Multiple CBMs on GTSE1 and adaptor interaction sites on CHC are required for the GTSE1–CHC interaction. (A) Sequence alignment of GTSE1 C-terminus (aa 661–727 of human GTSE1). Putative clathrin-binding motifs are in green; divergent motifs are italicized. Mutations in the “5xLID” mutant are shown in orange (last row). (B) GTSE1 fragment constructs. Numbers indicate amino acids. Binding of fragments to N-terminal CHC constructs is indicated. (C) Pulldown analysis of the interaction between GST-GTSE1 fragments and CHC(1–642)-6xHis. Immunoblot (anti-His) and Coomassie blue are from separate experiments. (D) AUC analysis of the stoichiometry of the complex between CHC(1–364) and GTSE1(639–739)-6xHis. (E) Structure of GTSE1 motif D interacting with site 3 on CHC. Superimposition (PyMOL) of the structures of CHC(1–364) in complex with GTSE1-C4 (dark green; PDB ID: 6QNN) or GTSE1-C5 (light green; PDB ID: 6QNP). A reference structure (PDB ID: 5M5S) of CHC bound to an amphiphysin peptide is presented in beige. (F) Structure of GTSE1 motif B (light green) interacting with CHC site 1 (PDB ID: 6QNP). A reference structure (PDB ID: 5M5S) of CHC bound to an amphiphysin peptide is presented in beige. (G) Pulldown analysis of the interaction between mutated GST-GTSE1-C3 fragments and CHC(1–642)-6xHis. Immunoblot (anti-His) and Coomassie blue gels are from separate experiments. (H) A model of the interaction between GTSE1 (green) and the CHC NTD. GTSE1 motifs B and D reside in site 1 and site 3 pockets, respectively. The darker green GTSE1 region was modeled manually using Coot.