Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

Abstract

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

Plant Lectins and Lectin Receptor-Like Kinases: How Do They Sense the Outside?

“…Modelling of the LecRK-I.9 lectin domain confirms the β-sandwich fold of legume lectins, the conserved residues involved in the Ca 2+ and Mn 2+ binding, and the potential carbohydrate-binding site [18,80,100]. The ASYY sequence which is supposed to bind the RGD motif of IPI-O is located in an exposed loop connecting strands β8 and β9, in a position allowing ligand recognition [80].…”

“…The ASYY sequence which is supposed to bind the RGD motif of IPI-O is located in an exposed loop connecting strands β8 and β9, in a position allowing ligand recognition [ 80 ]. The latter loop is only found in the Brassicaceae family [ 100 ]. Docking experiments with ATP show that the ATP binding site has the same location as that of carbohydrates, but the amino acid residues predicted to interact are different [ 100 ].…”

Cereal Root Interactions with Soilborne Pathogens—From Trait to Gene and Back

“…The action of extracellular ATP in Arabidopsis, acting through its receptor DORN1, has led us to consider characterizing the wheat orthologue of DORN1 in interactions with nematodes and fungal necrotrophic root pathogens. A collection of wheat and barley orthologues predicted to contain lectin-binding, kinase, and transmembrane domains were obtained from published literature [113,114]. Candidate wheat or barley orthologues were identified using HMMER 3.0 [115] and TMHMM [116] and databases IWGSC URGI and International Barley Sequencing Consortium genome coding sequences [110], for wheat and barley, respectively.…”

“…Boxes delineate a conserved transmembrane domain (TM) and kinase domains (KD). Domain KD-X is poorly conserved and is not indicated[113,114].…”

Purinoceptor P2K1/DORN1 Enhances Plant Resistance Against a Soilborne Fungal Pathogen, Rhizoctonia solani

“…In response to cellular disruption by wounding, necrotizing invasion or predation, ATP is released into the apoplast and perceived by the plant cell surface purinoceptor P2K1 (also known as DORN1) ( Choi et al, 2014 ). Upon ATP binding to the extracellular lectin domain of the purinoceptor ( Nguyen et al, 2016 ), its intracellular kinase domain is activated ( Chen et al, 2017 ), which subsequently results in the activation of a number of intracellular signaling pathways for the reprogramming of many plant defense-related genes ( Jewell et al, 2019 ). Overexpression of P2K1 enhances plant resistance against various foliar pathogens, such as Phytophthora brassicae (biotrophic oomycete), Pseudomonas syringae (hemibiotrophic bacterium), and Botrytis cinerea (necrotrophic fungus ) ( Bouwmeester et al, 2011 ; Balagué et al, 2017 ; Chen et al, 2017 ; Tripathi et al, 2018 ), suggesting that extracellular ATP plays an important role in plant defense against a broad range of pathogens.…”