Figure 1.
Structure of NHERF1 PDZ1 in complex with the CXCR2 C-terminal sequence TSTTL.
(A) Ribbon diagram of the PDZ1-CXCR2 structure, front view on the left and side view on the right. PDZ1 is shown in purple and the CXCR2 peptide shown in green. Secondary structures of PDZ1, α-helices and β-strands, are labeled and numbered according to their position in the sequence. Side chains of putative PDZ1 lipid-binding residues are depicted by balls-and-sticks in the side view of the structure. (B) Sequence alignment of selected PDZ domains. The alignment was performed by ClustalW [45], including human NHERF1, human NHERF2 and mouse PDZK1. Identical residues are shown as white on black, and similar residues appear shaded in cyan. Secondary structure elements are displayed above the sequences and labeled according to the scheme in Figure 1A. Sequence numbering is displayed to the left of the sequences, with every 10th residue marked by a dot shown above the alignment. (C) Sequence alignment of the last five residues of natural NHERF binding targets. The alignment includes CXCR2, CFTR, 2AR, PDGFR, PTHR, Npt2a (type 2 sodium-phosphate cotransporter), purinergic receptor P2Y1, CCR5 (C-C chemokine receptor type 5), and AQP9 (aquaporin 9). Protein names are shown at the left of the sequences. Position numbering is displayed above the alignment, with position 0 referring to the very C-terminal residue.
Figure 2.
Interactions between PDZ1 and CXCR2.
(A) Stereo view of the PDZ1 ligand-binding site bound to the CXCR2 C-terminal peptide. PDZ1 residues are represented by balls-and-sticks with their carbon atoms colored in purple. CXCR2 peptide is depicted by balls-and-sticks overlaid with 2Fo − Fc omit map calculated at 1.16 Å and contoured at 1.8 σ. Hydrogen bonds are illustrated as red broken lines. (B) Surface representation of the PDZ1 binding pocket with coloring according to the electrostatic potential: red, white, and blue correspond to negative, neutral and positive potential, respectively. The vacuum electrostatics/protein contact potential was generated by PyMOL. The CXCR2 peptide is depicted by balls-and-sticks overlaid by its transparent molecular surface.
Figure 3.
Structural comparison of PDZ domains.
(A) Superposition of the structures of PDZ1-CXCR2 (purple; PDB code: 4JL7), PDZ1-CFTR (orange; PDB code: 1I92) [12], PDZ1-2AR (cyan; PDB code: 1GQ4) [13], and PDZ1-PDGFR (yellow; PDB code: 1GQ5) [13]. PDZ domains are represented by ribbon, while residues in the ligands are displayed as sticks. (B) Superposition of the PDZ1 ligand binding pockets. Both PDZ1 and ligand residues are depicted by sticks and colored according to the scheme in Figure 3A. (C) Close-up views of structural differences of His29 (top) and Arg40 (bottom). The CXCR2 peptide is depicted by sticks overlaid with 2Fo − Fc omit map calculated at 1.16 Å and contoured at 2.0 σ. (D) Superposition of NHERF1 PDZ1 (purple) and PDZ2 (pink; PDB code: 2OZF) peptide binding pockets. CXCR2 peptide is shown in green and PDZ residues are depicted by balls-and-sticks.
Figure 4.
CXCR2 interacts with both PDZ1 and PDZ2 of NHERF1.
(A) GST pull-down of CXCR2 with NHERF1. Lysates of HEK293 cells overexpressing HA-tagged CXCR2 were used as prey. GST fusion proteins of NHERF1 PDZ1, PDZ2, and PDZ1-PDZ2 were used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblot using anti-HA antibodies. The amount of beads-immobilized GST proteins in each reaction is shown in the lower panel. (B) Biotin pull-down assays to detect direct interaction between CXCR2 and NHERF1. A biotinylated peptide corresponding to the last 13 residues of CXCR2 was used as bait, while purified GST-PDZ1, GST-PDZ2, GST-PDZ1-PDZ2 and GST alone as prey. Binding was resolved by SDS-PAGE and immunoblotted with anti-GST antibodies. (C) All experiments performed in (A) and (B) were repeated three times. The results were quantified using the CCD gel imager (UVP Chemidoc) and presented as mean±standard deviation. The asterisks indicate statistically significant differences (P < 0.05) between the values indicated by the brackets. Statistical analysis was performed using the two-tailed Student’s t-test. Top: GST pull-down of CXCR2 with NHERF1, and bottom: biotin pull-down of NHERF1 PDZ domains with the CXCR2 peptide.