Ligand-triggered allosteric ADP release primes a plant NLR complex

INTRODUCTION Nucleotide-binding (NB), leucine-rich repeat (LRR) receptor (NLR) proteins constitute a family of intracellular immune receptors in both animals and plants that detect the presence of pathogen molecules or host-derived signals. NLRs share a conserved tripartite domain structure with a conserved central NB and oligomerization domain (NOD), a C-terminal LRR domain, and a variable N-terminal domain. The NOD module can be further divided into an NB domain (NBD), a helical domain (HD1), and a winged-helix domain (WHD). In plants, direct or indirect recognition of pathogen effectors by NLRs induces numerous defenses, including programmed cell death called hypersensitive response, and restricts pathogens to the infection site. For instance, the coiled-coil (CC)–NLR HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) of the small mustard plant Arabidopsis thaliana forms a preactivation complex with resistance-related kinase 1 (RKS1, a pseudokinase belonging to receptor-like cytoplasmic kinase subfamily XII-2) and recognizes the uridylyltransferase effector AvrAC from the pathogen Xanthomonas campestris pv. campestris that is responsible for the black rot disease of crucifiers. AvrAC uridylates a number of host protein kinases, including the PBS1-like protein 2 (PBL2) kinase. PBL2 UMP , the version of the Arabidopsis protein uridylated by AvrAC, then acts as a ligand of the preformed ZAR1-RKS1 complex. NLRs are believed to function as a nucleotide [adenosine diphosphate (ADP) or adenosine triphosphate (ATP)]–operated molecular switch, with ADP- and ATP-bound forms corresponding to the “off” and “on” states, respectively, but the mechanism of how ADP is released from an NLR for exchange with ATP remains elusive. Structural elucidation of a full-length plant NLR protein and its recognition of modified self is lacking. RATONALE We reconstituted a ZAR1-RKS1 and a ZAR1-RKS1-PBL2 UMP complex and determined their cryo–electron microscopy (cryo-EM) structures at resolutions of 3.7 and 4.3 A, respectively. The structures were verified by biochemical, cell-based, and functional data. We determined how PBL2 UMP affects the ADP-binding activity of the ZAR1-RKS1 complex by radiolabeled assays. Structural comparison of the ZAR1-RKS1 and ZAR1-RKS1-PBL2 UMP complexes was used to probe the mechanism of PBL2 UMP -induced ADP release from ZAR1, which was further validated by biochemical assays. RESULTS The cryo-EM structure of the ZAR1-RKS1 complex revealed that intramolecular interactions within ZAR1 maintain the NLR protein in an inactive state. The inactive state is further stabilized by an ADP. The LRR domain of ZAR1 (ZAR1 LRR ) is positioned differently from LRR domains of animal NLRs but functions similarly to sequester ZAR1 in a monomeric state. ZAR1 CC appears to be kept in an inactive state via contacts with ZAR1 LRR , ZAR1 HD1 , and ZAR1 WHD . This contrasts with the flexible N-terminal domain of inactive apoptotic protease-activating factor 1 (Apaf-1). ZAR1 LRR mediates interaction with RKS1 in the preformed ZAR1-RKS1 complex. The ZAR1-RKS1-PBL2 UMP structure shows that RKS1 is exclusively responsible for the binding of PBL2 UMP . The two uridylyl moieties of PBL2 UMP interact with and consequently stabilize the activation segment of RKS1. Comparison of the two cryo-EM structures shows that the stabilized activation segment of RKS1 sterically clashes with the ADP-bound ZAR1 NBD from the ZAR1-RKS1 complex, resulting in conformational changes in the NBD but not other domains of ZAR1: ZAR1 NBD is rotated outward about 60° compared with that from the inactive ZAR1. Thus, PBL2 UMP allosterically induces release of ADP from the ZAR1-RKS1-PBL2 UMP complex. Indeed, radiolabeling assays showed that PBL2 UMP , but not PBL2, reduced the ADP-binding activity of the ZAR1-RKS1 complex. CONCLUSION Our study revealed the mechanisms of PBL2 UMP recognition by ZAR1-RKS1 and PBL2 UMP -induced priming of ZAR1, providing a structural template for understanding NLR proteins.