Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein

Abstract

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

Figure 1

Disruption of elongin B and elongin C interaction by an elongin C–derived peptide. (a) ³⁵S-labeled elongin B in vitro translate was incubated with glutathione Sepharose preloaded with GST-elongin C in the absence of peptide (lane 2), in the presence of increasing amounts of a synthetic peptide corresponding to elongin C residues 17–50 (lanes 3–5; 0.5, 2.5, 10 μg, respectively), or in the presence of 10 μg of a sequence-scrambled elongin (17–50) peptide. (b) ³⁵S-labeled elongin C in vitro translate was incubated with glutathione Sepharose preloaded with GST-elongin B in the absence (lane 2) or presence (lanes 3–6) of elongin C peptides as in a. Bound proteins were resolved by SDS-PAGE and detected by fluorography. Comparable recovery of the GST fusion proteins in each lane was confirmed by Coomassie blue staining. Twenty-five percent of the in vitro translate used per binding reaction was loaded directly in lane 1.

Figure 2

Binding of elongin B mutants to elongin C. ³⁵S-labeled wild-type elongin B (1–118) or the indicated elongin B mutants incubated with glutathione Sepharose preloaded with GST-elongin C (lanes 6–10) or GST-elongin C (16–50) (lanes 11–15). Comparable recovery of GST-elongin C in each lane was confirmed by Coomassie blue staining and bound proteins were detected by fluorography. Twenty percent of the in vitro translate used per binding reaction was loaded directly in lane 1–5.

Figure 3

Binding of elongin C deletion mutants to pVHL(157–177). Binding assays with GST-VHL(157–177) (upper panel) or GST (lower panel) and wild-type or mutant elongin C were performed as described in Methods. Elo, elongin.

Figure 4

Effect of single amino acid alanine substitutions on interaction of VHL(157–171) peptide with elongin B and C. The 786-O renal carcinoma cells were metabolically labeled with ³⁵S-methionine, lysed, and incubated with glutathione Sepharose preloaded with GST (lane 1) or GST-VHL(117–213) (lanes 2–18) in the absence (lanes 1–2) or presence (lanes 3–18) of the indicated VHL(157–171) peptides (final peptide concentration of 1 and 10 μM in upper and lower panels, respectively). The arrows at the top of the figure indicate which amino acid residue was changed to alanine in the mutated VHL(157–171) peptides. Bound proteins were resolved by SDS-PAGE and detected by fluorography. Comparable recovery of the GST fusion proteins in each lane was confirmed by Coomassie blue staining. NS = nonspecific.

Figure 5

Effect of tumor-derived single amino acid substitutions on interaction of VHL(157–171) peptide with elongin B and C. The 786-O renal carcinoma cells were metabolically labeled with ³⁵S-methionine, lysed, and incubated with glutathione Sepharose preloaded with GST (lane 1) or GST-VHL(117–213) (lanes 2–17) in the absence (lanes 1–2) or presence (lanes 3–17) of the indicated VHL(157–171) peptides. Wild-type VHL (0.1, 1, 10 μg of residues 157–171) was added in lanes 3–5, respectively. Mutant peptides were added to 10 μg. The single amino acid substitutions in the mutated VHL(157–171) peptides are indicated at the top of the figure. Bound proteins were resolved by SDS-PAGE and detected by fluorography. Comparable recovery of the GST fusion proteins in each lane was confirmed by Coomassie blue staining. NS = nonspecific.

Figure 6

Inhibition of elongin/SIII activity by pVHL-derived peptides. (a) Runoff transcription reactions containing elongin A and elongin BC were performed as described in Methods. All reactions included 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-³²P] CTP, and 2 μM UTP. The reaction in the first lane did not include VHL peptide. Reactions in the remaining lanes included 12 nM, 40 nM, 120 nM, or 400 nM of the indicated peptide. AdML indicates the position of the approximately 250 nucleotides runoff transcript initiated at the AdML promoter. (b) Runoff transcription reactions were performed in the presence of elongin A or elongin A and elongin BC as indicated, with 0, 100 nM, 200 nM, and 400 nM of the indicated peptide, with 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-³²P] CTP, and 2 μM UTP. Reactions labeled High NTPs were performed in the absence of elongin A and elongin BC and in the presence of 0 (–) or 600 nM (+) of the wild-type peptide and 50 μM ATP, 50 μM GTP, 10 μM CTP, 10 μCi [α-³²P] CTP, and 50 μM UTP. Elo, elongin.

Figure 7

Naturally occurring pVHL(157–171) domain mutants exhibit reduced binding to elongin B and Cul2. The 786-O renal carcinoma cells were transfected to produce HA-tagged versions of wild-type pVHL (lane 2) or the indicated pVHL mutants. Anti-HA immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Cul2 (top panel), anti-HA (middle panel), or anti–elongin B (lower panel).

Figure 8

Characterization of naturally occurring pVHL mutants with an intact pVHL(157–171) domain. The 786-O renal carcinoma cells were transfected to produce HA-tagged versions of wild-type pVHL (lane 2) or the indicated pVHL mutants. (a) Whole-cell extracts were resolved by SDS-PAGE and analyzed for Glut1 (top panel) and pVHL (lower panel) production by anti-Glut1 and anti-HA immunoblot analysis, respectively. (b) In parallel, anti-HA immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Cul2 antibodies (top panel) or anti-HA antibodies (lower panel).

Figure 9

Structure of pVHL-elongin C interface. The pVHL and elongin C secondary structure elements are shown in dark blue and fuchsia, respectively. The pVHL side chains contacting elongin C are shown in salmon, and elongin C side chains are shown in light blue. The pVHL residue T157 is not shown because it would obscure the depiction of some of the other contacts formed between pVHL and elongin C in this figure. Note that pVHL T157 makes low-density van der Waals contacts with elongin C residues C112 and Y76.