Date Published: August 27, 2018
Publisher: Public Library of Science
Author(s): Nicholas K. Hurlburt, Li-Hung Chen, Ioannis Stergiopoulos, Andrew J. Fisher, Yuanchao Wang.
Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4’s ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.
Effectors are intriguing and enigmatic proteins deployed by microbes during host-pathogen interactions [1, 2]. Although inhibition of plant immunity during host infection is the main function of these proteins, the manner by which individual effectors perform this task is poorly understood . One of the better understood effectors is CfAvr4, a 135-residue effector protein from the tomato pathogen Cladosporium fulvum, which utilizes a carbohydrate-binding module of family 14 (CBM14) to bind chitin present in fungal cell walls and protect it from hydrolysis by plant-derived chitinases during infection [3–5]. To date, functional orthologues of Avr4 have been identified in a number of fungal species within the Dothideomycete class of fungi and beyond, including the tomato pathogen Pseudocercospora fuligena , the banana pathogen Pseudocercospora fijiensis , and several others . The majority of Avr4 homologs share a similar cysteine-spacing pattern and contain a distinctive CBM14 domain in their structure, indicating that members of the Avr4 effector family have a conserved role in binding and protecting chitin in fungal cell walls against chitinases [4, 6]. Moreover, biochemical analysis between CfAvr4 and its PfAvr4 homolog from P. fuligena has shown that the specificity of these proteins extends further into binding the same length chito-oligosaccharide, i.e. (GlcNAc)6, suggesting that they share a similar binding-site topography and mechanism of interacting with the ligand .
During the past two decades, a great deal of effort has been placed towards deciphering the molecular determinants that define the structural basis of protein–carbohydrate interactions. Such interactions are ubiquitous in nature and at the heart of diverse biological processes of profound importance to human health, plant growth, and microbial disease . Although CBMs may interact with their oligosaccharide in various ways, generally they do not undergo conformational changes when binding to their ligands. Instead the tertiary structure provides a platform for substrate-binding. Binding-site topography is thus key to their binding mode and can be very diverse, ranging from planar surfaces with aromatic residues that stack against the pyranose rings of polysaccharides (Type A CBMs), to grooves or clefts that contain both aromatic and hydrogen-bonding interactions that accommodate long polysaccharide chains (Type B CBMs), and small pockets that bind short oligosaccharide ligands (Type C CBMs) . A recent refinement of these classes further proposed that Type B CBMs bind glycan chains internally (endo-type), whereas Type C modules interact with short mono-, di-, tri-saccharides or the termini of glycans (exo-type) .