Biochemical Characterization of RAR1 Cysteine- and Histidine-Rich Domains (CHORDs): A Novel Class of Zinc-Dependent Protein-Protein Interaction Modules

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• 作者：Charles T. Heise ; C&eacute ; cile S. Le Duff ; Marta Boter ; Catarina Casais ; Joanne E. Airey ; Andrew P. Leech ; B&eacute ; atrice Amigues ; Raphaë ; l Guerois ; Geoffrey R. Moore ; Ken Shirasu ; Colin Kleantho
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：February 13, 2007
• 年：2007
• 卷：46
• 期：6
• 页码：1612 - 1623
• 全文大小：436K
• 年卷期：v.46,no.6(February 13, 2007)
• ISSN：1520-4995

文摘

Disease resistance in plants requires the activation of defense signaling pathways to preventthe spread of infection. The protein Required for Mla12 Resistance (RAR1) is a component of suchpathways, which contains cysteine- and histidine-rich domains (CHORDs) that bind zinc. CHORDs are60 amino acid domains, usually arranged in tandem, found in almost all eukaryotes, where they are involvedin processes ranging from pressure sensing in the heart to maintenance of diploidy in fungi, and exhibitdistinct protein-protein interaction specificity. In the case of RAR1, CHORD-I is known to interact withheat-shock protein 90 (HSP90) and CHORD-II is known to interact with the Suppressor of the G2 alleleof Skp1 (SGT1). The focus of this work on RAR1 from barley and Arabidopsis was to address the paucityof 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 intramolecularinteractions. This was reinforced by denaturation experiments, where little difference between the stabilityof 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- andhistidine-containing motif (CCCH motif) located between the two CHORDs binds the fifth. Fluorescence,ultraviolet circular dichroism (UV CD), and nuclear magnetic resonance (NMR) spectroscopy furtherdemonstrated that zinc ions are essential for maintaining CHORD structure. Finally, we used isothermaltitratrion colarimetry to show that zinc is essential for the specific binding interactions of CHORD-IIwith SGT1. Our study provides the first biochemical and biophysical data on the zinc metalloproteinRAR1, defines its metal stoichiometry and that of its constituent CHORDs, and reveals that the metalions are essential for structural integrity and specific protein-protein associations.