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. 2013 Mar 5;21(3):342-54.
doi: 10.1016/j.str.2013.01.004. Epub 2013 Feb 7.

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics

Xu Liu  1 , Tyson R Shepherd, Ann M Murray, Zhen Xu, Ernesto J Fuentes
Affiliations

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics

Xu Liu et al. Structure. .

Abstract

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites.

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Figures

Figure 1
Figure 1. Tiam1 PDZ Domain Binding Affinity for Syndecan Family Proteins
(A) The C termini (C2 region) of syndecan family members and Caspr4. (B) Representative binding curves for the interaction between the Tiam1 PDZ domain and dansylated peptides derived from phosph-syndecan1 (▲), syn-decan1 (●), syndecan2 (○), syndecan3 (■), and syndecan4 (▼). *Data taken from Shepherd et al. (2010).
Figure 2
Figure 2. Thermodynamic Parameters of Tiam1 PDZ/Peptide Interactions Determined by ITC
(A) Thermograms and integrated titration curves are shown for phosphorylated syndecan1 (left panel), syndecan1 (middle panel), and Caspr4 (right panel). (B) Thermodynamic parameters for PDZ/peptide interactions at 25°C. The change in enthalpy (ΔH), association constant (Ka), and stoichiometry (n) were fit by nonlinear least squares analysis using a single-site binding model in ORIGIN software. The reported thermodynamics parameters are the average of three individual experiments. See also Figure S1 and Supplemental Experimental Procedures.
Figure 3
Figure 3. Structures of the Tiam1 PDZ Domain Bound to Syndecan1 and Phospho-Syndecan1
(A and B) Stick models showing side-chain and backbone interactions in the Tiam1 PDZ/SDC1 and PDZ/pSDC1 complexes, respectively. PDZ-domain residues involved in peptide binding are labeled and colored yellow. Dotted lines indicate hydrogen-bond interactions. (C and D) Space-filling models of the Tiam1 PDZ domain bound to SDC1 and pSDC1 peptides (shown as stick models), respectively. The peptide is colored green; PDZ-domain residues involved in peptide binding are labeled and colored red. See also Table S2 and Figures S1 and S2.
Figure 4
Figure 4. Structural Features of the pSDC1 Phosphotyrosine Binding Pocket
(A) The structures of the Tiam1 PDZ domain (gray) bound to SDC1 (green) or pSDC1 (cyan) are overlaid. Side chains involved in forming the S−1 and S−2 binding pockets are represented as stick lines. This view highlights the 90° rotation of the P−1 tyrosine upon phosphorylation. (B) The electrostatic potential surface of the PDZ domain in the Tiam1 PDZ/pSDC1 complex. The electrostatic surface is colored continuously from red to blue (−1.0 to +1.0 keV). The electrostatic potential calculation was performed in PyMol (v1.4) using the APBS module. (C) Structure-based amino acid alignment of PDZ domains that bind to syndecan proteins. See also Table S2 and Figure S5.
Figure 5
Figure 5. The Pico- to Nanosecond Timescale Dynamics of the Free Tiam1 PDZ-Domain Main Chain and Its Response to Syndecan1 Binding
(A and B) The order parameter (S2), timescale of motion (τe, ●), and chemical exchange (Rex, ▲) of the free Tiam1 PDZ domain plotted for amides along the backbone. Arrows (β strand) and rectangles (α helix) indicate secondary structure of the PDZ domain. Error bars represent the uncertainty as derived from Monte Carlo simulations. (C) The change in backbone order parameter (ΔS2) caused by SDC1 binding. Residues that experience significant changes in this parameter are colored black. See also Figures S3 and S4.
Figure 6
Figure 6. Dynamics of the Methyl Side Chains of Tiam1 PDZ Domain Complexes
(A) The change in S2axis and τe caused by SDC1-binding. (B) The change in S2axis and τe caused by pSDC1-binding. (C) The change in S2axis and τe caused by Caspr4-binding. Three regions that showed significant changes in ΔS2axis (ΔS2axis = S2axis,bound − S2axis,apo) and Δτe (Δτe = τe,bound − τe,apo) were the β1-β2 loop (i.e., carboxylate-binding loop) (shaded gray), the β3-α1 region (shaded gray), and the peptide-binding site (the remaining black bars). The error bars represent propagated uncertainty, as derived from Monte Carlo simulations.
Figure 7
Figure 7. Distinct Dynamics Responses of Methyl Groups of Tiam1 PDZ/Ligand Complexes
(A–C) Side-chain methyl groups with significant changes in dynamics parameters are mapped onto structural models of the PDZ/SDC1, PDZ/pSDC1, and PDZ/Caspr4 complexes, respectively. The methyl groups (spheres) are colored in a continuous gradient from red to blue, with their intensity scaling to the magnitude of ΔS2axis. Methyl groups colored yellow had a significant Δτe. The PDZ/SDC1 crystal structure was used as a template to model the PDZ/Caspr4 complex. Residues Y858 and F860 discussed in the text are colored yellow and shown as sticks.
Figure 8
Figure 8. A Model Depicting Ligand-Dependent Dynamic Communication between Regions in the Tiam1 PDZ Domain
The left panel shows the Tiam1 PDZ/SDC1 structure and the three regions whose dynamics were perturbed upon ligand binding (red, β1-β2 loop; blue, β3-α1 region; gold, SDC1-binding site). Phosphorylation of the SDC1 P−1 tyrosine residue induces a conformational change that flips this residue into a groove at the junction of α1 helix and the β1-β2 loop (right panel). The conformational switch decouples the dynamics in the β3-α1 region from those at the ligand-binding site.
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