title: Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein.
abstract: The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.
authors.affiliation: Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xu
authors.forename: Karl I
authors.initials: KI
authors.lastname: Devlin
authors.forename: Michael G
authors.initials: MG
authors.lastname: Ford
authors.forename: Jay C
authors.initials: JC
authors.lastname: Nix
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.forename: Saurav
authors.initials: S
authors.lastname: Misra
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; C116331:HSPBP1 protein, human; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C510550:stub1 protein, zebrafish