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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.

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In vivo and in vitro interaction between Stardust/Pals1 and Crumbs/Crb1. (a) Schematic for Pals1 and Crumbs domain organization. EGF, epidermal growth factor; LamAG, laminin AG-like; TM, transmembrane; ICD, intracellular; L27, lin-2, lin-7 homology; PDZ, postsynaptic density 95, disc-large, zona occludens homology; SH3, sarcoma homology 3; GUK, guanylate kinase. The PDZ domain of Pals1 interacts with Crumbs through its ICD. (b) The amino-acid sequence of the Crb17 peptide used for biochemical experiments and for crystallization is underlined; the PDZ-binding motif is coloured. (c) D. melanogaster Crumbs (shown in red) localizes to the apical membrane of the follicle cells of egg chambers. Wild-type follicle cells (WT) are marked by the presence of green fluorescent protein (GFP). (d) Deletion of the four C-terminal residues of Crumbs (CrbΔERLI) causes a failure of Crb to localize to the apical plasma membrane of the follicle cells and disrupts polarity. Mutant cells are marked by the absence of GFP; some GFP-positive cells can be seen. (e) Representative binding curves for fluorescein-labelled Crb17 for Pals1PDZ and (f) Crb17 mutants measured by fluorescence polarization (Crb17, green; Crb17 L1405A, orange; Crb17 R1404A, pink; Crb17 E1403A, blue; Crb17 M1402A, red). The affinity between Crb17 I1406A and Pals1PDZ could not be reliably determined. (g) Summary of dissociation constants (in µM) between Crb17 and Pals1PDZ [colour-coded as in (f); Crb17 II406A, brown, Crb6, light blue] measured by Fp with fluorescein-labelled peptides. All values shown were measured in parallel from the same Pals1 preparation (except for the Crb6 peptide, which was measured using a different Pals1PDZ preparation; the Kd between Crb17 and Pals1PDZ from this preparation was 12.4 ± 2.4 µM). These data were representative of three independent experiments.
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fig1: In vivo and in vitro interaction between Stardust/Pals1 and Crumbs/Crb1. (a) Schematic for Pals1 and Crumbs domain organization. EGF, epidermal growth factor; LamAG, laminin AG-like; TM, transmembrane; ICD, intracellular; L27, lin-2, lin-7 homology; PDZ, postsynaptic density 95, disc-large, zona occludens homology; SH3, sarcoma homology 3; GUK, guanylate kinase. The PDZ domain of Pals1 interacts with Crumbs through its ICD. (b) The amino-acid sequence of the Crb17 peptide used for biochemical experiments and for crystallization is underlined; the PDZ-binding motif is coloured. (c) D. melanogaster Crumbs (shown in red) localizes to the apical membrane of the follicle cells of egg chambers. Wild-type follicle cells (WT) are marked by the presence of green fluorescent protein (GFP). (d) Deletion of the four C-terminal residues of Crumbs (CrbΔERLI) causes a failure of Crb to localize to the apical plasma membrane of the follicle cells and disrupts polarity. Mutant cells are marked by the absence of GFP; some GFP-positive cells can be seen. (e) Representative binding curves for fluorescein-labelled Crb17 for Pals1PDZ and (f) Crb17 mutants measured by fluorescence polarization (Crb17, green; Crb17 L1405A, orange; Crb17 R1404A, pink; Crb17 E1403A, blue; Crb17 M1402A, red). The affinity between Crb17 I1406A and Pals1PDZ could not be reliably determined. (g) Summary of dissociation constants (in µM) between Crb17 and Pals1PDZ [colour-coded as in (f); Crb17 II406A, brown, Crb6, light blue] measured by Fp with fluorescein-labelled peptides. All values shown were measured in parallel from the same Pals1 preparation (except for the Crb6 peptide, which was measured using a different Pals1PDZ preparation; the Kd between Crb17 and Pals1PDZ from this preparation was 12.4 ± 2.4 µM). These data were representative of three independent experiments.

Mentions: Crb consists of a large extracellular domain (ECD), a transmembrane region (TM) and a 37-amino-acid intracellular domain (ICD). It has been shown that the Crb ECD (CrbECD) can oligomerize to mediate cell adhesion in the retina (Zhou & Hong, 2012 ▶; Fletcher et al., 2012 ▶). The CrbICD contains two protein-binding motifs: a juxtamembrane FERM (band 4.1, ezrin, radixin, moesin)-binding motif (FBM) and a carboxy-terminal PDZ-binding motif. Crb recruits Pals1 to the apical membrane via an interaction between CrbICD and Pals1PDZ, and this interaction is required for Crb localization at the apical membrane (Bachmann et al., 2001 ▶; Fig. 1 ▶a). Pals1 belongs to the MPP family of proteins (membrane protein, palmitoylated), also known as the p55 subfamily of membrane-associated guanylate kinases (MAGUKs). Pals1 acts as a scaffold protein, recruiting other proteins to the apical domain of the cells: the L27 domains of Pals1 bind to Patj and Lin7, while the amino-terminal regions of Pals1 can interact with the PDZ domain of Par6, linking the Crb and Par complexes (Hurd et al., 2003 ▶). It has also been shown that the PDZ domain of Par6 can bind directly to the PDZ-binding motif (PBM) of Crb (Hurd et al., 2003 ▶; Kempkens et al., 2006 ▶).


