Fig 1.
Minimal interacting domains of MYC:TRRAP.
(A) The indicated regions of TRRAP were cloned into a CMV-FLAG expression vector. Proteins were co-expressed with PYO-tagged full-length MYC (1–439) and then MYC was IPed with anti-PYO beads. Co-IP was assessed by western blot with anti-FLAG. The most critical binding domain is within residues 1997–2088. (B) Full-length TRRAP (1–3830) and TRRAP Δ2033–2088 were cloned into a CMV-FLAG expression vector and transfected into HEK293T cells. Proteins were co-expressed with PYO-tagged full-length MYC and then MYC was IPed with anti-PYO beads. Co-IP was evaluated by western blot. TRRAP Δ2033–2088 shows reduced binding to MYC. (C) Full-length MYC, MYC ΔMB2, MYC 1–190, and MYC 1–190 ΔMB2 were cloned into a CMV-PYO expression vector and transfected into HEK293T cells. Proteins were co-expressed with FLAG-tagged TRRAP 2033–2283 then MYC was IPed with anti-PYO beads. Co-IP was evaluated by western blot. TRRAP 2033–2288 shows equal co-IP with full-length MYC as with MYC 1–190, and both require MB2.
Fig 2.
A MYC:TRRAP complex does not form in vitro.
(A) Coomassie-stained SDS-PAGE of a TS tag pulldown of TRRAP 2033–2088 mixed with MYC 1–190 and MYC 1–190 ΔMB2 at 50 μM each. This result demonstrates that MYC 1–190 and TRRAP 2033–2088 do not interact when mixed in vitro. (B-C) CD spectra of MYC 1–190 and MYC 1–190 ΔMB2 mixed in vitro with TRRAP 2033–2088 at 10 μM each. CD spectra show no gain in secondary structure after mixing either MYC 1–190 or MYC 1–190 ΔMB2 with TRRAP 2033–2088.
Table 1.
The effect of additives on MYC:TRRAP.
Fig 3.
Effects of ethylene glycol on MYC and TRRAP.
(A-C) CD spectra of MYC 1–190, TRRAP 2033–2088, and BSA. Solid lines represent measurements taken in 1X PBS; dotted lines represent measurements taken in 30% EG. A significant increase in the α-helical character of MYC and (to a lesser extent) TRRAP is observed in the presence of EG. However, BSA (a highly α-helical well-folded protein) appears unaffected by the presence of EG. D) SEC λ280 spectra of MYC 1–190 (black), TRRAP 2033–2088 (grey), and MYC 1–190 mixed with TRRAP 2033–2088 (black dotted) in 30% EG all at 100 μM. Neither MYC nor TRRAP showed any variation in their expected hydrodynamic radius as measured in 1X PBS. The mixed sample did not have any measurable tertiary peak that would indicate an association between the MYC and TRRAP.
Fig 4.
The effect of EG on MYC-TRRAP.
(A) Coomassie-stained SDS-PAGE of a pulldown of two 3C protease-cleavable fusion proteins. MYC 1-190-TRRAP 2033-2088-TS and MYC 1–190 ΔMB2-TRRAP2033-2088-TS incubated in either 1X PBS or 30% EG. After 3C cleavage of the linker, the TRRAP domain was pulled down with StrepTactin® beads and the EG was washed away with 1X PBS. MYC 1–190 showed enhanced binding to TRRAP 2033–2088 in the presence of 30% EG but not in PBS, when compared to MYC 1–190 ΔMB2 in 30% EG. (B) CD spectra of two MYC-TRRAP fusion proteins in 30% EG: MYC 1-190-TRRAP 2033–2088 in black and MYC 1–190 ΔMB2-TRRAP 2033–2088 in red. The effects of EG on the fusion protein containing MB2 are more profound and are indicative of a specific gain in α-helical character.
Fig 5.
The environment of W135 in MYC 120–161 vs MYC 120-161-TRRAP 2033–2088.
(A-B) 1H, 15N-HSQC spectra of MYC 120–161 (black) and MYC 120-161-TRRAP 2033–2088 (red) in 30% TFE-d2. The peak shifts of W135 in the MYC-TRRAP fusion spectrum is indicative of a binding event. The splitting of the peak suggests two stable conformations: bound and unbound states.