Tumorigenic Mutations in VHL Disrupt Folding In Vivo by Interfering with Chaperonin Binding  Douglas E. Feldman, Christoph Spiess, Daniel E. Howard, Judith Frydman  Molecular Cell  Volume 12, Issue 5, Pages 1213-1224 (November 2003) DOI: 10.1016/S1097-2765(03)00423-4

Figure 1 Two Hydrophobic β Strands in VHL Mediate Stable Association with TRIC (A) A panel of 35S-labeled VHL mutants, each harboring four consecutive alanine residues (Ala-4), was translated in reticulocyte lysate. TRiC binding was evaluated either by anti-TRiC immunoprecipitation (B) or by nondenaturing PAGE analysis (C). (B) TRiC binding of VHLΔC Ala-4 variants (lanes 1–14) analyzed by immunoprecipitation with TRiC-specific antibodies (top panel). As controls for antibody specificity, wild-type VHLΔC was immunoprecipitated with either nonimmune serum (NI, lane 15) or anti-TRiC antibody (lane 16). Bottom panel (Total) shows input for all reactions. (C) Nondenaturing PAGE analysis of full-length VHL Ala-4 mutants. Full-length wild-type VHL (lane 2), VHL-L158P (lane 1), or VHL Ala-4 substitution mutants (lanes 3–8), translated in reticulocyte lysate (bottom panel), were resolved by nondenaturing PAGE (top panel). The migration of the VHL-TRiC complex (top arrow), folded VBC (bottom arrow), and putative misfolded VBC complexes (*) are indicated. (D) Location of Box 1 and Box 2 in VHL structure. Folded VHL (in the VBC complex) contains a β domain (cyan), consisting of antiparallel β strands, and an α domain that binds elongin BC. Boxes 1 and 2 (red) are in adjacent β strands buried within the folded β domain. L63 and E204, the N- and C-terminal boundaries of the known VHL structure, are indicated. (E) Fine mapping of TRiC binding interface in Box 1 and Box 2. Equivalent amounts of wild-type VHLΔC (amino acids 1–160) (lane 1), VHLΔC variants containing the indicated Ala-4 substitutions (lanes 2, 7, and 8), or the indicated single alanine substitutions in Box 1 (lanes 3–6) and Box 2 (lanes 10–15) were immunoprecipitated with anti-TRiC antibodies (top panel) or analyzed by SDS-PAGE (middle panel) or nondenaturing PAGE (bottom panel). The VHL-TRiC complex is indicated (bottom panels, arrow). (F) Structural basis of TRiC binding sites in Box 1 and Box 2. Amino acids with side chains required for stable association with TRiC, identified in (E) and highlighted in red, are shown in two views, as they appear in the native VHL structure. Key TRiC binding residues contain hydrophobic side chains that together project in the same direction, revealing a putative hydrophobic binding interface with the chaperonin. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 2 Hydrophobic Nature of the VHL-TRiC Interaction (A) The effect of the indicated cations, arranged according to their chaotropic properties, on TRiC binding to VHLΔC (amino acids 1–160) (B) or the VHL[99–155]-DHFR fusion protein (C) was analyzed either by anti-TRiC immunoprecipitation (B) or by gel filtration chromatography (C). (B) 35S-labeled VHLΔC was immunoprecipitated with anti-TRiC antibody, and immune complexes were washed with buffer supplemented with the indicated salts, shown in order of ascending chaotropic stringency. (C) 35S-labeled VHL[99–155]-DHFR, translated in reticulocyte lysate, was subjected to gel filtration chromatography in buffer supplemented with the indicated salts. Top panel: Elution profile for VHL[99–155]-DHFR fusion analyzed under three different salt conditions. A high molecular weight complex of TRiC-bound VHL-DHFR (11.5–13 ml elution volume) remains intact in the presence of NaCl (lower panel) but is dissociated in buffer supplemented with MgSO4, which destabilizes hydrophobic interactions (bottom panel). Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 3 Tumor-Causing Mutations in VHL Box 1 and Box 2 Impair Binding to TRiC (A) Schematic representation of VHL primary structure indicating the position of Box 1, Box 2, the elongin BC binding site, and some of the tumorigenic mutations used in this study. (B) VHL tumor mutants in Box 1 and 2 destabilize association with TRiC. 35S-labeled wild-type VHLΔC (lane 1) or VHLΔC tumor-derived point mutant variants (lanes 2–9), translated in reticulocyte lysate (bottom panel), were analyzed by anti-TRiC immunoprecipitation (top panel). (C) Impairment of TRiC binding by VHL tumor mutants in vivo. HEK293 cells expressing Flag-tagged wild-type VHL (lane 2) or tumor mutant variants (lanes 3–10) or containing the backbone vector plasmid (lane 11) were labeled with 35S-methionine, lysed, and immunoprecipitated with anti-TRiC (lane 1) or anti-Flag (lane 2–11) antibodies. TRiC, Hsp/Hsc70, VHL, and VHL-Δexon 2 are indicated. An uncharacterized 50 K band that also associates with VHL is also indicated (*). (D) Direct binding of VHL Box 1 and Box 2 to TRiC is destabilized by tumorigenic mutations. Pure TRiC (0.2 μM) was incubated with 2 μM purified GST (Ctrl, lanes 1 and 6) or with GST fused to a short VHL peptide containing either Box 1 (amino acids 112–121, lane 3) or Box 2 (amino acids 145–155, lane 8) or tumor-causing mutations in Box 1 (lanes 4 and 5) and Box 2 (lanes 9 and 10). Following gel filtration chromatography, the fractions containing TRiC (11–13 ml) were analyzed by anti-GST immunoblotting (top panel). As controls, equivalent amounts of GST-Box 1(lane 2) or GST-Box 2 (lane 7) fusions were incubated in buffer alone and analyzed in a similar manner. Bottom panel shows 5% of total input material. