Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein

Abstract

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.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization

“…Previous biochemical analysis revealed that Avr4 monomers require a stretch of at least three exposed GlcNAc residues for binding, and positive allosteric interactions among Avr4 molecules occur during chitin binding to facilitate the shielding of cell wall chitin against host chitinases [ 21 ]. Based on X-ray crystallography it was recently shown that two Avr4 molecules interact through their chitohexaose ligand to form a three-dimensional molecular sandwich that encapsulates two chitohexaose molecules within the dimeric assembly [ 23 ]. A crystal structure of Ecp6 revealed chitin-induced dimerization of two of the three LysM domains, resulting in the formation of an ultrahigh affinity (pM) chitin-binding groove, conferring the capacity to outcompete plant receptors for chitin binding [ 20 ].…”

The invisibility cloak: Chitin binding protein ofVerticillium nonalfalfaedisguises fungus from plant chitinases

“…These are thought to stabilize the interaction and contribute to submicromolar chitin binding affinity, as determined by SPR. Similarly, the recently solved crystal structure of fungal effector CfAvr4, a CBM14 lectin, in complex with chitin hexamer (Hurlburt et al 2018) has revealed that two effector molecules form a sandwich structure, which encloses two parallel stacked chitin hexamer molecules, shifted by one sugar ring, in an extended chitin binding site. In this complex, the interaction is mediated through aromatic residues and numerous hydrogen bonds with both side chains and main chains.…”

Proteinaceous effector discovery and characterization in filamentous plant pathogens

“…The C. fulvum effector Avr4 was one of the first to be characterized from a family of effectors that bind to and protect fungal cell‐wall chitin from host chitinase (Joosten et al ., 1997; van den Burg et al ., 2006). Recently the crystalline structure of Avr4 in complex with its chitin ligand (resolved to 1.95Å) has highlighted the residues required for this function (Hurlburt et al ., 2018). Structural mutant studies have also shown that recognition of the Avr4 by the cognate Cf‐4 immune receptor does not depend on the same ligand binding as previously thought (Hurlburt et al ., 2018).…”

“…Recently the crystalline structure of Avr4 in complex with its chitin ligand (resolved to 1.95Å) has highlighted the residues required for this function (Hurlburt et al ., 2018). Structural mutant studies have also shown that recognition of the Avr4 by the cognate Cf‐4 immune receptor does not depend on the same ligand binding as previously thought (Hurlburt et al ., 2018).…”