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

Nicholas K. Hurlburt, Li Hung Chen, Ioannis Stergiopoulos, Andrew J Fisher

Research output: Contribution to journalArticle

5 Citations (Scopus)

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)6binding 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)6molecules 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)6positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6enabled 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.

Original languageEnglish (US)
Article numbere1007263
JournalPLoS Pathogens
Volume14
Issue number8
DOIs
StatePublished - Aug 1 2018

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Cladosporium
Ligands
Chitin
Proteins
Carbohydrates
Chitinases
X Ray Crystallography
Lycopersicon esculentum
Site-Directed Mutagenesis
Proteolysis
Immunity
Amino Acids
Infection
chitohexaose

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein. / Hurlburt, Nicholas K.; Chen, Li Hung; Stergiopoulos, Ioannis; Fisher, Andrew J.

In: PLoS Pathogens, Vol. 14, No. 8, e1007263, 01.08.2018.

Research output: Contribution to journalArticle

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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 1.95{\AA} resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6binding 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)6molecules 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)6positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6enabled 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.",
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