Biophysical characterization of elongin C from Saccharomyces cerevisiae

Elongin C (ELC) is an essential component of the mammalian CBCVHL E3 ubiqui tin ligase complex. As a step toward understanding the role of ELC in assem bly 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 con junction with size exclusion chromatography showed that Elc1 is a nonglobul ar monomer over a wide range of concentrations. Pronounced line broadening in H-1, N-15-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformation ally labile. Measurement of N-15 NMR relaxation parameters including T-1, T -2, and the {H-1-N-15} nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange co ntributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical ex change contributions, suggesting that stabilization of helix 4 suppresses t he tendency of Elc1 to undergo conformational exchange on a micro- to milli second time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost complet ely eliminated chemical exchange processes, but induced substantial conform ational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins inclu ding the VHL protein by an induced fit mechanism involving the conformation ally flexible carboxy-terminal helix 4.