title: Biochemical characterization of RAR1 cysteine- and histidine-rich domains (CHORDs): a novel class of zinc-dependent protein-protein interaction modules.
abstract: Disease resistance in plants requires the activation of defense signaling pathways to prevent the spread of infection. The protein Required for Mla12 Resistance (RAR1) is a component of such pathways, which contains cysteine- and histidine-rich domains (CHORDs) that bind zinc. CHORDs are 60 amino acid domains, usually arranged in tandem, found in almost all eukaryotes, where they are involved in processes ranging from pressure sensing in the heart to maintenance of diploidy in fungi, and exhibit distinct protein-protein interaction specificity. In the case of RAR1, CHORD-I is known to interact with heat-shock protein 90 (HSP90) and CHORD-II is known to interact with the Suppressor of the G2 allele of Skp1 (SGT1). The focus of this work on RAR1 from barley and Arabidopsis was to address the paucity of biochemical information on RAR1 and its constituent CHORDs, particularly the role of the metal ion. Sedimentation experiments indicated RAR1 to be an extended monomer in solution with few intramolecular interactions. This was reinforced by denaturation experiments, where little difference between the stability of the individual domains and intact RAR1 could be detected by intrinsic tryptophan fluorescence. Electrospray ionization-mass spectrometry and atomic absorption showed that, contrary to previous reports, RAR1 binds five zinc ions; each CHORD binds two, and the plant-specific, 20 amino acid cysteine- and histidine-containing motif (CCCH motif) located between the two CHORDs binds the fifth. Fluorescence, ultraviolet circular dichroism (UV CD), and nuclear magnetic resonance (NMR) spectroscopy further demonstrated that zinc ions are essential for maintaining CHORD structure. Finally, we used isothermal titratrion colarimetry to show that zinc is essential for the specific binding interactions of CHORD-II with SGT1. Our study provides the first biochemical and biophysical data on the zinc metalloprotein RAR1, defines its metal stoichiometry and that of its constituent CHORDs, and reveals that the metal ions are essential for structural integrity and specific protein-protein associations.
authors.affiliation: Department of Biology, University of York, Post Office Box 373, York, YO10 5YW, United Kingdom.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Heise
authors.forename: Cécile S
authors.initials: CS
authors.lastname: Le Duff
authors.forename: Marta
authors.initials: M
authors.lastname: Boter
authors.forename: Catarina
authors.initials: C
authors.lastname: Casais
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Airey
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Leech
authors.forename: Béatrice
authors.initials: B
authors.lastname: Amigues
authors.forename: Raphaël
authors.initials: R
authors.lastname: Guerois
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
authors.forename: Ken
authors.initials: K
authors.lastname: Shirasu
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005964:Glucosyltransferases / Q000737:chemistry; D001467:Hordeum / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic; D014461:Ultracentrifugation; D015032:Zinc / Q000737:chemistry*
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C499581:PBS2 protein, Arabidopsis; D010940:Plant Proteins; C499582:Rar1 protein, Hordeum vulgare; D005964:Glucosyltransferases; C515668:SGT1 protein, Arabidopsis; D015032:Zinc