Modeling and Mutation of NHERF1 Dimerization Domains

Abstract

Na/H Exchange Regulatory Factor-1 (NHERF1) is a cytoplasmic scaffolding protein that regulates parathyroid hormone receptor (PTHR) ligand bias, trafficking, and signaling. Mice lacking NHERF1 present a constellation of mineral ion abnormalities and are osteopenic. NHERF1 consists of 2 tandem PDZ domains and a MERM domain that itself terminates with a PDZ ligand (EB). Recently, 3 NHERF1 mutations were described (N Engl J Med 359: 1128-1135, 2008) in patients who exhibited pathologies similar to the mice. Two mutations (R153Q, E225K) are in PDZ2, and the third, L110V is in the linker region between PDZ1 and PDZ2. In earlier work we showed that the PTHR binds productively to either PDZ1 or PDZ2. Therefore, we hypothesized that the described mutations interfere with NHERF1 dimerization. To probe such interactions we performed computational mutations using thermodynamic integration method with atomistic Molecular Dynamics (MD) simulations to quantitatively assess these predictions. We investigated interactions of EB with PDZ2, R153Q PDZ2 or E225K PDZ2 mutant. For the PDZ2-EB system it was assumed that the C-terminus LFSNL (L) of EB interacts with PDZ2. We confirmed that EB has a higher affinity to R153Q-PDZ2 than to PDZ2 or E225K-PDZ2. In the simulations the complex of EB with the mutated R153Q-PDZ2 is predicted to be more stable than that with the wild type PDZ2 by approximately 2 kcal/mol. Molecular biological methods show that wild-type NHERF1 dimerizes. NHERF1-R153Q abolishes dimerization; NHERF1-E225K reduces dimerization; and NHERF1-L110V dimerizes normally. Dimerization is enhanced in the presence of the PTHR carboxy terminus, which includes the PDZ ligand. Importantly, NHERF1 mutations do not disrupt interaction with the PTH1R. We conclude that NHERF1-R153Q and NHERF1-E225K may undermine PTHR action because of compromised dimerization. NHERF1-L110V interferes with PTHR action through a different mechanism.