Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites

Abstract

PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

Figure 1

The overall structure of the ZO-1 PDZ2/Cx43 peptide complex. (A) Superposition plot of the ¹H–¹⁵N HSQC spectra of ZO-1 PDZ2 with increasing molar ratios of the Cx43 peptide. The arrows indicate the peptide binding-induced peak shifts and the dotted boxes highlight the peptide binding-induced peak broadenings. (B) Stereo view of the final crystal structure model of the ZO-1 PDZ2/Cx43 peptide complex shown in the ribbon diagram. The Cx43 peptides are drawn in purple. The secondary structures are labelled following the scheme of the canonical PDZ domains. (C) The F_O−F_C map of the bound Cx43 peptide showing that the densities of all nine residues can be clearly assigned. The map is calculated by omitting the peptide from the final PDB file and contoured at 2.5σ.

Figure 2

Domain-swapped dimerization is required for ZO-1 PDZ2 to bind to Cx43. (A) ¹H–¹⁵N HSQC spectrum of the GGGA insertion mutant of ZO-1 PDZ2. The well-dispersed spectrum indicates that the mutant is well folded. (B) Sedimentation equilibrium analysis showing that the GGGA insertion mutant ZO-1 PDZ2 is a stable monomer. The insert shows a schematic model illustrating the GGGA insertion-induced conversion of the ZO-1 PDZ2 dimer into a monomer. (C) Plot of backbone amide chemical shift differences as a function of the residue number of ZO-1 PDZ2 between the wild-type dimer and the GGGA insertion monomer. The two residues (Lys209 and Glu238) forming inter-domain salt bridges in the wild-type ZO-1 PDZ2 are highlighted using red stars. The secondary structures of PDZ2 are also indicated at the top of the figure. The ribbon diagram shows the shift changes mapped onto the 3D structure of the ZO-1 PDZ2 dimer. In this representation, the combined ¹H and ¹⁵N chemical shift changes are defined as: where Δδ_HN and Δδ_N represent chemical shift differences of amide proton and nitrogen chemical shifts of the each residue of ZO-1 PDZ2. The scaling factor (α_N) used to normalize the ¹H and ¹⁵N chemical shift is 0.17. (D) Fluorescence-based measurement of the binding affinities of the wild-type ZO-1 PDZ2 domain and the GGGA insertion mutant towards the Cx43 peptide. (E) Comparison of the cellular localizations of the GFP-tagged full-length wild-type ZO-1 and the ZO-1 mutant with the GGGA insertion in its PDZ2 in HeLa cells. The overexpressed wild-type ZO-1 forms plaques at the contact regions between transfected cells, whereas the ZO-1 mutant transfected cells lack such intercellular ZO-1 punta.

Figure 3

The domain-swapped dimer interface of ZO-1 PDZ2 provides a distinct specificity-determining binding site for Cx43. (A) Structure-based sequence alignment of ZO-1 PDZ2 from different species. In this alignment, the conserved hydrophobic residues are shown in orange, negatively charged residues in magenta, positively charged residues in blue and the rest of the highly conserved residues in cyan. The residues that are directly involved in the binding to the Cx43 peptide are boxed and highlighted with red stars. (B) Stereo view showing the detailed interactions of the Cx43 peptide with the residues from the swapped dimer of ZO-1 PDZ2. The hydrogen bonds involved in the binding are shown as the dotted lines. (C) Surface representation showing the binding interface between the ZO-1 PDZ2 and the Cx43 peptide. In this presentation, the hydrophobic amino-acid residues in PDZ2 surface model are drawn in yellow, the positively charged residues in blue, the negatively charged residues in red and the uncharged polar residues in grey. The Cx43 peptide is shown in the stick model. (D) The combined stick-dot model and the ribbon representation showing the charge–charge interaction network formed in the ZO-1 PDZ2–Cx43 peptide complex.

Figure 4

The interaction between Cx43 and ZO-1 PDZ2 could be regulated by the phosphorylation of Cx43. (A) Amino-acid sequence alignment of the C-terminal tail of Cx43 from different species. The highly conserved Pro(−5) is highlighted with a red star. Three well-conserved SSR repeats are boxed. Ser(−9) and Ser(−10) of Cx43 are highlighted with red triangles. (B) Overlay plot of a selected region of the ¹H–¹⁵N HSQC spectrum of free ZO-1 PDZ2 and the protein in the presence of saturating amounts of short and long Cx43 peptides. (C) Superposition plots of the ¹H–¹⁵N HSQC spectrum of free ZO-1 PDZ2 and with excess amount of the short, long, long S(−9)/E, long S(−10)/E, and phosphor-S(−9) Cx43 peptides, For clarity, only three well-resolved peaks are shown.

Figure 5

The specific interaction between ZO-1 PDZ2 and Cx43 is required for the formation of Cx43 GJs. (A1–C4) Comparison of the cellular localizations of the wild-type Cx43 and the S(−10)E, S(−9)E mutant of Cx43 in HeLa cells. The endogenous ZO-1 was stained with an anti-ZO-1 antibody and various forms of Cx43 were visualized by fluorescence signals from GFP. DAP staining (A3, B3, and C3) was used to show nuclei of cells. Colocalization of the wild-type Cx43 signal with the endogenous ZO-1 plaques between two adjacent transfected cells is indicated by arrowheads (A1–A4). Empty arrows indicate ZO-1 plaques between two adjacent cells that were transfected by GFP vector control or the GFP-Cx43 S(−10)E, S(−9)E mutant. The arrows highlight the endogenous ZO-1 plaques between two adjacent, untransfected cells. (D) A structural model showing six Cx43 pack together to form a connexon through their transmembrane helices. The Cx43 connexon model is built based on the connexon structure (PDB id: 1TXH). The cytoplasmic side of the connnexon points to readers and the distance between the two helix tails from two adjacent Cx43 is labelled. (E) The ribbon diagram representation showing the distance between two Cx43 peptides in the ZO-1 PDZ2–Cx43 peptide complex structure. In this drawing, the distance between the Cα atoms of Arg(−8) in Cx43 peptides is labelled. (F) A cartoon model showing the synergistic interactions between multimerized ZO-1 and polyvalent Cx43 connexons. In this mode, the hexameric Cx43 connexon can bind to the domain-swapped dimer of ZO-1 PDZ2, thereby greatly enhancing both the affinity and specificity of the binding of Cx43 connexon to ZO-1. Multimerization of ZO-1 through its SH3-GuK module can further enhance the interaction avidity and specificity.