Binding of Crumbs to the Par-6 CRIB-PDZ Module Is Regulated by Cdc42

Abstract

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by NMR that Par-6 binds a Crumbs carboxyl terminal peptide and report the crystal structure of the PDZ-peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ-peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding.

Emerging Themes in PDZ Domain Signaling

“…Interestingly, the region involved in this regulation spanned the a1 helix to the carboxyl-binding loop and a2 helix, consistent with the dynamics and energetic pathway proposed by NMR-and in silico-based studies (Fuentes et al, 2004;Lockless and Ranganathan, 1999). Moreover, the Crumbs ligand binding to Par-6 was also regulated by the Cdc42/CRIB-mediated allostery, suggesting a common mechanism for C-terminal ligands in Par-6 PDZ (Whitney et al, 2016). In contrast, Cdc42 did not regulate binding to the Pals1 protein that binds the Par-6 PDZ domain through an internal sequence (Peterson et al, 2004).…”

Structural basis for the interaction between the cell polarity proteins Par3 and Par6

“…Thus, both the PDZ domain and the PBM contribute to the correct localization of Par6 in vivo, with deletion of both domains resulting in Par6 mislocalization. However, this effect may reflect an indirect association between Par3 and Par6 through aPKC as suggested previously (7,10) and/or Par6 recruitment by other epithelial cell polarity regulators such as Crumbs or Stardust that bind to the Par6 PDZ domain (22,(31)(32)(33)(34)(35).…”

The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity

“…However, unknown input(s) stimulate aPKC to phosphorylate Par-3, reducing their mutual affinity, and allowing Crumbs to out-compete Par-3 for binding to Par-6/aPKC (Morais- de-Sá et al, 2010;Walther and Pichaud, 2010). Cdc-42 may promote this segregation by binding Par-6 and increasing its affinity for Crumbs (Whitney et al, 2016). Together, these interactions promote the accumulation of Par-6/ aPKC with Crb and Cdc-42 on the apical surface, while restricting Par-3 to adherens junctions.…”