Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor

Abstract

Na⁺/H⁺ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition.

Figure 1

The structure of PDZ1 and PDZ2 in complex with PTHRct-9. The PDZ1 and PDZ2 domains are highlighted in wheat cartoon, whereas the peptide is represented in green sticks. The key residues stabilizing the complex are highlighted and labeled on the structure. Plausible electrostatic interactions involved Glu⁻³, Glu⁻⁵ and Glu⁻⁶ are discussed in the text. The dotted line represents a hydrogen bond or salt bridge between a hydrogen atom and acceptor with a distance labeled in Å. Hydrogen atoms are white, oxygens are red, and nitrogens are blue. N-terminal Leu and Gln of PTHRct-9 are not shown for simplicity.

Figure 2

(A) MS-MS spectrum of the C-terminus of PTHR. The peak heights show the relative abundances of the corresponding fragmentation ions, with the annotation of the identified matched amino terminus-containing b ions in blue and the C-terminus-containing y ions in red. (B) In FP competition assays a mixture of FITC-PTHRct-9 (0.5 μM) and PDZ1 (7 μM) was incubated with increasing concentrations of the unlabeled PTHRct-9, Cys-PTHRct-9 or PTHR-pThrct-9 (competitors). (C) PTHR-Cys591 exhibited similar cAMP formation in response to challenge with PTH(1–34) as wild-type PTHR.

Figure 3

Binding curve (left) and sensorgrams (right) from SPR experiments for b-PTHRct-22 bound PDZ1 and PDZ2 with the affinity reported in Table 2. SPR experiments were carried out three times; standard errors are presented in Table 2.

Figure 4

Thermograms (top) and binding isotherms (bottom) from the ITC experiments measuring the binding of the PTHRct-8 peptide with PDZ1 and PDZ2 with the affinity reported in Table 2.

Figure 5

Linear plot of ΔH° vs. ΔS° for the binding of the wild-type and the ensemble of Ala variants of PTHRct-8 to the PDZ1 and PDZ2 domains (using the data from Table 4). Experimental data and linear regression line are blue and red, respectively.