Sequence requirement and subtype specificity in the high-affinity interaction between human frizzled and dishevelled proteins

Abstract

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N-terminal and C-terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high-affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz-Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/beta-catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype-specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides.

Figure 1

A: Sequences of the Fz7-related peptides used in this study, aligned on the KTXXXW motif. B: Alignment of all human Fz C-terminal peptides spanning the KTXXXW motif that were used in this study.

Figure 2

Alphascreen assays to determine the minimum sequence requirement for specific binding of Fz7 with Dvl1 PDZ. A: Comparison of Fz7-#2 peptide displacement by self (▪), by Fz7-#1 (▴), and by a nonspecific peptide (CLQEKHRILHKLLQNGNSPA, •). B: Comparison of Fz7-#2 peptide displacement by self (▪), by Fz7-N-fragment (▴), and by Fz7-C-fragment (•]). Values shown are the mean ± standard error.

Figure 3

CD spectra showing secondary structure formation by Fz7 peptides. A: Different Fz7 peptides used in the study. Similar spectra were obtained at a range of concentrations (20–80 μM). B: Mutant Fz7 peptides with single residues replaced by Ala.

Figure 4

Lowest energy conformation of mutated 23 amino-acid peptides of Fz7 calculated by molecular modeling. (A) Wild-type Fz7#2, (B) W547A, (C) W557A, (D) K552A, (E) H562A, and (F) T553A mutants.

Figure 5

Comparison of the energy-minimized conformations of PDZ-binding and nonbinding Fz subtypes. The peptide regions shown are the 14 amino acids corresponding to the region F546 – R559 of Fz7. A–C: PDZ binding subtypes containing a looped N-terminal region followed by an extended KTXXXW motif (orange). D–F: Fz subtypes that do not bind to PDZ, containing an extended N-terminal region and/or a coiled KTXXXW motif.