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)

In vivo and in vitro interaction between Stardust/Pals1 and Crumbs/Crb1. (a) Schematic for Pals1 and Crumbs domain organization. EGF, epidermal growth factor; LamAG, laminin AG-like; TM, transmembrane; ICD, intracellular; L27, lin-2, lin-7 homology; PDZ, postsynaptic density 95, disc-large, zona occludens homology; SH3, sarcoma homology 3; GUK, guanylate kinase. The PDZ domain of Pals1 interacts with Crumbs through its ICD. (b) The amino-acid sequence of the Crb17 peptide used for biochemical experiments and for crystallization is underlined; the PDZ-binding motif is coloured. (c) D. melanogaster Crumbs (shown in red) localizes to the apical membrane of the follicle cells of egg chambers. Wild-type follicle cells (WT) are marked by the presence of green fluorescent protein (GFP). (d) Deletion of the four C-terminal residues of Crumbs (CrbΔERLI) causes a failure of Crb to localize to the apical plasma membrane of the follicle cells and disrupts polarity. Mutant cells are marked by the absence of GFP; some GFP-positive cells can be seen. (e) Representative binding curves for fluorescein-labelled Crb17 for Pals1PDZ and (f) Crb17 mutants measured by fluorescence polarization (Crb17, green; Crb17 L1405A, orange; Crb17 R1404A, pink; Crb17 E1403A, blue; Crb17 M1402A, red). The affinity between Crb17 I1406A and Pals1PDZ could not be reliably determined. (g) Summary of dissociation constants (in µM) between Crb17 and Pals1PDZ [colour-coded as in (f); Crb17 II406A, brown, Crb6, light blue] measured by Fp with fluorescein-labelled peptides. All values shown were measured in parallel from the same Pals1 preparation (except for the Crb6 peptide, which was measured using a different Pals1PDZ preparation; the Kd between Crb17 and Pals1PDZ from this preparation was 12.4 ± 2.4 µM). These data were representative of three independent experiments.
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Related In: Results  -  Collection

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fig1: In vivo and in vitro interaction between Stardust/Pals1 and Crumbs/Crb1. (a) Schematic for Pals1 and Crumbs domain organization. EGF, epidermal growth factor; LamAG, laminin AG-like; TM, transmembrane; ICD, intracellular; L27, lin-2, lin-7 homology; PDZ, postsynaptic density 95, disc-large, zona occludens homology; SH3, sarcoma homology 3; GUK, guanylate kinase. The PDZ domain of Pals1 interacts with Crumbs through its ICD. (b) The amino-acid sequence of the Crb17 peptide used for biochemical experiments and for crystallization is underlined; the PDZ-binding motif is coloured. (c) D. melanogaster Crumbs (shown in red) localizes to the apical membrane of the follicle cells of egg chambers. Wild-type follicle cells (WT) are marked by the presence of green fluorescent protein (GFP). (d) Deletion of the four C-terminal residues of Crumbs (CrbΔERLI) causes a failure of Crb to localize to the apical plasma membrane of the follicle cells and disrupts polarity. Mutant cells are marked by the absence of GFP; some GFP-positive cells can be seen. (e) Representative binding curves for fluorescein-labelled Crb17 for Pals1PDZ and (f) Crb17 mutants measured by fluorescence polarization (Crb17, green; Crb17 L1405A, orange; Crb17 R1404A, pink; Crb17 E1403A, blue; Crb17 M1402A, red). The affinity between Crb17 I1406A and Pals1PDZ could not be reliably determined. (g) Summary of dissociation constants (in µM) between Crb17 and Pals1PDZ [colour-coded as in (f); Crb17 II406A, brown, Crb6, light blue] measured by Fp with fluorescein-labelled peptides. All values shown were measured in parallel from the same Pals1 preparation (except for the Crb6 peptide, which was measured using a different Pals1PDZ preparation; the Kd between Crb17 and Pals1PDZ from this preparation was 12.4 ± 2.4 µM). These data were representative of three independent experiments.
Mentions: Crb consists of a large extracellular domain (ECD), a transmembrane region (TM) and a 37-amino-acid intracellular domain (ICD). It has been shown that the Crb ECD (CrbECD) can oligomerize to mediate cell adhesion in the retina (Zhou & Hong, 2012 ▶; Fletcher et al., 2012 ▶). The CrbICD contains two protein-binding motifs: a juxtamembrane FERM (band 4.1, ezrin, radixin, moesin)-binding motif (FBM) and a carboxy-terminal PDZ-binding motif. Crb recruits Pals1 to the apical membrane via an interaction between CrbICD and Pals1PDZ, and this interaction is required for Crb localization at the apical membrane (Bachmann et al., 2001 ▶; Fig. 1 ▶a). Pals1 belongs to the MPP family of proteins (membrane protein, palmitoylated), also known as the p55 subfamily of membrane-associated guanylate kinases (MAGUKs). Pals1 acts as a scaffold protein, recruiting other proteins to the apical domain of the cells: the L27 domains of Pals1 bind to Patj and Lin7, while the amino-terminal regions of Pals1 can interact with the PDZ domain of Par6, linking the Crb and Par complexes (Hurd et al., 2003 ▶). It has also been shown that the PDZ domain of Par6 can bind directly to the PDZ-binding motif (PBM) of Crb (Hurd et al., 2003 ▶; Kempkens et al., 2006 ▶).

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