Dynamic Intra-Ligand Binding Reveals a New Function for PDZ Domains

Abstract

PDZ domains canonically interact with the C-termini of partner proteins; however, they also bind internal motifs. In this issue of Structure, Kumar et al. ¹ reveal a novel function of PDZ domains, revealing a dynamic binding mode that alternates between a C-terminal and an internal motif within the same ligand.

PDZ (PSD-95/Discs-large/ZO-1) domains are small protein-protein interaction modules that play crucial roles in various cellular processes, including cell adhesion, synapse formation, and signal transduction. These domains are highly prevalent, forming a family of approximately 200 human proteins that collectively contain nearly 260 PDZ domains. Their primary function is to bind specific protein targets and localize signaling complexes to subcellular regions essential for biological activity ². Traditionally, PDZ domains are known to recognize and bind the extreme C-terminal residues of their partner proteins. Although though less common, they also interact with internal sequences. Class III PDZ ligands are characterized by a C-terminal motif of X-D/E-G-Φ_COOH, where Φ denotes a hydrophobic residue. The role of the acidic D/E residue in PDZ binding has been relatively understudied, yet it may confer additional flexibility to the binding mechanism. This is due to the carboxylate side chain of D/E, which could facilitate interactions with internal ligand sequences. This possibility suggests that PDZ/class III ligand interactions may possess greater biochemical and biological complexity than previously appreciated.

The Wnt signaling pathway plays a crucial role in embryonic development, cell proliferation, polarity, migration, and tissue morphogenesis. It consists of two main branches: canonical and non-canonical. The core signaling module includes the Frizzled (FZD) receptor, various co-receptors, the Wnt protein ligand, and the Dishevelled (DVL) protein, which contains a PDZ domain (Figure 1A). In the canonical pathway, Wnt signaling regulates the degradation of β-catenin. In the absence of Wnt ligands, β-catenin is targeted for degradation, thereby inhibiting cell proliferation. When Wnt is present, DVL is recruited to FZD via its PDZ domain and interacts with Axin, a key component of the β-catenin destruction complex. This interaction precludes β-catenin degradation, allowing it to accumulate and translocate to the nucleus, where it activates transcription of Wnt target genes. The non-canonical pathway, also activated by Wnt ligands, operates independently of β-catenin. It involves distinct co-receptors, DVL, and other signaling proteins to regulate processes such as intracellular calcium signaling and planar cell polarity ^2,3.

Figure 1.

The Wnt signaling pathway and the Dishevelled (DVL) PDZ domain dual binding mode switching mechanism.

(A) A simplified schematic of the canonical and non-canonical branches of the Wnt signaling pathway. The Frizzled co-receptors LRP5/6 and ROR2 are depicted. Adapter proteins such as GSK, CK and DAAM1 can interact with DVL alone or when in complex with Frizzled.

(B) The dual-binding mode switching mechanism found in the DVL PDZ domain.

(C) A possible mechanism for the regulation of DVL activity by PDZ domain dual mode switching. Dual mode switching allows for C-terminus availability to post-translational modifications such as polyglutamylation and phosphorylation, which may allosterically regulate transition into the DVL active state.

In this study, the authors investigate Dishevelled (DVL), a protein that contains a PDZ domain known for binding a wide array of partners through both C-terminal and internal motifs ^3,4. Importantly, the PDZ domain of DVL can interact with its own C-terminus, a self-association that plays a crucial role in maintaining its autoinhibited conformation and dictating downstream signaling outcomes ⁵. Additionally, DVL undergoes polyglutamylation – a reversible post-translational modification that appends variable numbers of glutamate residues to its C-terminus. This modification has been shown to enhance non-canonical Wnt signaling and reduce the formation of biomolecular condensates (i.e., phase separation), which is associated with subcellular localization and tissue morphogenesis ⁶.

In this issue of Structure, Kumar et al. report the crystal structure of the DVL PDZ domain bound to a peptide derived from its own C-terminus. Unexpectedly, they discovered that this interaction occurs via two distinct binding modes: the classical C-terminal mode and a novel internal mode. This surprising finding forms the cornerstone of their study.

