Origin and Evolution of GALA-LRR, a New Member of the CC-LRR Subfamily: From Plants to Bacteria? Andrey V. Kajava1*, Maria Anisimova2,3, Nemo Peeters4 1 Centre de Recherches de Biochimie Macromole´culaire, CNRS, University of Montpellier 1 and 2, Montpellier, France, 2 Institute of Computational Science, ETH Zurich, Zurich, Switzerland, 3 Swiss Institute of Bioinformatics, Lausanne, Switzerland, 4 Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR 441-2594 (INRA-CNRS), Castanet-Tolosan, France Abstract The phytopathogenic bacterium Ralstonia solanacearum encodes type III effectors, called GALA proteins, which contain F- box and LRR domains. The GALA LRRs do not perfectly fit any of the previously described LRR subfamilies. By applying protein sequence analysis and structural prediction, we clarify this ambiguous case of LRR classification and assign GALA- LRRs to CC-LRR subfamily. We demonstrate that side-by-side packing of LRRs in the 3D structures may control the limits of repeat variability within the LRR subfamilies during evolution. The LRR packing can be used as a criterion, complementing the repeat sequences, to classify newly identified LRR domains. Our phylogenetic analysis of F-box domains proposes the lateral gene transfer of bacterial GALA proteins from host plants. We also present an evolutionary scenario which can explain the transformation of the original plant LRRs into slightly different bacterial LRRs. The examination of the selective evolutionary pressure acting on GALA proteins suggests that the convex side of their horse-shoe shaped LRR domains is more prone to positive selection than the concave side, and we therefore hypothesize that the convex surface might be the site of protein binding relevant to the adaptor function of the F-box GALA proteins. This conclusion provides a strong background for further functional studies aimed at determining the role of these type III effectors in the virulence of R. solanacearum. Citation: Kajava AV, Anisimova M, Peeters N (2008) Origin and Evolution of GALA-LRR, a New Member of the CC-LRR Subfamily: From Plants to Bacteria? PLoS ONE 3(2): e1694. doi:10.1371/journal.pone.0001694 Editor: Cathal Seoighe, University of Cape Town, South Africa Received July 5, 2007; Accepted January 24, 2008; Published February 27, 2008 Copyright: 2008 Kajava 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: MA was supported by the BBSRC (UK) grant to Prof Ziheng Yang and, at finalizing stage of this study, by the ETH, Zurich. This work is partly supported by a 2007 ANR jeune-chercheur (FR) grant to NP. Competing Interests: The authors have declared that no competing interests exist. *E-mail: andrey.kajava@crbm.cnrs.fr Introduction Leucine-rich repeats (LRRs) are 20–29-residue sequence motifs present in a number of proteins with diverse functions [1,2]. In the 3D structures, each LRR corresponds to one coil of the solenoidal fold. The coils consist of a b-strand and mostly a-helical elements (can also be 310 helix or polyproline helix) connected by loops. The coils are arranged so that all the strands and helices are parallel to a common axis, resulting in a non-globular, horseshoe-shaped molecule with a curved parallel b-sheet lining the inner circumference of the horseshoe and the helices flanking the outer circumference. In LRR proteins, a six-residue motif LxxLxL is conserved (x can be any amino acid and L-positions can be occupied by Leu, Val, Ile, and Phe), and in the known structures corresponds to a turn and a consecutive b-strand; whereas the remaining parts of repeats may be very different. While the invariant motif of the b-region is a characteristic feature of the entire LRR superfamily, the consensus sequences of the variable part suggest several specific subfamilies. LRR proteins can be subdivided into at least seven subfamilies [1,3]. The repeats from different subfamilies retain a similar solenoidal fold and non- globular horseshoe shape but differ by 3D structures of individual repeats. Based on sequence analysis, it was concluded that LRRs from different subfamilies never occur concomitantly within one LRR protein [3]. This observation is explained by mutually exclusive inter-coil packing arrangement of LRRs from different subfamilies [3]. Such a relationship for LRRs suggests that LRR proteins of different subfamilies most probably have emerged independently during evolution rather than descended from a common ancestor. In line with this conclusion, the described LRR subfamilies could be assigned to a specific subgroup of eukaryotes or prokaryotes, and share similar functions and cellular locations [3]. For example, the bacterial LRR subfamily with the shortest known LRRs contains only extracellular proteins of Gram- negative bacteria. The Plant-Specific LRR (PS-LRR) subfamily has exclusively extracellular proteins from plants. Proteins of ribonuclease inhibitor-like LRR (RI-LRR) subfamily are intracel- lular and all belong to the Metazoa kingdom. Since 1998, when these conclusions were formulated, a large number of new LRR proteins have been identified and several new 3D structures of LRR proteins have been determined [1]. After a lapse of nine years, the classification of the LRRs and most of the previously made conclusions, including the mutual exclusive rule, withstand the test of time. At the same time, the analysis of some newly identified LRRs shows that their assignment within the existing classification of the LRR subfamilies may lead to confusion. Recently, it was shown that the phytopathogenic bacterium Ralstonia solanacearum encodes several type III effectors, called GALA proteins, that contain F-box and LRR domains [4,5]. The F-box domain enables the interaction with SKP1 in the SCF-type E3 ubiquitin ligase protein complex [6]. Their LRRs (hereafter PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1694