Limits...
Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove.

Ivanova ME, Fletcher GC, O'Reilly N, Purkiss AG, Thompson BJ, McDonald NQ - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs).Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove.Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology Laboratories, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3LY, England.

ABSTRACT
Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

Show MeSH

Related in: MedlinePlus

Overall structure and molecular interactions between Pals1PDZ and Crb17. (a) Difference electron density (green) for the Crb17 peptide bound to Pals1PDZ prior to fitting. The map is a σA-weighted Fo − Fc electron-density OMIT map (contoured at σ = 3.0). (b) Electron density for the Crb17 peptide bound to Pals1PDZ after refinement. The map is shown as a σA-weighted 2Fo − Fc electron-density OMIT map (contoured at σ = 1.0). (c) Schematic representation of Crb17 (green) bound to Pals1PDZ (grey cartoon). Secondary-structure elements are labelled starting from the N-terminus of Pals1PDZ. α, α-Helix; β, β-­strand. (d) Close-up of the Crb17 peptide (green sticks) bound to Pals1PDZ (grey cartoon). Pals1 residues involved in the recognition of Crumbs are shown as sticks. Pink dashed lines represent hydrogen bonds that are established between the Crb17 peptide and Pals1PDZ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4356366&req=5

fig2: Overall structure and molecular interactions between Pals1PDZ and Crb17. (a) Difference electron density (green) for the Crb17 peptide bound to Pals1PDZ prior to fitting. The map is a σA-weighted Fo − Fc electron-density OMIT map (contoured at σ = 3.0). (b) Electron density for the Crb17 peptide bound to Pals1PDZ after refinement. The map is shown as a σA-weighted 2Fo − Fc electron-density OMIT map (contoured at σ = 1.0). (c) Schematic representation of Crb17 (green) bound to Pals1PDZ (grey cartoon). Secondary-structure elements are labelled starting from the N-terminus of Pals1PDZ. α, α-Helix; β, β-­strand. (d) Close-up of the Crb17 peptide (green sticks) bound to Pals1PDZ (grey cartoon). Pals1 residues involved in the recognition of Crumbs are shown as sticks. Pink dashed lines represent hydrogen bonds that are established between the Crb17 peptide and Pals1PDZ.

Mentions: To define the molecular interaction between human Pals1PDZ and Crb17, we successfully crystallized the complex and determined its structure at 1.23 Å resolution. Data-processing and refinement statistics for the structure are presented in Table 1 ▶. Electron density for residues 1393–1406 of Crb17 was observed, allowing the fitting of all but three residues of the Crb17 peptide (Figs. 2 ▶a and 2 ▶b). A single Pals1PDZ–Crb17 complex is present within the asymmetric unit. The Pals1PDZ adopts a canonical PDZ fold consisting of two β-sheets flanked by two α-helices. The carboxyl-terminus of the Crb17 peptide binds as an antiparallel β-strand extending the smaller PDZ β-sheet between strand β2 and helix α2 (Fig. 2 ▶c). The total buried surface area between Pals1PDZ and Crb17 is ∼500 Å2. The carboxy-terminal carboxylate group of Ile1406 in Crb17 forms hydrogen bonds to main-chain amides from the invariant Pals1 residues Leu267, Gly268 and Ala269 that define the canonical carboxylate-binding loop between strands β1 and β2 of Pals1PDZ (Fig. 2 ▶d). Leu321 of Pals1 adopts a strained side-chain rotamer on binding the ligand, allowing the side chain of Ile1406 from Crb17 to point into a deep hydrophobic pocket in Pals1PDZ. Van der Waals contacts between Crb17 Leu1405 and Pals1 Pro266 stabilize a closed carboxylate-binding loop enveloping the peptide ligand. Charged interactions with the ERLI motif are also found; Crb17 Glu1403 forms a salt bridge with Pals1PDZ Arg282 and also forms hydrogen bonds to the side chains of Thr270 and Ser281. The Crb17 Arg1404 side chain makes a cation–π interaction with Phe318 and also forms hydrogen bonds to the Asn315 side chain (Fig. 2 ▶d). Overall, the structure explains the tight conservation of each side chain of the Crb ERLI motif through selective interactions with the Pals1PDZ domain, consistent with the binding affinities measured in solution by fluorescence polarization.


Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove.

