Structure of Crumbs tail in complex with the PALS1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical Crumbs complex

Abstract

The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ∼ 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems.

Keywords: MAGUK; PBM; Stardust; cell polarity; supramodule.

Fig. 1.

PALS1-PSG binds to Crb-CT with high affinity. (A) Schematic diagrams of the domain organization of PALS1 and Crb. The PALS1-PSG–Crb-CT interaction is indicated by a two-way arrow. Note that, unlike Crb1 and Crb2, vertebrate Crb3 lacks the large extracellular domains. (B and C) Analytical gel filtration chromatography showing the binding profiles of Crb-CT to PALS1-PDZ (B) and PALS1-PSG (C). mAU, UV absorbance at 280 nm. (D and E) Isothermal titration calorimetry (ITC)-based measurements quantifying the binding of Crb-CT to PALS1-PDZ (D) and PALS1-PSG (E).

Fig. 2.

Overall structure of the PALS1-PSG–Crb-CT complex. (A) Two PALS1-PSG–Crb-CT heterodimers form a dimer related by a twofold axis. The disordered loops are indicated by dotted lines. The deletion site in PALS1-PSG_ΔHOOK is denoted by a red star. (B) Surface representation of the Crb-PBM binding site in the PDZ–SH3 tandem. The surfaces are semitransparent to show that Crb-PBM is partially buried by two residues, F318 and E368, from the PDZ and SH3 domains, respectively. (C) Schematic cartoon showing the PALS1-PSG–Crb-CT interaction as well as the PSG interdomain interactions for the dimer of dimers in the crystal. (D) The SH3–GK tandem of DLG4 is shown with the SH3 aligned in the same view as that of PALS1. The cartoon shows the corresponding assembly mode for the SH3–GK tandem in DLG4.

Fig. 3.

Molecular details of PDZ–SH3 coupling. (A) Stereoview of the binding of Crb-PBM to PALS1 PDZ–SH3. Salt bridges and hydrogen bonds are indicated by dashed lines. (B) Sequence alignment of the cytoplasmic regions of different Crb proteins (dm, Drosophila melanogaster; dr, Danio rerio; hs, Homo sapiens; mm, Mus musculus; xt, Xenopus tropicalis). In this alignment, residues that are absolutely conserved and highly conserved are highlighted in red and yellow, respectively. The residues involved in the PDZ–PBM and GK–GBM interactions are indicated by blue and orange circles, respectively. (C) ITC-based measurements summarizing the binding affinities of Crb-CT to PALS1-PSG and several of its mutants characterized in the study. (D) Molecular details of the PDZ–SH3 interdomain interaction.

Fig. 4.

Crb-CT–PALS1-GK interaction. (A) Molecular details showing how Crb-GBM interacts with PALS1-GK. (B) The phosphorylation-dependent interaction between the phosphor-LGN peptide and the DLG1 GK domain (36). (Inset) Sequence alignment of the GK-binding peptides from Crb-CT and LGN. The arrow points to the phospho-Ser–mimicking residue Glu.

Fig. 5.

PSG tandem of PALS1 is essential for its role in apical–basal cell polarity in MDCK cysts. (A) MDCK cells stably expressing the Venus-tagged wild-type PALS1 (WT), PALS1 mutant with the SH3–GK tandem deleted (PDZ), and PALS1 PDZ–SH3 uncoupling mutant (D386K) were cultured in Matrigel. Cysts were fixed and stained with rhodamine-conjugated phalloidin (red), anti-GFP antibody (green) against Venus, and DNA dye (blue), respectively. (B) Lentivirus-mediated knockdown of endogenous PALS1 (KD-PALS1) leads to defective cystogenesis, as indicated by the abnormal multilumen formation, and such defects can be rescued by expressing shRNA-resistant WT-PALS1 (top two panels). Knockdown of endogenous PALS1 led to more severe cystogenesis defects in cells stably expressing shRNA-resistant PALS1-PDZ or PALS1-D386K (bottom two panels). Control MDCK cells (Top) and MDCK cells stably expressing WT-PALS1, PALS1-PDZ, or PALS1-D386K were transduced by lentiviruses expressing mCherry (red) and shRNA against canine PALS1. mCherry-expressing cells are considered virus-transduced, PALS1 knockdown cells. Virus-transduced cells were cultured in Matrigel for cystogenesis. Cysts were fixed and stained with Alexa 488-conjugated phalloidin (green) and DNA dye (blue). (C and D) Quantification of the cysts with abnormal lumen formation in A and B, respectively. Values are mean ± SD from three independent experiments (n > 100 for each group). *P < 0.01, **P < 0.001.

Fig. 6.

Other MAGUKs may also form PSG supramodules. (A) Schematic diagrams showing the domain organization of MAGUKs. (B) Sequence alignment showing the residues in the PDZ–SH3 interface among PALS1 and other MAGUKs. Residues involved in the PDZ–SH3 coupling and Crb-PBM binding are highlighted with triangles and circles, respectively. (C) ITC-based measurements comparing the binding of the neurexin tail to CASK-PSG and CASK-PDZ.