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Biochemistry. 2017 May 23;56(20):2584-2593. doi: 10.1021/acs.biochem.7b00078. Epub 2017 Apr 14.

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

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Institute for Molecular Bioscience, The University of Queensland , St. Lucia, Brisbane, Queensland 4072, Australia.
Department of Chemistry, City College of New York , New York, New York 10031, United States.


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.

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