origin, but also proteins from pathogenic or non- pathogenic microorganisms such as bacteria (Parachlamydia sp.), protista (Dictyostelium) and fungi (Cryptococcus, Candida albicans, Candida glabrata, Neurospora). The sequence profiles and search results can be viewed on a dedicated web-page http://bioinfo. montp.cnrs.fr (Tools.Profiles.Show Profiles). Interestingly, about 600 of them have an F-box domain. This strongly supports a similar role for these specific LRRs in binding the protein substrate that is then recruited by the SCF-type E3-ubiquitin ligase. Structural implications The knowledge of even one 3D protein structure in a given sequence alignment provides a powerful means to test the correctness of the whole alignment. In our case, the 3D structure of one CC-LRR protein, the human F-box protein Skp2 is known [9,10] and was used to verify the alignment of GALA- and GL- LRRs with CC-LRRs. The analysis shows that all conserved and apolar residues of GALA- and GL-LRRs in the suggested alignment (positions 5, 10, 13, 19, 22 and 24) correspond to the residues of Skp2 CC-LRRs that form the hydrophobic core inside of the structure (Figures 1B and 2A). In the conserved position 3, GALA-LRRs have an Asn residue and GL-LRRs have a Thr residue instead of a Cys in CC-LRRs. This is an additional support for the alignment, because, in general, position 3 of LRRs tolerates a few amino acid residues including mentioned Asn, Thr and Cys. These residues being in position 3 can form specific hydrogen bonds with the peptide groups of the backbone. The two extra residues in the typical 26-residue-long CC-LRRs compared to the 24-residue-long GALA- and GL-LRRs, in the alignment are located in the loop regions of CC-LRRs connecting a-helices and b-strands (Figure 1B). The LRRs of Skp2 are variable in length and some of them are 1–2 residues shorter than typical 26-residue CC-LRR. The superposition of the 3D structures of these LRRs revealed that the loops are the most variable regions. In particular, missing residues of the short 24- and 25-residue LRRs of Skp2 are located in the loops. These structures represent good examples of how each of two loops of the CC-LRR can accommodate the loss of one residue. One of these short LRRs from Skp2 crystal structure (residue 2211 to 2235) was used as a template for construction of the GALA-LRR model (see Methods for details). Figure 2 shows structural models of a single GALA-LRR and a complete set of LRRs (12 repeats) from GALA4 of Ralstonia solanacearum (strain MolK2, personal communication C. Boucher and S. Genin). In Figure 2, the superposition of the GALA-LRR model and the crystal structure of Skp2 demonstrates that the difference between them is in the loops connecting a-helices and b- strands. The conserved asparagine of GALA-LRR (position 3 in Evolution of GALA Proteins Figure 2) similarly to the majority of the other LRR structures, located right after the b-strand so that it is able to form a network of specific hydrogen bonds with these NH and CO groups, thus satisfying their hydrogen-bonding potential in the hydrophobic core of the structure. The conserved bulky aliphatic residues form the hydrophobic core of the structure. The conserved small Ala and Gly residues allow a tighter side-by-side packing of a-helices. The modeling also shows that GALA-, GL-, and CC-LRRs can be packed well together and therefore, in contrast to the other LRR subfamilies, do not have mutually exclusive relationships with CC-LRRs in terms of inter-LRR packing (Figure 3). This conclusion is based on the following analysis. The conserved b- structural parts of the known crystal structures of LRR domains from different subfamilies and the model of GALA-LRRs were superimposed with the CC-LRR domain and the side-by-side packing of variable LRR fragments was analyzed (see Methods for details). The analysis shows that only the a-helices of GALA-LRR Figure 2. Structural model of GALA-LRR. (A) Ca-trace superposi- tion of a modeled GALA-LRR and the known CC-LRR from human Skp2 protein [10] and RI-LRR from porcine ribonuclease inhibitor [46]. GALA- LRR model is shown in a ball-and-stick representation, CC-LRR is shown by a blue trace and RI-LRR by a magenta trace. Numbering of the conserved GALA-LRR residues is taken from Figure 1. Numbers in red point to positions inferred to be under positive selection. The carbon atoms are in green, oxygen in red, nitrogen in blue. (B) A ribbon diagram of a structural model of the C-terminal LRR domain of GALA4 type III effector protein from R. solanacearum (strain MolK2, region 170 to 460, accession code ZP_00946474). The figure was generated with Pymol [47]. The atomic coordinates of the model are available on request. doi:10.1371/journal.pone.0001694.g002 PLoS ONE | www.plosone.org 3 February 2008 | Volume 3 | Issue 2 | e1694