LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains Laura Helft1, Vignyan Reddy2, Xiyang Chen3¤, Teresa Koller3, Luca Federici4, Juan Ferna´ ndez-Recio5, Rishabh Gupta2, Andrew Bent1,3* 1 Program in Cellular and Molecular Biology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America, 2 Department of Electrical and Computer Engineering, University of Wisconsin - Madison, Madison, Wisconsin, United States of America, 3 Department of Plant Pathology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America, 4 Dipartimento di Scienze Biomediche, Universita` ‘G. d’Annunzio’, Chieti, Italy, 5 Life Sciences Department, Barcelona Supercomputing Center, Barcelona, Spain Abstract Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/ RCM, and the approach should be modifiable for use with other types of repeat protein domains. Citation: Helft L, Reddy V, Chen X, Koller T, Federici L, et al. (2011) LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains. PLoS ONE 6(7): e21614. doi:10.1371/journal.pone.0021614 Editor: Miguel A. Blazquez, Instituto de Biolog´ıa Molecular y Celular de Plantas, Spain Received March 11, 2011; Accepted June 3, 2011; Published July 18, 2011 Copyright: 2011 Helft et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by U.S. Department of Energy Basic Energy Sciences Grant DE-FG02-02ER15342 to AB, and by an National Science Foundation Predoctoral Fellowship to LH. Support from MIUR of Italy (PRIN2007K7KY8Y) to LF is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: afbent@wisc.edu ¤ Current address: Department of Computer Science, Purdue University, West Lafayette, Indiana, United States of America Introduction The conservative nature of evolution causes selection of stable structures that nevertheless are modifiable for operation in varied processes. Proteins carrying repetitive domains such as leucine rich repeats (LRRs), ankyrin repeats or tetratricopeptide repeats are one common solution to these evolutionary demands [1–3]. A single class of repeat domain can interact with a wide array of chemically distinct ligands, yet each particular repeat protein shows high specificity for particular ligands. Consensus amino acid motifs have been identified for these repeat domains [1–5]. The consensus amino acids within a particular type of repeat provide a regular, stable scaffold to the domain, while non-consensus residues within the repeat allow variability in function [1]. The characteristic structure formed by a particular type of repetitive motif is identifiable by comparison of multiple solved protein structures, and can be used to predict the overall configuration of other protein domains sharing that repetitive motif. However, methods to query the variable portions of these repeat domains, to identify and understand the sites that control specialized functions, are less well developed. LRR domains are a protein-ligand interaction domain found in many types of prokaryotic, eukaryotic, and viral proteins, including ubiquitin ligases, hormone receptors, enzyme inhibitors, and immune receptors in plants and animals [1,6–12]. As anno- tated by Pfam, more than 500 different proteins encoded by the human genome contain LRRs, and there are over 1000 types of LRR-containing proteins in individual plants such as Arabidopsis thaliana or rice [4]. The ubiquity of this domain may be due to its ability to interact with a wide range of substrates including proteins, nucleic acids, lipids, and small molecule hormones. It is particularly compelling that in jawless vertebrate adaptive immune systems, antibodies are produced by shuffling hypervariable LRR repeats [13,14]. The resultant receptors recognize a wide range of substrates with high affinity. Any single LRR domain can poten- tially interact with several different molecules, either simulta- neously or asynchronously. There are multiple sub-families of LRR domain types, each with slightly different consensus amino acid motifs, but all share the tendency to form a solenoid structure with approximately 20– 30 amino acids per repeat where each repeat forms one turn of the helix (Figure 1A; an example of an LRR consensus motif is PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e21614