Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C

Abstract

Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

Figure 1

Amino acid sequences and alignment of yeast Elc1 (Yeast C) with rat elongin C (Rat C) and yeast Skp1 (Yeast Skp1). (A) blast alignment of rat elongin C and yeast Elc1. Identical residues are indicated by a colon. Similar residues are indicated by a period. The symbols [ ], ( ), and { } indicate the sites in rat elongin C that were previously reported to be involved in elongin B binding, elongin A/B/C complex formation, and elongin A activation, respectively (13). (B) Alignment of yeast Skp1 with yeast Elc1. Identical residues are indicated by a colon. Similar residues are indicated by a period. The yeast Elc1 construct used in our studies has an additional three residues, GSH, at the N terminus for a total of 102 residues.

Figure 2

¹⁵N HSQC spectra of ¹⁵N-labeled Elc1. (A) Free. (B) VHL(157–171)-bound. (C) Ela1(1–143)-bound. (D) Ela1(3–17)-bound. Peaks assigned are labeled. Samples A, B, and D are 0.7–1.5 mM in 10 mM NaP_i, pH 7.0/100 mM NaCl/10 μM ZnSO₄/100 μM EDTA/7.5 mM DTT in 90% H₂O/10% D₂O. Sample C is 0.7 mM in similar buffer but at pH 7.5 and 500 mM NaCl. Experiments were run at 30°C (A, B, and D) or 25°C (C).

Figure 3

Plots of consensus CSI values for free Elc1 (A) and VHL(157–171)-bound Elc1 (B). CSI values of +1 reflect residues with helical backbone conformation, and values of −1 reflect regions with extended (β-type) backbone conformation. Unassigned residues are given the value of +0.2. Predicted helices are represented by coiled ribbons and β-strands by arrows.

Figure 4

Schematic representation of β-strand interactions in Elc1. Inter-strand long-range NOEs are indicated by arrows.