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 cellgrowth 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 MadinDarby 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.cell polarity | supramodule | MAGUK | PBM | Stardust

AP-2 complex-mediated endocytosis of Drosophila Crumbs regulates polarity via antagonizing Stardust

“…Alternatively, the accessibility of the PDZ domain of Sdt/Pals1 to Crb could be modulated. In fact, a ∼100-fold stronger binding of the PDZ domain of Pals1 to the Crb tail is achieved upon intra-or inter-molecular interactions between the Src homology 3 (SH3)-and the guanylate kinase (GUK)-domain of Pals1 (Kantardzhieva et al, 2005;Li et al, 2014). Similarly, the affinity of the PDZ domain of the postsynaptic density protein PSD-95 to its ligand is reduced upon phosphorylation of a tyrosine residue in a linker region between the third PDZ domain and the subsequent SH3-domain, thereby weakening the intramolecular interaction between the PDZ and the SH3 domain (Murciano-Calles et al, 2014;Zhang et al, 2011).…”

MAGUKs end a tale of promiscuity

“…Although full-length MAGUKs generally display a high degree of binding specificity, binding studies with isolated PDZ domains often show the ability to interact with numerous partners (2). In PNAS, Li et al (3) provide a conclusive structural answer to this discrepancy. The authors have solved the crystal structure of a PALS1/Crb complex that demonstrates why the PDZ-SH3-GK supramodule of PALS1, but not the isolated PDZ domain, binds with an extraordinarily high affinity to the C terminus of Crb (3).…”

“…In PNAS, Li et al (3) provide a conclusive structural answer to this discrepancy. The authors have solved the crystal structure of a PALS1/Crb complex that demonstrates why the PDZ-SH3-GK supramodule of PALS1, but not the isolated PDZ domain, binds with an extraordinarily high affinity to the C terminus of Crb (3). Domains in the supramodule are arranged in a way that a peptide comprising the last 17 residues of Crb binds simultaneously to the PDZ, the SH3 and, most surprisingly, to the GK domain using two distinct sites.…”

“…Interestingly, residues of the SH3 domain collaborate in peptide binding by building a kind of a clasp. Using site-directed mutagenesis, Li et al (3) prove that the clasp accounts for the high affinity and specificity of Crb binding to PALS1. This discovery has immediate consequences for other MAGUKs, for example at synapses in the brain, because the authors propose that sequence variations within the clasp-loop are responsible for defining their specificity.…”

“…Unexpectedly, the PALS1/Crb structure shows an additional participation of the GK domain in Crb binding (3). Crb binds in a manner comparable to the complex of a phosphorylated peptide p-LGN with the GK domain of DLG1/SAP97 (4).…”