Pull-down assay on TCS variants suggests that K173, R174 and K177 are involved in binding P2

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> TCS () or its variants (–) were loaded to a P2-coupled NHS-Sepharose pre-equilibrated with binding buffer. Bound protein was eluted with 1 M NaCl in 20 mM Tris/HCl buffer pH 8.0. Fractions containing unbound protein collected during washing (W) and bound protein collected during elution (E) were analysed in 15% SDS-PAGE stained with Coomassie blue. As indicated by the presence of TCS in the wash fraction, substitution of alanine at K173, R174 and K177 positions decreases the binding of TCS on P2-coupled column (b–d). Triple-alanine substitutions in these residue positions resulted in a TCS variant (K173A/R174A/K177A) that was unable to bind P2 ()

Interaction between TCS and ribosome was compromised by K173A/R174A/K177A triple-alanine substitutions

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> () . Rat ribosome was loaded to NHS-Sepharose coupled with TCS or its triple-alanine (K173A/R174A/K177A) variants. After extensive washing, the bound proteins were eluted with 1 M NaCl, and detected by western blot using anti-P antibody. Ribosomal proteins P0, P1 and P2 were pull-down by wild-type TCS (lane 2), while the interaction between ribosome and the triple-alanine variants (lane 1) was greatly reduced to that similar to the control (lane 3), in which the faint band of P0 was due to non-specific interactions between ribosome and the uncoupled resins. () . After rat ribosome was incubated with TCS or the triple-alanine variants in room temperature for 20 min, DSS was added to induce cross-linking between TCS and ribosomal proteins, and cross-linking product was detected by western blot using anti-P or anti-TCS antibodies. A protein band at ∼66 kDa, corresponding to the size of TCS–P0 complex, was detected by both anti-P and anti-TCS antibodies when ribosome was cross-linked with wild-type TCS (lane 2), but not with the triple-alanine variants (lane 5) and in other negative controls (lanes 1 and 4: without addition of ribosome; lanes 3, 6 and 8: without addition of DSS; lanes 7 and 8: without addition of TCS or its variants)

P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> () H–N correlation spectra of TCS in the absence (black contours) and in the presence (red contours) of equal molar ratio of P2 were compared, and () changes in chemical shifts, ▵ppm(HN) and ▵ppm(N), of amide resonances of TCS were measured. Residues with ▵ppm(HN) >0.075 ppm or ▵ppm(N) >0.5 ppm are indicated in (a) and (b), and colour-coded magenta in the stereo diagram of TCS in (). These residues are localized in or near the C-terminal domain (173–247, colour-coded green) of TCS. Scanning alanine mutagenesis was performed on all charge residues in the C-terminal domain and E123, which are indicated in (c)