Ivanova ME, Fletcher GC, O'Reilly N, Purkiss AG, Thompson BJ, McDonald NQ - Acta Crystallogr. D Biol. Crystallogr. (2015)

Overall structure and molecular interactions between Pals1PDZ and Crb17. (a) Difference electron density (green) for the Crb17 peptide bound to Pals1PDZ prior to fitting. The map is a σA-weighted Fo − Fc electron-density OMIT map (contoured at σ = 3.0). (b) Electron density for the Crb17 peptide bound to Pals1PDZ after refinement. The map is shown as a σA-weighted 2Fo − Fc electron-density OMIT map (contoured at σ = 1.0). (c) Schematic representation of Crb17 (green) bound to Pals1PDZ (grey cartoon). Secondary-structure elements are labelled starting from the N-terminus of Pals1PDZ. α, α-Helix; β, β-­strand. (d) Close-up of the Crb17 peptide (green sticks) bound to Pals1PDZ (grey cartoon). Pals1 residues involved in the recognition of Crumbs are shown as sticks. Pink dashed lines represent hydrogen bonds that are established between the Crb17 peptide and Pals1PDZ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356366&req=5

fig2: Overall structure and molecular interactions between Pals1PDZ and Crb17. (a) Difference electron density (green) for the Crb17 peptide bound to Pals1PDZ prior to fitting. The map is a σA-weighted Fo − Fc electron-density OMIT map (contoured at σ = 3.0). (b) Electron density for the Crb17 peptide bound to Pals1PDZ after refinement. The map is shown as a σA-weighted 2Fo − Fc electron-density OMIT map (contoured at σ = 1.0). (c) Schematic representation of Crb17 (green) bound to Pals1PDZ (grey cartoon). Secondary-structure elements are labelled starting from the N-terminus of Pals1PDZ. α, α-Helix; β, β-­strand. (d) Close-up of the Crb17 peptide (green sticks) bound to Pals1PDZ (grey cartoon). Pals1 residues involved in the recognition of Crumbs are shown as sticks. Pink dashed lines represent hydrogen bonds that are established between the Crb17 peptide and Pals1PDZ.
Mentions: To define the molecular interaction between human Pals1PDZ and Crb17, we successfully crystallized the complex and determined its structure at 1.23 Å resolution. Data-processing and refinement statistics for the structure are presented in Table 1 ▶. Electron density for residues 1393–1406 of Crb17 was observed, allowing the fitting of all but three residues of the Crb17 peptide (Figs. 2 ▶a and 2 ▶b). A single Pals1PDZ–Crb17 complex is present within the asymmetric unit. The Pals1PDZ adopts a canonical PDZ fold consisting of two β-sheets flanked by two α-helices. The carboxyl-terminus of the Crb17 peptide binds as an antiparallel β-strand extending the smaller PDZ β-sheet between strand β2 and helix α2 (Fig. 2 ▶c). The total buried surface area between Pals1PDZ and Crb17 is ∼500 Å2. The carboxy-terminal carboxylate group of Ile1406 in Crb17 forms hydrogen bonds to main-chain amides from the invariant Pals1 residues Leu267, Gly268 and Ala269 that define the canonical carboxylate-binding loop between strands β1 and β2 of Pals1PDZ (Fig. 2 ▶d). Leu321 of Pals1 adopts a strained side-chain rotamer on binding the ligand, allowing the side chain of Ile1406 from Crb17 to point into a deep hydrophobic pocket in Pals1PDZ. Van der Waals contacts between Crb17 Leu1405 and Pals1 Pro266 stabilize a closed carboxylate-binding loop enveloping the peptide ligand. Charged interactions with the ERLI motif are also found; Crb17 Glu1403 forms a salt bridge with Pals1PDZ Arg282 and also forms hydrogen bonds to the side chains of Thr270 and Ser281. The Crb17 Arg1404 side chain makes a cation–π interaction with Phe318 and also forms hydrogen bonds to the Asn315 side chain (Fig. 2 ▶d). Overall, the structure explains the tight conservation of each side chain of the Crb ERLI motif through selective interactions with the Pals1PDZ domain, consistent with the binding affinities measured in solution by fluorescence polarization.

Bottom Line: Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs).Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove.Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology Laboratories, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3LY, England.

ABSTRACT
Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

Show MeSH
Related in: MedlinePlus