title: LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains.
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.
authors.affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: Helft
authors.forename: Vignyan
authors.initials: V
authors.lastname: Reddy
authors.forename: Xiyang
authors.initials: X
authors.lastname: Chen
authors.forename: Teresa
authors.initials: T
authors.lastname: Koller
authors.forename: Luca
authors.initials: L
authors.lastname: Federici
authors.forename: Juan
authors.initials: J
authors.lastname: Fernández-Recio
authors.forename: Rishabh
authors.initials: R
authors.lastname: Gupta
authors.forename: Andrew
authors.initials: A
authors.lastname: Bent
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D000090263:Leucine-Rich Repeat Proteins; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051192:Receptors, Pattern Recognition / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid
chemical_list: D029681:Arabidopsis Proteins; C545106:EFR protein, Arabidopsis; D000090263:Leucine-Rich Repeat Proteins; D008024:Ligands; C081479:PGIP protein, plant; D010940:Plant Proteins; D011506:Proteins; D051192:Receptors, Pattern Recognition; D011494:Protein Kinases; C411853:FLS2 protein, Arabidopsis