title: Biophysical characterization of elongin C from Saccharomyces cerevisiae.
abstract: Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.
authors.affiliation: Department of Molecular Cell Biology, Max Planck Institute for Biochemistry, Martinsried, Germany [corrected].
authors.forename: A
authors.initials: A
authors.lastname: Buchberger
authors.forename: M J
authors.initials: MJ
authors.lastname: Howard
authors.forename: S M
authors.initials: SM
authors.lastname: Freund
authors.forename: M
authors.initials: M
authors.lastname: Proctor
authors.forename: P J
authors.initials: PJ
authors.lastname: Butler
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
authors.forename: M
authors.initials: M
authors.lastname: Bycroft
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D000075065:Elongin; D006801:Humans; D008025:Ligases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012996:Solutions; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014270:Trifluoroethanol / Q000494:pharmacology; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014461:Ultracentrifugation; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D006623:von Hippel-Lindau Disease / Q000378:metabolism
chemical_list: C000616395:ELOC protein, human; D000075065:Elongin; D010446:Peptide Fragments; D011506:Proteins; D012996:Solutions; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014270:Trifluoroethanol; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human