SGEF interacts with Scribble PDZ domain through a novel PBM. (A) Schematic representation of SGEF and Scribble constructs used in this figure. LRR, leucine-rich repeat. (B–G) Lysates from HEK293FT cells expressing the indicated constructs were immunoprecipitated (IP) using GFP antibodies (GFP-trap nanobodies). In all experiments, the precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc or anti-His to detect the potential interacting partner. (H) Sequence alignment comprising the Scribble binding domain of SGEF in vertebrates (aa 32–55 in human). (I) The indicated boxed regions within the Scribble binding domain in SGEF were mutagenized to Ala in full-length SGEF. Lysates from HEK293FT cells expressing the myc-tagged WT-SGEF or the indicated Ala mutants and GFP-Scribble were immunoprecipitated using GFP antibodies (GFP-trap nanobodies). The precipitates were immunoblotted with anti-GFP antibodies to detect the immunoprecipitated protein and with anti-myc to detect my-tagged SGEF mutants. Green boxes show the sequence that when mutated still maintain an interaction with Scribble whereas the red boxes denote sequence that when mutated lead to loss of interaction with Scribble. (J) Crystal structure of the Scribble PDZ1 domain in complex with an SGEF iPBM peptide. The crystal structure of the apo Scribble PDZ1 domain is shown in gray, while the complex with the SGEFpeptide (KSPNGLLITDFP) is shown in red (left panel). The right panel is a surface representation of the PDZ1/SGEF-PDZpeptide complex.