To understand the requirements for stable PDZ domain interactions with either C-terminal or internal motifs, the authors developed a bacterial expression system to produce a peptide comprising the C-terminal 24 amino acids of DVL. This system enabled isotopic labeling with either ¹⁵N or ¹³C-methyl precursors for detection in solution nuclear magnetic resonance (NMR) experiments. Using this peptide, the authors engineered variants containing both the terminal and internal (D714) carboxylates, either carboxylate alone, or neither. The D714T variant removed the internal carboxylate, while the addition of 12 C-terminal glutamates (12E) mimicked polyglutamylation, effectively eliminating the C-terminal carboxylate of the PDZ-binding motif. Next, the team employed solution NMR-based two-dimensional ¹H-¹⁵N Heteronuclear Single Quantum Correlation (HSQC) titration experiments to analyze binding and interaction dynamics. These experiments revealed that at least one carboxylate moiety is essential for DVL PDZ/ligand interaction. Intriguingly, the data also suggested that in the native peptide sequence both binding modes may coexist and dynamically exchange. However, due to extreme peak broadening in the NMR spectra – likely caused by exchange between free and ligand bound forms of the PDZ domain – quantitative analysis of the binding dynamics could not be performed.

Quantitative analysis of the exchange between two binding modes required minimizing contributions from ligand binding and unbinding to the DVL PDZ domain. To achieve this, the C-terminal ligand was fused directly to the DVL PDZ domain – a biochemical strategy previously employed to resolve PDZ domain complex structures ⁷. This fusion maintains a high local ligand concentration, effectively saturating the PDZ domain with ligand and suppressing binding/unbinding exchange contributions. Over the past 25 years, NMR-based relaxation techniques have been developed to quantitatively probe exchange processes occurring on the millisecond to microsecond timescale, such as those between distinct binding modes ⁸. Kumar et al. applied advanced relaxation dispersion experiments using the C-terminal fusion strategy. Their native-like construct, containing both C-terminal and internal carboxylates, exhibited pronounced dispersion profiles indicative of conformational exchange. In contrast, both the D714T and 12E fusion constructs showed no such profiles, suggesting stable, non-exchanging complexes. Quantitative analysis revealed that the two binding modes are approximately equally populated, with an exchange rate (k_ex) of 477 s⁻¹. These findings demonstrate that the DVL class III ligand dynamically interconverts between at least two conformations (Figure 1B).

Collectively, Kumar et al.’s data provide new insights into class III PDZ ligands. Notably, they show that a class III ligand can bind a PDZ domain via either a canonical C-terminal or an internal binding mode, and that these modes interchange on a biologically relevant timescale. Furthermore, polyglutamylation modulates this dynamic behavior. Their crystal structure elucidated the molecular basis for dual binding, explaining how the DVL PDZ domain accommodates both modes. However, there remain some open questions. With nearly 700 predicted class III ligands in the human genome, the generality of this dual binding phenomenon is unclear ⁹. While this study defines the structural basis for DVL’s interaction with its own C-terminus and internal carboxylate, other sequence, structural, and dynamic features likely influence PDZ/class III interactions across the proteome. Future bioinformatic and structural studies are needed to uncover the broader principles governing these interactions.

The findings by Kumnar et al. also have implications for Wnt signaling regulation. The DVL PDZ domain plays a pivotal role in selecting between canonical and non-canonical Wnt pathways. In its autoinhibited state, DVL adopts a closed conformation via intramolecular PDZ/C-terminal binding, and disruption of this interaction promotes non-canonical signaling. Post-translational modifications, including phosphorylation and polyglutamylation, regulate this switch. Phosphorylation at multiple sites, including the PDZ domain, disrupts PDZ/ligand binding, relieving autoinhibition and promoting activation. It also facilitates the dissolution of DVL condensates ⁶. Similarly, polyglutamylation disrupts condensates but through a distinct mechanism. Significantly, the DVL E710/D714A mutant cannot be polyglutamylated in vivo, resulting in a persistently autoinhibited state ¹⁰. This highlights an additional regulatory role for the D714 residue in polyglutamylation. Kumar et al. further show that polyglutamylation favors the internal binding mode, though a direct link between binding mode switching and functional activation remains to be established. It is possible that the presentation of both carboxylate motifs provides access to post-translational modifications, like polyglutamylation, that act as allosteric regulators of activation (Figure 1C). Further studies using DVL ligand variants will be essential to fully elucidate the functional significance of this exchange mechanism.

In summary, Kumar et al. describe a novel dual binding mode-switching mechanism that introduces a new functional element to PDZ domains. This discovery expands upon the well-established C-terminal and internal binding modes and challenges the traditional binary classification of PDZ binding modes. Importantly, it introduces dynamic conformational exchange between binding modes within the same ligand as another fundamental mechanism for regulation of PDZ domain signal transduction. Given the abundance of class III PDZ ligands in the human proteome, it is likely that additional variations on the dual mode-switching theme identified by Kumar et al. remain to be uncovered. More than 30 years after their discovery, PDZ domains continue to reveal unexpected and fascinating complexities.