Structural and functional study Of key proteins in animal and plant innate immunity

Abstract

Innate immunity is used by both plants and animals to defend themselves against pathogens and prevent diseases. Plants, which lack an adaptive immune system present in animals, rely exclusively on the innate immune system. In this study, we used structural biology to investigate important proteins in the innate immune systems of both plants and animals. Plants are engaged in a continuous battle against plant pathogens, which affects human activities, especially our agriculture. As an outcome of these battles, plants have evolved a sophisticated immune system to detect pathogens. As a result of the immune response, plant resistance (R) proteins recognize specific pathogen proteins (effectors) to trigger the effector-triggered immune response (ETI). The biological functions of effector proteins and the molecular basis of how R proteins are activated and signal, are poorly understood. A major sub-family of R proteins contain a Toll/interleukin-1 receptor (TIR) domain at the N-terminus. Structural and functional analysis of the R proteins L6 and RPS4 has previously shown that the TIR domain region is both necessary and sufficient for triggering ETI, and that TIR domain self-association is required for signalling. Here I report the crystal structure of the TIR domain from the Arabidopsis R-like protein SNC1. Analysis of the structure combined with site-directed mutagenesis reveals two distinct dimerization interfaces. Both interfaces have recently been shown to be important for signaling, but this is the first time that these two interfaces have been shown to exist in the same R protein. The structure therefore provides a unique model for TIR domain association. The molecular functions of most fungal effector proteins have not been identified. AvrP from flax-rust is recognised by the flax resistance protein P and is a small secreted cysteine-rich protein of unknown function with sequence similarity to disulfide-containing Kazal protease inhibitors. However, homology modeling and biochemical studies suggest that AvrP is not disulfide-bonded and may instead be structurally similar to plant homeodomain (PHD) zinc-finger proteins. Using zinc as an additive, we obtained crystals of AvrP diffracting to 2.5 A resolution and determined the three-dimensional structure using experimental phasing. The structure of AvrP reveals a novel zinc-binding fold with some limited similarities to DNA-binding proteins. The zinc-coordinating region of the structure displays a positively charged surface. The polymorphic residues in the AvrP family that are associated with R protein recognition differences map to the surface of AvrP and form surface patches that may mediate host recognition. The structure of AvrP provides insights into possible pathogen-associated functions of this protein in the plant-associated immune response. In animals, Toll-like receptors (TLRs) recognize invading pathogens and initiate innate immune responses. TLRs protect the host from diverse diseases, but are also associated with a range of disorders including inflammatory diseases, making them attractive therapeutic targets. To initiate the innate immune response, TLRs associate with adaptor proteins through TIR domain interactions. The TIR-containing adaptor family of the TLR signaling pathway consists of five key members including MyD88, Mal, TRIF, TRAM and SARM. SARM (sterile alpha and armadillo-motif containing) protein consists of N-terminal armadillo motifs (ARM) domain, two central sterile-a motif (SAM) domains and a C-terminal TIR domain. SARM has been shown to be involved in the negative regulation of Toll-like receptor signaling, inflammation-driven apoptosis and axon degeneration. However the molecular functions and signaling pathways of SARM remain poorly understand. To investigate the structural basis of SARM functions, we set out to determine the crystal structure of SARM. Constructs were designed to produce the full-length human SARM protein and fragments containing individual domains in E coli. Protein constructs of both the tandem SAM domains and the TIR domain have been produced in a soluble form using the E. coli expression system, and they have been purified to homogeneity. The tandem SAM domains form a stable octamer in solution, while the TIR domain is monomeric. Crystals of the tandem SAM domains have been obtained, and a dataset (2.5 A resolution) has been collected at the Australian Synchrotron. Our attempts to solve this structure by molecular replacement have so far been unsuccessful, and we will use experimental phasing methods to determine the structure.