Newly identified S+1 sub-pocket of PDZ domain: Scribble PDZ1 – SGEF interaction

Titus Hou


Scribble PDZ1 is an important component in the formation of the polarity complex composed of Discs Large 1 (Dlg1), Scribble, and Src homology 3 domain-containing Guanine nucleotide Exchange Factor (SGEF). While the role of Scribble PDZ1 in the regulation of SGEF enzymatic activity is unclear, Scribble PDZ1 defies expectations by binding to an internal PDZ binding motif (iPBM). PDZ domains are known to bind C-terminal segments of protein and only two PDZ-iPBM interactions have been structurally characterized. However, the determinants of PDZ-iPBM interactions remain unclear. High resolution crystal structures of Scribble PDZ1 bound to a C-terminal ligand and SGEF iPBM help elucidate the structural determinants of these interactions. NMR HSQC titration experiments identified key residues utilized in the interaction. Binding affinities were measured using fluorescence-based anisotropy and isothermal calorimetry experiments. Combined, these data provide an understanding of a new S+1 binding region utilized in Scribble-SGEF interaction not found in C-terminal PDZ binding motifs. This information was then used to design a Scribble PDZ1 mutant that selectively abrogated SGEF iPBM-Scribble PDZ1 interaction while leaving C-terminal PDZ binding motifs unaffected. In this study, we characterized the ligand binding specificities of all four Scribble PDZ domains to help understand the role of the Dlg1-SGEF-Scribble complex. Additionally, this work may provide a fundamental basis for the rational design of PDZ domains.