Figure S2. CBMs on the GTSE1 C-terminus... | Rockefeller University Press

Figure S2.

$CBMs on the GTSE1 C-terminus interact with adaptor binding sites on the CHC NTD. (A) GST-pulldown analysis of the interaction between GST-CHC(1–364) and GTSE1 fragments fused to a His-tag (GTSE1-His). Numbers indicate amino acids present in the fragments. Immunoblot is with anti-His antibody. (B) Yeast two-hybrid analysis of the interaction between CHC(1–330) fused to activation domain (AD) and different GTSE1(463–739) fragments fused to the DNA binding domain (BD). Letters A–E indicate mutated motifs in GTSE1 (see Fig. 3 A for sequences). Interaction was visualized by yeast growth on −His selection plates. (C) Gel filtration profile (SuperDex200 10 300 GL column) of CHC(1–364) in complex with GTSE1(639–739) fused to 6 His tag. The indicated (pink box) peak protein fraction was run on SDS-PAGE (Coomassie blue stained). (D) Fractions containing CHC(1–364) in complex with GTSE1(639–739)-6xHis were pooled and submitted to AUC-SV. (E) AUC-SV data best-curve-fitting results with root mean square difference (RMSD) value in top. (F) Diagrams showing the amino acids involved in the interactions between CBMs on GTSE1 and sites 1 and 3 on the CHC NTD. The diagrams were designed using Ligplot based on the crystal structures of GTSE1-C4(661–726; upper left panel, PDB ID: 6QNN) and GTSE1-C5(653–719; upper right and lower panels, PDB ID: 6QNP) in complex with CHC(1–364). Site 3 is occupied by motif D in both crystals, while site 1 is occupied by motif E in the GTSE1-C4 crystal and by motif B or motif C in the GTSE1-C5 crystal. Amino acids constituting CBMs are highlighted. (G) Difference electron density maps after refinement of the CHC inthe absence of any atoms of the peptides. The A–E peptide is bound to site 1, and the A–D peptide is shown in site 3.$

CBMs on the GTSE1 C-terminus interact with adaptor binding sites on the CHC NTD. (A) GST-pulldown analysis of the interaction between GST-CHC(1–364) and GTSE1 fragments fused to a His-tag (GTSE1-His). Numbers indicate amino acids present in the fragments. Immunoblot is with anti-His antibody. (B) Yeast two-hybrid analysis of the interaction between CHC(1–330) fused to activation domain (AD) and different GTSE1(463–739) fragments fused to the DNA binding domain (BD). Letters A–E indicate mutated motifs in GTSE1 (see Fig. 3 A for sequences). Interaction was visualized by yeast growth on −His selection plates. (C) Gel filtration profile (SuperDex200 10 300 GL column) of CHC(1–364) in complex with GTSE1(639–739) fused to 6 His tag. The indicated (pink box) peak protein fraction was run on SDS-PAGE (Coomassie blue stained). (D) Fractions containing CHC(1–364) in complex with GTSE1(639–739)-6xHis were pooled and submitted to AUC-SV. (E) AUC-SV data best-curve-fitting results with root mean square difference (RMSD) value in top. (F) Diagrams showing the amino acids involved in the interactions between CBMs on GTSE1 and sites 1 and 3 on the CHC NTD. The diagrams were designed using Ligplot based on the crystal structures of GTSE1-C4(661–726; upper left panel, PDB ID: 6QNN) and GTSE1-C5(653–719; upper right and lower panels, PDB ID: 6QNP) in complex with CHC(1–364). Site 3 is occupied by motif D in both crystals, while site 1 is occupied by motif E in the GTSE1-C4 crystal and by motif B or motif C in the GTSE1-C5 crystal. Amino acids constituting CBMs are highlighted. (G) Difference electron density maps after refinement of the CHC inthe absence of any atoms of the peptides. The A–E peptide is bound to site 1, and the A–D peptide is shown in site 3.