examples, RIBONUCLEASE INHIBITOR (RI), and TRANS- PORT INHIBITOR-RESPONSIVE 1 (TIR1) are discussed here; the maps and analyses of the other nine protein groups are provided as Figure S1. For all of these RCM analyses, proteins were compared to at least three high-scoring homologs identified through BLAST and/or literature searches. An amino acid was considered to be interacting with ligand if identified in the article(s) accompanying the crystal structure(s) [66–76]. RI is one of the most extensively studied LRR-containing proteins, with many reported crystal structures and mutagenesis studies (for a review, see [77]). RI prevents ribonucleases (RNases) from acting by binding to their catalytic domains with femtomo- lar affinities. There are two solved structures of RI bound to ribonucleases, human RI complexed with angiogenin, and porcine RI complexed with RNaseA. Binding of angiogenin also induced homodimerization of RI. These two RIs, along with RI from rat and mouse, were used to generate the conservation map in Figure 2A. Residues involved in any of the three interactions (hRI- angiogenin, sRI-RNAse A, hRI-hRI) are highlighted in Figure 2A with asterisks. Although many ligand contact sites could be seen in the crystal structure, kinetic analyes of mutants in these regions demonstrated that mutation of the locations identified by RCM as most conserved, that is, the residues in the b-strand, b-turn region of LRRs 10, 12, and 14–16, have the largest impact on ligand binding [77]. TIR1 is an auxin receptor in Arabidopsis and other plants; it ubiquitinates target proteins when it binds auxin hormones in a pocket formed by the LRR domain of the protein and the cofactor inositol-6 phosphate (InsP6). To investigate recognition of auxin by these receptors, we constructed a conservation map of Arabidopsis TIR1 with its five paralogs. In this case, the five Arabidopsis paralogs (AUXIN SIGNALNG F-BOX (AFB) 1–5) have all been implicated in auxin signaling [78–81]. Again, RCM successfully highlighted two patches on the surface of the LRR (Figure 2B): one where TIR1 binds auxin, and a second site where TIR1 binds auxin as well as the cofactor InsP6 [68]. For comparison, it can be valuable to see the individual residue conservation scores for the RI and TIR1 protein sets (Figure S2), which are obtained after alignment of primary amino acid sequences, but before sliding- window calculation of regional conservation scores that are shown in Figure 2. Visual inspection of the RCM maps for the other validation proteins (Figure S1) again indicates successful identifi- cation by RCM of sites involved in LRR+ligand interactions, in follicle stimulating hormone receptor, glycoprotein 1b alpha, Skp2, Slit, and TLR3. Poor success was obtained for TLR1, TLR2, TLR4 and TLR6. However, the ligand specificity is not known for many of the homolog sequences that were used, and they may not have been appropriate proteins to compare via RCM (see Discussion). Continuing with the above validation, combined data were analyzed for all conservation maps generated for the above proteins with known LRR+ligand structures. The regional conservation scores for residue positions known to be involved in LRR+ligand interactions were significantly higher than the set of all other residue scores in each RCM map generated (Student’s T- test, p-value 0.005 or less). We also ranked the scores for each map into deciles and then determined the distribution of regional conservation scores, and individual residue conservation scores, for known LRR+ligand contact positions. Despite the presence of many potentially misleading TLR comparisons in the dataset, the distribution of scores for LRR+ligand interaction residues is weighted towards the highest ten percent of the RCM scores on their respective maps (Figure 3). Importantly, the proportion of scores in this decile increases when weighted pairwise comparisons Repeat Conservation Mapping Figure 2. Validation of RCM by mapping LRR domains for which there are solved crystal structures with ligand. For all maps, each row represents a single repeat of the LRR, with each colored box representing a solvent exposed (non-consensus) amino acid position. Each column corresponds to a position within the LRR consensus sequence, as denoted at the top of each map. The color in each box reports the center-weighted regional conservation score for the 565 set of boxes that centers on that box (see text for details); dark red indicates the most conserved regions and blue indicates the most divergent regions (see scale bar in Fig. 1). Bold black vertical lines delineate the five residues in each row that comprise the b-strand, b- turn region (the convex face of the LRR domain). White asterisks were added after RCM and indicate amino acid positions that are LRR-ligand contact points in solved crystal structures. (A) RCM output for Ribonuclease inhibitor (RI) from human, rat, mouse and pig ribonucle- ase inhibitor (RI) LRRs. (B) RCM output for TIR1 and AFB1-5 (auxin receptors) from Arabidopsis. doi:10.1371/journal.pone.0021614.g002 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e21614