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 4 Tumor-Causing Mutations in VHL Box 1 and Box 2 Impair Folding of Newly Synthesized VHL (A) VHL Box 1 and Box 2 mutants are in a protease-labile conformation. 35S-labeled full-length wild-type VHL (lane 1) or VHL tumor mutant variants (lanes 2–9), translated in rabbit reticulocyte lysate (bottom panel) and subjected to limited protease digestion with either chymotrypsin (top panel) or trypsin (middle panel). (B) Nondenaturing PAGE analysis of VHL Box 1 and Box 2 tumor mutants. Wild-type VHL (lane 1) and a panel of tumor-derived VHL mutants (lanes 2–9) translated in reticulocyte lysate as in (A) (bottom panel) were analyzed by nondenaturing gel electrophoresis (top panel). A no-mRNA control translation, where no translated protein is generated, is also included (lane 10) to highlight the background radioactivity (probably free [35S]methionine) retained at the origin. The migration of TRiC-VHL (top arrow), folded VBC, and unidentified complexes of mutant VHL (a–c) are indicated. (C) VHL Box 1 and Box 2 tumor mutants do not assemble into a correctly folded VBC complex. 35S-labeled wild-type VHL (lane 2) or VHL tumor mutant variants (lanes 1 and 3–12) were translated in lysate supplemented with BC-His6 (10 μM) (bottom panel). The extent of incorporation of [35S]VHL variants into correctly folded VBC-His6 (top panel) was assessed by stringent pull-down with metal affinity resin. (D) VHL Box 1 and Box 2 tumor mutants disrupt VBC complex formation in vivo. HEK293 cells coexpressing additional elongin BC (lanes 3–13) and Flag-tagged wild-type VHL (lanes 2 and 3) or VHL tumor mutant variants (lanes 4–12) or containing the backbone vector plasmid (lanes 1 and 13) were labeled with 35S-methionine, lysed, and immunoprecipitated with anti-TRiC (lane 1) or anti-Flag (lane 2–13) antibodies. TRiC, Hsp/Hsc70, elongin BC, and VHL are indicated. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 5 VHL Box 1 and Box 2 Tumor Mutants Are Associated with Hsp70 In Vivo HEK293 cells expressing Flag-tagged wild-type VHL (A) or tumor mutant variants G114R (B) or I151S (C) were labeled as above and analyzed by gel filtration chromatography. VHL and VHL-associated proteins were detected by immunoprecipitation with anti-Flag antibodies. Ve: elution volume (ml). The uncharacterized endogenous 50 kDa protein associated with mutant VHL (*) elutes in lower molecular weight fractions. TRiC, Hsp/Hsc70, elongin BC, and VHL are indicated. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 6 Tumor-Causing Mutations in VHL Box 1 and Box 2 Can Fold Correctly in a Chaperonin-Independent Manner (A) Chaperonin-independent refolding of wild-type VHL (lane 1) and of tumor-causing mutants in the elongin BC binding site (L158P, lane 2) or in Box 1 (lanes 3–7) and Box 2 (lanes 8–10). [35S]VHL variants were refolded as described in the Experimental Procedures, and formation of correctly folded VBC complexes was determined by metal affinity pull-downs (B), mild protease treatment (C), and analysis on nondenaturing PAGE (D). (B) VHL Box 1 and Box 2 tumor mutants are folded into a tight VBC complex. Incorporation of [35S]VHL variants into a stable complex with BC-His6 was evaluated as in Figure 4C. The amounts of total input VHL (lower panel) and VHL incorporated into VBC-His6 (upper panel) are shown. (C) Protease susceptibility of refolded VBC complexes. The protease susceptibility of wild-type VHL (lane 1) and VHL tumor mutants (lanes 2–10) refolded as in (A) was assessed as in Figure 4A. (D) Nondenaturing gel analysis of in vitro refolding reactions. The position of the folded VBC complex is indicated. In addition to the discrete VBC complex, all reactions contained a variety of putative misfolded VHL conformers (misfolded VHL). (E) Thermal stability of refolded VBC complexes. The stability of wild-type and mutant VBC complexes to thermal denaturation was assessed by incubation at a range of physiological temperatures, followed by a protease susceptibility assay carried out at the same temperature. Wild-type VHL (lane 1) and VHL tumor mutants (lanes 2–5) refolded as in (A) were analyzed as in Figure 4A. A control reaction using mutant L158P (lane 5) was also analyzed. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)

Figure 7 Effect of Tumor-Causing Mutations on VHL Folding In Vivo and In Vitro In the cell (left panel), newly translated VHL associates with TRiC/CCT in an Hsp70-dependent manner (Melville et al., 2003). The chaperonin recognizes Box 1 and Box 2, two hydrophobic strands in the β domain of VHL (highlighted as ribbons). Class A mutations in these sites destabilize binding to TRiC, leading to misfolding of VHL. TRiC-mediated VHL folding is coupled to incorporation into the VBC complex. Mutations that destabilize the folded VBC structure constitute another group of folding mutants (Class B) that cannot be productively released from TRiC. Class C mutants can fold but lack key surface residues that specify binding to cellular targets of the VHL ligase. In vitro refolding by dialysis from denaturant (right panel) can bypass the chaperone requirements and yield correctly folded VBC. Under these conditions, wild-type VHL and Class A mutants can reach the native state, whereas Class B mutants are still misfolded. The structure of VBC indicates the α and β domains of VHL (in red). See text for details. Molecular Cell 2003 12, 1213-1224DOI: (10.1016/S1097-2765(03)00423-4)